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FUNDAMENTALS  OF  CYTOLOGY 


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FUNDAMENTALS 

of 

CYTOLOGY 


BY 

LESTER  W.  SHARP 

Professor  of  Botany,  Cornell  University 


First  Edition 


McGRAW-HILL  BOOK  COMPANY,  Inc. 

NEW     YORK     AND     LONDON 

19  4  3 


fundamentals  of  cytology 

Copyright,   1943,  by  the 
McGraw-Hill  Book  Company,  Inc. 


PRINTED    IN    THE    UNITED    STATES    OF    AMERICA 

.1//  rights  reserved.   This  book,  or 

parts  thereof,  may  not  be  reproduced 

in  any  form  without  permission  of 

the  pvblishers. 


THE  MAPLE  PRESS  COMPANY,  YORK,  PA. 


To  my  wife 
MABEL  GUNTHER  SHARP 


PREFACE 

THIS  book  is  intended  for  use  in  connection  with  college  and  university 
courses  in  the  biological  sciences.  It  is  not  a  new  edition  of  the 
author's  "Introduction  to  Cytolog.y,"  which  was  designed  to  serve  both 
as  a  reference  work,  of  which  plant  cj^tology  was  in  need,  and  as  a  general 
textbook.  This  is  a  textbook  only.  Although  it  deals  again  with 
various  principal  topics  of  the  older  work  and  uses  some  of  its  illustrations 
along  with  manj^  new  ones,  the  present  text  is  much  briefer,  simpler  in 
treatment,  and,  it  is  hoped,  better  adapted  to  the  needs  of  students  whose 
curiosity  concerning  cytology  has  been  newly  aroused  in  elementary 
courses  in  animal  and  plant  science.  The  title  of  the  oldei-  book,  in  fact, 
might  well  have  been  reserved  for  this  one.  Where  subjects  not  essential 
to  an  introductory^  treatise  are  included,  it  is  not  with  the  thought  that 
they  should  be  mastered  now,  but  rather  to  indicate  further  fields  of 
inquiry  open  to  students  of  cytology. 

No  attempt  has  been  made  to  maintain  the  connection  between 
statement  and  source  characteristic  of  the  "Introduction  to  Cytology." 
Each  chapter  has,  however,  been  provided  with  a  short  list  of  books  and 
recent  papers  for  the  use  of  those  who  wish  to  begin  an  examination  of 
the  literature,  but  to  avoid  distraction  the  text  has  been  left  unencum- 
bered by  specific  references  to  them.  Anyone  who  prepares  a  book  of 
this  kind  knows  well  that  he  is  presenting  mainly  the  work  of  authors 
whose  names  do  not  appear  on  the  title  page,  and  he  only  trusts  that  he 
does  this  with  accuracy  and  fairness. 

The  selection,  arrangement,  and  treatment  of  the  various  topics 
have  been  determined  by  experience  in  cytology  courses  having  a  geneti- 
cal  and  phylogenetical  bearing.  In  the  interest  of  simplicity,  emphasis 
has  been  placed  upon  "typical"  cytological  phenomena.  Care  has  been 
taken,  however,  to  suggest  the  great  diversity  in  cytological  constitution 
and  behavior  exhibited  by  plants  and  animals  and  to  indicate  the  many 
uncertainties  in  a  field  where  growth  is  rapid  and  opinions  are  subject  to 
change  with  new  evidence.  It  is  unfortunate  that  the  terminology 
could  not  be  further  simplified  without  impairing  the  usefulness  of  the 
book  for  those  preparing  to  consult  the  literature.  As  every  experienced 
teacher  of  natural  science  is  aware,  the  student's  actual  knowledge  of  the 
subject  is  gained  primarily-  in  the  field  and  laboratory.  It  is  there, 
by  thoughtful  observation  and  discussion,   that   his   comprehension  is 


viii  PREFACE 

developed;  hence,  a  textbook  .such  as  this  one  should  be  regarded  as  an 
adjunct  to  this  process  and  never  as  a  substitute  for  it. 

It  is  a  pleasant  duty  to  acknowledge  indebtedness  to  authors  and 
commercial  firms  who  have  so  generousl}^  contributed  illustrations. 
The  author  is  especially  grateful  to  his  associates  at  Cornell  University 
for^ their  willing  help:  to  Professors  Lewis  Knudson,  Arthur  J.  Eames, 
Lowell  F.  Randolph,  Robert  T.  Clausen,  and  Dr.  Victor  M.  Cutter  for 
criticisms  of  certain  chapters,  to  Miss  Louise  Raynor  for  executing  the 
original  drawings  and  preparing  the  index,  and  to  Miss  Lillian  Piepei- 
for  her  assistance  with  the  manuscript. 

Lester  W.  Shakp. 
Ithaca,  New  York, 
June,  1943. 


CONTENTS 


Page 

Preface vii 

CHAPTER  I 

The  Position  of  Cytology  in  Biological  Science 1 

Biology — Cytology — The  Classic  Period  of  Cytology — Cytology  in  the 
Twentieth  Century — What  Cytology  Is. 

CHAPTER  11 

The  Organism  and  the  Cell 11 

The  Typical  Protoplast — Growth — Differentiation — ('orrelatif>n — Tissues 
and  Organs — Conclusions. 

CHAPTER  111 

The  Structural  Components  of  Protoplasts 22 

Cytoplasm — Nucleus — Centrosome — Plastids — Chondriosomes — Vacuoles 
— Golgi  Material — Ergastic  Matter. 

CHAPTER  IV 
Protoplasm 44 

Physical  Characters — Protoplasm  as  a  Colloidal  System — Chemical  Nature 
of  Protoplasm — The  Staining  of  Protoplasm — Conclusions. 

CHAPTER  V 

Division  of  the  Protoplast ."JG 

Somatic  Cell  Division  in  Plants:  Mitosis  and  Cytokinesis — Somatic  Cell 
Division  in  Animals:  ^Mitosis  and  Cytokinesis — Further  Aspects  of  Cell 
Division:  C-auses  of  Anaphasic  Chromosome  Movement — Time  Occupied  hj- 
Cell  Division — The  Shape  of  Cells  in  Tissues. 

CHAPTER  VI 

The  Cell  Walls  of  Plants 75 

Development  of  the  Wall — Minute  Structvire  of  the  Cell  Wail — Plasmodesms 
— Formation  of  Cellulose  by  the  Protoplast. 

CHAPTER  VII 

The  Chromosomes 84 

Somatic  Chromosomes — Meiotic  Chromosomes — Salivary-gland  ( 'liromo- 
somes — Chromo.some  Complements — Conclusions. 

CHAPTER   VI II 

IMeiosis 102 

Distribution  of  the  Chromosomes  in  the  Meiotic  Divisions — Detailed  Ac- 
count of  the  Phases  of  Moiosis — Problems  of  Mciosis. 


.>7819 


X  CONTENTS 

I'aoe 

CHAPTER  IX 
Cytology  of  Reproduction  in  Animals 118 

The  Germ  Cells — Spermatogenesis — Oogenesis — Synganiy — Cleavage — 
Parthenogenesis — Protozoa. 

CHAPTER  X 

Cytology  of  Reproduction  in  Angiosperms 136 

The  Flower — Microsporogenesis  and  the  ]\Iale  Gametopyhte — Megasporo- 
genesis  and  the  Female  Gametophyte — Syngamy — Embryogeny  and  Seed 
Development — Aberrations  of  the  Reproductive  Process — Conclusions. 

CHAPTER  XI 

Cytology  of  Reproduction  in  Plants  Other  than  Angiosperms  148 
Gymnosperms — Ferns — Mosses    and    Liverworts — Algae — Fnngi — Conclu- 
sions. 

CHAPTER  XII 

Cytology  and  Mendelian  Heredity 168 

Examples  of  Mendelian  Heredity — Terminology — Explanation  in  Terms 
of  Chromosomes — Assignment  of  Genes  to  a  Chromosome — Recombination 
and  Crossing  Over — Position  of  Genes  in  the  Chromosome — The  Special 
Case  of  Sex — Sex-linkage — Summary  and  Conclusions. 

CHAPTER  XIII 

Chromosomal  Aberrations 193 

Deletion — Inversion — Translocation — Duplication — Aberrations    and     the  . 
Nature  of  the  Gene. 

CHAPTER  XIV 

Chromosome  Numbers  and  Their  Alteration 204 

Terminology — Tetraploidy — Triploidy — Higher  Degrees  of  Polyploidy — 
Other  Types  of  Heteroploidy — Significance  of  Autoheteroploidy  in  Nature. 

CHAPTER  XV 

Cytological  Aspects  of  Hybridity 216 

Chromosome  Behavior  in  Diploid  Hybrids — Hybridity  Involving  Polyploid}' 
— Amphidiploidy — Cytological  Types  of  Hybriditj' — Conclusions. 

CHAPTER  XVI 

The  Role  of  the  Cytoplasm  in  Development  and  Heredity.    .   227 
Reciprocal  Crosses — Chlorophyll  Characters — Conclusions. 

CHAPTER  XVII 

Cytology  and  Taxonomy 234 

Cytological  Evidence  of  Relationship:  Chromosome  Number — Chromo- 
some Morphology — Chromosome  Behavior  in  Crosses — Aberrations  in 
Reproduction — The  Role  of  Chromosomal  Changes  in  Speciation — Experi- 
mental Taxonomy — Conclusions. 

Suggested  Rk.vding 251 

Index 261 


FUNDAMENTALS  OF 
CYTOLOGY 

CHAPTER  I 
THE  POSITION  OF  CYTOLOGY  IN  BIOLOGICAL  SCIENCE 

Man  has  been  led  by  his  native  curiosity  and  his  desire  to  understand 
the  world  in  which  he  lives  to  examine  ever  smaller  natural  objects. 
Rocks,  animals,  plants,  and  even  the  stars  themselves  he  has  regarded 
first  as  wholes,  then  as  organizations  of  visible  parts,  later  as  systems  of 
invisible  molecules  and  atoms,  and  eventually  as  vast  arrays  of  subatomic 
units.  In  the  earlier  stages  of  his  investigations,  he  depended  solely 
upon  his  unaided  senses,  whereas  the  later  steps  were  possible  only  after 
he  had  devised  special  instruments,  such  as  the  microscope,  telescope, 
spectroscope,  chemical  balance,  vacuum  tube,  and  cyclotron.  With 
these  powerful  aids,  he  has  been  able  to  extend  his  observations  into  the 
realms  of  the  infinitely  remote  and  the  infinitely  small;  and  despite  the 
indirectness  of  approach  involved,  they  have  led  him  to  knowledge  of 
the  utmost  significance. 

Biology. — Animals  and  plants  must  have  engaged  primiti\'e  man's 
attention  from  his  earliest  days.  In  his  struggle  to  keep  alive,  he  was 
inevitably  led  to  the  discovery  that  certain  constituent  parts  of  these 
organisms  would  serve  him  well  as  food,  clothing,  and  fibers.  Even 
his  superstitions  may  have  contributed  something  to  his  knowledge,  for 
in  practicing  the  art  of  divination  he  could  scarcely  have  failed  to  observe 
much  in  the  anatom}^  of  animals  that  was  new  to  him. 

A  more  scientific  interest  in  the  constitution  of  organisms  had  become 
well  developed  in  Greece  by  the  fourth  century  B.C.,  when  Aristotle  and 
Theophrastus  produced,  respectively,  their  famous  treatises  on  animals 
and  plants.  One  can  well  imagine  the  wish  of  these  men  for  keener 
vision.  In  Aristotle's  work  on  the  generation  of  animals  it  is  all  too 
evident  that  without  more  trustworthy  information  regarding  minute 
structures  further  speculation  could  hardly  lead  him  nearer  to  a  true 
solution  of  the  fundamental  biological  problems  that  held  his  interest. 

It  was  not  until  many  centuries  later  that  the  small  tissue  elements 
lying  beyond  the  reach  of  unaided  vision  could  be  investigated.     vSimple 

1 


2  FUNDAMENTALS  OF  CYTOLOGY 

lenses  were  probably  first  used  seriously  as  optical  instruments  sometime 
in  the  eleventh  century,  while  the  first  compound  microscopes  (and 
telescopes)  were  devised  at  the  end  of  the  sixteenth  and  the  beginning  of 
the  seventeenth  century.  A  number  of  models  of  simple  and  compound 
instruments  soon  became  available,  and  these  in  the  hands  of  many  men, 
professional  and  amateur,  made  possible  a  number  of  researches  on  the 
minute  anatomy  of  living  beings  that  still  excite  our  admiration. 

Even  when  aided  by  the  microscope,  however,  vision  is  limited.  As 
long  as  the  objects  studied  lie  within  the  visible  range,  direct  observation 
with  or  without  the  aid  of  lenses  may  suffice,  but  when  they  are  smaller 
than  this,  one  must  employ  the  very  different  analytical  methods  famil- 
iar to  the  chemist'  and  physicist.  With  these  methods  have  been  devel- 
oped clear  concepts  of  minute  structure  in  terms  of  molecules  and  atoms. 

Obviously  the  organism  confronts  the  worker  with  many  problems 
which  require  more  than  one  mode  of  approach.  For  example,  in  the 
study  of  the  development  of  an  embryo  or  the  process  of  secretion  in  a 
cell  one  encounters  structural  alterations  visible  with  the  microscope, 
while  accompanying  these  are  invisible  alterations  in  functional  activity 
that  must  be  investigated  by  chemical  means.  The  question  of  the  rela- 
tive priority  of  functional  and  structiu'al  change  is  frequently  debated, 
but,  if  atoms  and  electrons  could  be  as  easily  seen  as  muscles  and  cells,  so 
that  every  functional  reaction  would  appear  also  as  a  visible  change  in 
molecular  structure,  the  question  as  stated  would  probably  not  arise. 

Every  major  biological  problem  thus  comprises  several  partial  prob- 
lems which  must  be  solved  through  the  use  of  different  techniques.  One 
can  no  more  solve  such  a  major  problem  with  a  single  technique  than  a 
carpenter  can  build  a  modern  house  with  a  single  tool.  The  fact  that  the 
organism  sets  such  complex  problems  before  us  began  long  ago  to  resolve 
the  science  of  biology  into  a  number  of  ''fields,"  each  of  them  charac- 
terized by  a  particular  class  of  observations  to  be  made  and  hence  by  a 
particular  set  of  requisite  techniques.  Morphologists,  physiologists,  and 
taxonomists,  for  example,  asked  the  organism  for  three  different  classes  of 
data,  and  they  employed  three  different  methods  of  inducing  the  organism 
to  yield  them.  This  diversification  of  the  science,  which  proceeded 
rapidly  in  the  nineteenth  century,  gave  rise  to  groups  of  specialists  who 
scarcely  understood  the  language  of  other  groups,  yet  all  agreed  in 
employing  one  general  underlying  method- — the  scientific  method  of 
controlled  observation,  formulation  of  hypotheses,  and  verification. 
Upon  this  method  they  will  continue  to  rely,  for  it  is  chiefly  responsible 
for  the  rapid  increase  of  dependable  biological  knowledge  in  recent  times. 

Cytology. — Cytology  as  a  specialized  branch  of  biology  arose  from 
attempts  to  see  more  clearly  the  microscopic  structure  of  plants  and 
animals.     That  the  tissues  of  organisms  have  a  cellular  organization  was 


THE  POSITION  OF  CYTOLOGY  IN  BIOLOGICAL  SCIENCE  3 

demonstrated  b,y  a  number  of  students  of  minute  anatomy  during  tlie 
seventeenth  and  eighteenth  centuries.  The  earliest  published  picture  of 
such  structure  appeared  in  1665  in  a  book  by  Robert  Hooke.  In  the 
-works  of  this  period  the  term  cells  meant  two  things:  simply  cavities 
bounded  by  walls  like  cells  in  a  honeycomb,  or  globules  of  numerous 
unrelated  kinds.  For  the  most  part  they  were  looked  upon  as  subor- 
dinate components  of  tissues  rather  than  imj^ortant  individualized  units. 
Early  in  the  nineteenth  century,  attention  shifted  to  the  "juice,"  or 
"slime,"  which  had  often  been  observed  in  the  cells.     By  the  middle  of 


Fig.  1. — Living  cells  as  they  appear  when  illuminated  against  a  dark  background. 
a,  cell  from  hair  of  squash  plant.  The  cytoplasm  forms  a  thin  sheet  against  the  cell  wall, 
a  series  of  streaming  strands  through  the  cell  sap,  and  a  large  mass  about  the  nucleus. 
{After  M.  Heidenhain.)  b,  chick-embryo  cell  growing  on  glass  surface  in  tissue  culture. 
In  the  cytoplasm  are  fat  globules  and  filamentous  chondriosomes.  (After  T.  S.  P.  Strange- 
ways  and  R.  G.  Canti.) 

the  century  it  had  become  evident  that  this  unique  fluid,  or  'protoplasm 
as  it  came  to  be  called,  was  the  substance  actually  manifesting  the 
phenomena  of  life,  the  thick  walls  observed  in  so  many  plant  tissues 
being  a  product  of  its  activit3^  In  1831,  Robert  Brown  had  pointed  out 
the  nucleus  as  a  normal  and  characteristic  constituent  of  cells.  This 
made  it  easier  to  describe  the  typical  cell  as  a  mass  of  cytoplasm  enclosing 
a  nucleus,  to  distinguish  it  thus  from  other  globules  \\\i\\  which  it  had  so 
often  been  confused,  and  to  regard  it  as  an  important  unit  of  organization. 
To  this  unit  the  more  appropriate  term  protoplast  has  since  been  applied, 
although  cytology's  ow^n  name  remains  as  a  reminder  of  an  earlier  con- 
ception of  the  cell  (kytos  =  hollow  place). 


FUNDAMENTALS  OF  CYTOLOGY 


From  this  point  onward  cytology  stood  out  more  clearly  as  a  distinct 
field  of  study,  and  its  own  problems  became  more  sharply  defined. 
Among  these  w^as  the  task  of  determining  to  what  extent  the  protoplast 
actually  is  "the  unit  of  structure  and  function"  as  was  claimed,  a  question 
to  which  we  shall  revert  in  the  next  chapter.  It  was  soon  discovered 
that  many  very  small  organisms  have  the  structure  of  a  single  protoplast, 
that  gametes  and  spores  are  likewise  single  protoplasts,  and  that  the 
protoplast,  w^hether  constituting  a  whole  organism  or  only  a  portion  of 
one,  multiplies  regularly  by  division  (Fig.  2).  These  discoveries, 
together  with  others  that  followed,  made  it  clear  that  many  kinds  of 
biological   problems  would   have  to  be  attacked  directly   or  indirectly 


^    "^    :':-<," '^-M       \ 


Fig.  2. — Figures  of  division  of  Tradescantia  niicro.sporocytes  published  by  Wilhelm 
Hofmeister  in  1848.  This  was  a  quarter  of  a  century  before  chromosomes,  shown  plainly 
here,  appeared  prominently  in  biological  literature  and  came  to  be  recognized  as  indi- 
vidualized nuclear  units. 

through  the  cell  unit.  To  the  extent  that  such  problems  require  this 
approach,  they  are  cytological.  As  a  matter  of  fact,  much  of  the  signifi- 
cant knowledge  of  the  protoplast  gained  in  recent  years  has  developed 
in  connection  with  questions  that  did  not  at  first  arise  within  the  field  of 
cytology  itself. 

The  Classic  Period  of  Cytology. — The  latter  portion  of  the  nineteenth 
century  Avitnessed  striking  advances  in  biology  and  the  other  sciences. 
It  has  become  customary  to  refer  to  the  last  quarter  of  the  century  as  the 
"classic  period"  of  cytology  because  of  its  many  fundamental  discoveries. 
Some  of  the  principal  accomplishments  of  this  period  are  enumerated  in 
the  following  paragraphs. 

Much  was  learned  concerning  the  structure  of  cells  and  nuclei,  their 
reproduction  by  division,  and  the  behavior  of  their  several  components 
during  the  various  phases  of  cell  activity.     Special  attention  was  focused 


TlIK  J'OSITION  OF  CYTOLOGY  IN  BIOLOGICAL  SCIENCE  5 

ui)()ii  the  remarkable  process  of  mitosis,  the  chromosomes  occupying  an 
csijecially  prominent  place  in  researches  of  the  period.  Noteworthy 
progress  was  also  made  in  studies  of  the  physiological  processes  occurring 
in  cells  and  the  relation  of  these  to  the  activities  of  the  organism  of  which 
they  are  parts.  As  examples  may  be  mentioned  the  advances  in  knowl- 
edge of  mineral  nutrition,  enzymes,  osmotic  concentrations  in  cells,  and 
the  permeability  of  the  plasma  membrane. 

The  life  cycles  of  various  groups  of  plants  and  animals  were  minutely 
described  in  terms  of  the  multiplication  and  differentiation  of  cells.  The 
details  of  cell  and  nuclear  behavior  during  the  organism's  reproductive 
phases  were  examined  with  special  care,  for  it  was  realized  that  two 
successive  generations  of  individuals  are  connected  by  a  minute  single 
cell — in  some  cases  a  spore,  in  others  a  fertilized  egg — and  that  this 
single  cell  must  therefore  contain  in  its  organization  some  kind  of  basis 
for  the  type  of  organism  developing  from  it.  A  discovery  of  cardinal 
importance  was  that  of  the  fusion  of  two  nuclei,  one  from  each  parent,  in 
the  process  of  fertilization  (1875). 

It  was  observed  further  that  certain  organisms  do  not  consist  of  cells 
in  the  usual  sense,  but  are  extensive  and  continuous  masses  of  cytoplasm 
containing  large  numbers  of  nuclei.  Notwithstanding  this  lack  of  internal 
cell  partitions  or  of  fixed  nuclear  positions,  these  organisms  develop 
characteristic  body  forms  and  internal  specializations.  Moreover, 
certain  organisms  mainly  cellular  in  structure  pass  through  a  non- 
cellular  stage  at  some  period  in  their  development  or  possess  certain 
noncellular  tissues  in  the  mature  body. 

Discoveries  like  the  foregoing  not  onh"  furnished  partial  solutions  to 
the  problems  originally  attacked,  Imt  the}^  also  made  it  possible  to 
formulate  with  the  necessary  precision  those  further  special  questions', 
requiring  answers  before  the  problems  in  their  broader  aspects  could  be 
regarded  as  solved.  It  is  thus  bit  by  bit,  rather  than  by  one  stroke,  that 
an  understanding  of  fundamental  biological  processes  is  reached.  Even 
now,  a  centur}^  after  researches  on  the  division  of  cells  began,  we  have 
arrived  at  no  complete  explanation  of  that  remarkable  process. 

Mention  should  be  made  of  technical  advances  made  during  the 
classic  period.  Far  better  methods  for  fixing  and  staining  tissues  were 
devised.  With  the  aim  of  preserving  cells  with  a  more  nearly  natural 
appearance,  careful  studies  were  carried  out  on  the  effects  of  many  rea- 
gents upon  the  various  parts  of  the  protoplast.  Other  investigations 
revealed  the  usefulness  of  coal-tar  dyes  which  were  then  being  produced 
for  the  first  time.  Formerly  onh'-  a  few  natural  dyes  such  as  carmine  and 
hematoxjdin  were  employed,  and  although  these  are  still  extremely 
valuable  today,  the  coal-tar  dj^es  have  added  greatly  to  the  variety  and 
effectiveness  of  staining  procedures.     Vastly  improved  section-cutting 


6  FUNDAMENTALS  OF  CYTOLOGY 

machines,  or  microtomes,  yielded  perfect  and  uniformly  thin  sections 
at  relatively  high  speed.  All  these  aids  were  welcomed  by  cytologists 
and  histologists  whose  work  they  greatlj'  facilitated,  though  they  too 
often  had  the  undesirable  effect  of  diverting  attention  from  living 
material. 

Compound  microscopes  were  brought  to  a  very  high  level  of  efficiency 
during  the  nineteenth  century.  During  the  classic  period,  apochromatic 
objectives,  highly  corrected  against  chromatic  and  spherical  aberrations 
and  with  high  resolving  power,  were  perfected,  and  these,  employed 
with  newl}^  devised  eyepieces  and  condensers,  furnished  the  cytologist 
with  the  finest  images  of  highl}^  magnified  objects  he  had  yet  seen. 

The  closing  years  of  the  century  also  saw  the  formulation  of  theories 
that  gave  direction  to  many  of  the  investigations  yet  to  come.  For 
example,  there  were  biologists,  notably  August  Weismann,  who  pro- 
pounded theories  of  the  mechanism  of  individual  development,  heredity, 
and  evolution  largely  on  the  basis  of  what  had  recently  been  learned 
about  the  behavior  of  cells,  nuclei,  and  chromosomes  throughout  the 
life  cycles  of  organisms.  Some  of  the  concepts  embodied  in  Weismann 's 
theory  of  inheritance  continued  as  a  part  of  the  framework  of  our  modern 
theory,  although  his  theory  of  development  has  been  abandoned.  In 
the  field  of  cellular  physiology  the  theoretical  interpretations  and  laws 
pertaining  to  solutions,  which  grew  out  of  researches  on  osmotic  pressure 
and  permeability  in  cells  and  nonliving  systems,  also  continue  as  strong 
influences  in  the  present  century. 

By  way  of  summary  we  may  say  that  the  nineteenth  century  con- 
tributed to  the  twentieth  a  number  of  fundamental  observations,  certain 
intriguing  problems,  an  array  of  useful  techniques,  and  certain  suggestive 
guiding  theories.  Observation,  technical  skill,  and  theory  are  all 
required  in  scientific  research,  and  progress  is  surest  when  all  three  are 
utilized  in  the  correct  proportions. 

Cytology  in  the  Twentieth  Century. — Cytology  in  the  present 
century  is  fortunate  in  having  several  new  and  extremely  valuable 
technical  aids.  Methods  for  the  successful  cultivation  of  living  animal 
and  plant  tissues  under  controlled  conditions  in  glass  containers  have 
been  devised.  For  instance,  there  are  living  in  flasks  today  (1942) 
healthy  cells  which  are  the  descendants  of  similar  cells  removed  from  the 
heart  of  a  chick  embryo  in  1912  (Fig.  3).  Such  cultures  enable  the 
worker  to  learn  many  things  concerning  the  capacities  of  cells  in  a  direct 
manner  rather  than  by  a  series  of  inferences  from  fixed  preparations  or 
even  from  similar  cells  living  in  the  complex  environment  within  the 
body.  Direct  study  is  also  facilitated  by  the  micromanipulator,  with 
which  one  can  dissect  or  inject  normal  living  cells  under  the  best  high- 
powered  objectives  (Fig.  4).     Striking  permanent  records  of  observations 


THE  POSITION  OF  CYTOLOGY  IN  BIOLOGICAL  SCIENCE  7 

of  this  character,  e.g.,  those  on  cell  division,  have  been  made  with  the 
motion-picture  camera. 

Another  tool  now  in  common  use  among  cytologists  is  the  X-ray  tube. 
The  irradiation  of  living  material  is  now  one  of  the  best  experimental 
means  of  inducinji-  alterations  in  chromosonK^s  for  the  ]-)urposo  of  anal3'zing 


Fig.  3. 


^        \:m^mmit^ if 

-Cells  (fibrobla.sts  from  heart  of  chick  embryo)   descended  from  a  tissue  culture 
begun  bj'  Carrel  in  1912.      (Photograph  by  A.  Carrel;  after  W.  Seifriz.) 


the  role  of  these  bodies  in  development  and  heredity.  X  rays  have  also 
served  to  reveal  the  ultramicroscopic  structure  of  plant  cell  walls. 
Radiations  of  various  other  types  are  being  employed  with  success  in 
connection  \\\ih.  a  number  of  such  fundamental  problems. 


Fig.  4. — Photographs  of  cells  being  operated  upon  with  a  micromanipulator.  A,  B,  C, 
stages  in  the  stretching  of  a  red  blood  corpuscle  from  an  amphibian  between  two  micro- 
needles.     D,  a  micro-needle  entering  a  living  plant  cell.      {After  W.  Seifriz.) 

The  chief  advance  in  microscopy  so  far  in  this  century  is  the  invention 
of  the  electron  microscope.  Instead  of  making  use  of  visible  light  focused 
by  a  series  of  glass  lenses  as  in  the  ordinary  microscope  (photomicro- 
scope),  this  new  instrument  employs  electron  streams  brought  to  a  focus 
by  a  series  of  magnetic  fields.     The  result  is  a  greatly  increased  resolving 


8  FUNDAMENTALS  OF  CYTOLOGY 

power,  i.e.,  the  ability  to  render  fine  detail  in  an  image.  The  photo- 
microscopes  used  for  direct  visual  observation  by  cytologists  reach  the 
limit  of  their  resolving  power  somewhere  between  0.2  and  0.1  ju/  two 
minute  objects  closer  together  than  this  appearing  as  one.  A  single 
particle  somewhat  below  this  limit  may  appear  as  a  dark  or  light  spot, 
depending  upon  the  method  of  illumination,  but  its  true  form  is  not  fully 
revealed.  When  ultraviolet  light,  quartz  lenses,  and  photographic  plates 
are  used,  the  resolving  power  is  about  twice  as  great,  for  it  varies  with 
the  frequency  of  the  radiation  employed.  With  the  electron  microscope, 
however,  the  resolving  power  is  increased  20  to  50  times.  The  material 
to  be  examined  must  be  dry  for  the  best  results,  the  interior  of  the  instru- 
ment where  it  is  placed  for  observation  is  occupied  by  a  vacuum,  and  the 
image  obtained  is  a  shadow  picture  only;  hence  there  are  great  difficulties 
attending  the  study  of  biological  objects.  Some  of  the  results  already 
obtained,  such  as  those  with  viruses,  together  with  the  prospect  of 
ingenious  improvements,  warrant  the  hope  that  the  instrument  will  lead 
to  further  discoveries  of  immense  importance. 

Some  of  the  fixing  and  staining  techniques  devised  half  a  centmy  ago 
still  survive  in  the  laboratory  \x\i\\  little  modification,  some  have  been 
greatly  altered  and  improved,  and  some  have  been  replaced  by  new  ones 
of  greater  value.  Advances  in  physical  chemistry  and  microchemistry 
have  enabled  the  technician  to  go  about  his  tasks  with  an  increased 
understanding  of  what  is  going  on  in  the  tissues  before  him,  even  though 
much  is  still  there  that  has  not  been  fully  explained.  The  cause  of  one 
of  his  chief  perplexities  has  been  very  largely  removed  by  the  standardiza- 
tion of  biological  stains,  an  accomplishment  of  a  cooperative  group  of 
biologists,  manufacturers,  chemists,  and  other  interested  workers. 

Cytologists  as  well  as  investigators  in  other  fields  are  now  making 
increasing  use  of  statistical  methods  in  the  evaluation  of  their  data. 
Many  valuable  contribu^ons  have  been  made  in  past  years  with  little 
conscious  attention  to  the  mathematical  aspects  of  the  problems  in  hand, 
but  it  is  now  more  widely  recognized  that  inferences  drawn  from  observa- 
tions may  stand  or  fall  with  the  results  of  mathematical  analysis  and 
that  the  true  significance  of  what  has  been  observed  may  not  appear 
unless  such  methods  are  employed. 

By  far  the  most  significant  and  encouraging  development  in  twentieth- 
century  cytolog.y  has  l^een  its  more  definite  alliance  with  neighboring 
fields  of  biology.  For  many  decades  the  various  fields  had  given  promise 
of  becoming  more  mutually  helpful,  and  now  that  promise  is  being  ful- 
filled: the  nineteenth  century's  subdivision  of  biology  is  being  succeeded 

^  The  symbol  ix  stands  for  micron.  This  equals  0.001  millimeter,  or  1/25,400 
inch,  and  is  the  unit  of  length  most  often  used  in  cytologj'.  The  angstrom  unit  (A), 
also  used  at  times,  equals  0.0001^1. 


THE  POSITION  OF  CYTOLOGY  IN  BIOLOGICAL  SCIENCE  9 

by  a  reunion  of  the  parts  into  a  more  efficient  whole.  As  each  field  has 
<'xtcnded,  its  borders  have  come  into  contact  with  those  of  other  fields 
from  which  it  ac(iuires  aid  for  its  own  further  development.  Indeed, 
1he  n^gions  formerly  separating  the  fields  are  often  found  to  be  the  most 
fertile  of  all. 

The  most  conspicuous  and  so  far  the  most  profitable  of  such  alliances 
is  that  between  cytology  and  genetics.  Although  much  of  the  founda- 
tion for  this  union  was  laid  bj^  nineteenth-century  cytologists  and  students 
of  heredity,  modern  cytogenetics  dates  rather  definitely  from  1900  and 
shortly  thereafter,  when  Mendel's  laws  of  inheritance  were  rediscovered 
and  shown  to  have  a  physical  basis  in  the  known  behavior  of  chromosomes 
through  successive  life  cycles.  The  remarkable  results  of  this  alliance 
will  receive  special  consideration  in  later  chapters. 

A  second  alliance  now  undergoing  development  is  that  between 
cytolog\^  and  taxonomy.  It  has  been  found  that  characters  useful  in 
classification  can  often  be  recognized  in  the  number  and  the  form  of  the 
chromosomes.  Not  only  do  the  chromosomal  data  aid  in  the  grouping 
of  the  species  and  ^'arieties,  but  they  often  furnish  strong  suggestions  as 
to  the  manner  in  which  certain  taxonomic  units  have  arisen  during  the 
course  of  evolution.  The  bulk  of  such  work  has  so  far  been  done  with 
plants,  although  the  method  has  proved  useful  with  certain  animal  groups 
also. 

The  study  of  chromosomes  in  related  plants  has  led  to  the  discovery 
that  diiTerences  in  chromosome  number  and  form  sometimes  show  a 
significant  correlation  with  differences  in  geographical  range  or  ecological 
habitat.     Here  again  workers  in  different  fields — ecology,   phj'-siology, 
and  cytology- — are  discovering  the  need  for  further  cooperative  endeavor. 

vSince  disease  is  primarily  an  abnormal  activity  in  cells  and  tissues, 
the  close  relation  between  cytology  on  the  one  hand  and  pathology  and 
medicine  on  the  other  should  be  obvious.  One  needs  only  to  mention 
the  imruly  growth  of  cancer  cells  or  the  effects  of  viruses  on  cell  structure 
and  function  to  indicate  the  importance  of  cooperative  studies  on  diseased 
tissues. 

The  major  problems  of  biology  are  thus  joint  problems,  and  coopera- 
tion is  the  modern  wa}^  of  solving  them.  The  benefits  of  the  various 
alliances  are  manifold.  Cytology  itself  has  been  furnished  with  new  and 
effective  tools;  it  has  become  more  experimental  in  nature;  its  findings 
have  taken  on  new  meanings.  The  other  allied  fields  have  been  furnished 
by  cytology  with  a  better  conception  of  the  physical  basis  of  the  phenom- 
ena observed  within  their  borders.  The  workers  in  all  fields  have  been 
made  more  fully  aware  of  their  dependence  upon  one  another  and  of  their 
ignorance  of  much  that  assumes  new  importance  for  them ;  this  all  makes 
for    tolerance    and   humility.     Cooperation    may   make    o\n-   individual 


10  FUNDAMENTALS  OF  CYTOLOGY 

ignorance  more  painfully  evident,  but  at  the  same  time  it  gives  us  hope 
of  avoiding  some  of  the  consequences  of  ignorance. 

What  Cytology  Is. — In  view  of  what  has  been  set  forth  above  and  what 
is  to  follow  in  later  chapters,  cytology  may  be  defined  as  the  branch  of 
scientific  biology  that  deals  more  or  less  directly  with  the  structural  and 
functional  organization  of  protoplasm,  usually  in  single  or  closely  asso- 
ciated protoplasts,  and  with  the  relation  of  this  organization  to  the  phe- 
nomena of  metabolism,  growth,  differentiation,  heredity,  and  evolution. 

Cytology  thus  broadly  defined  would  appear  to  occupy  a  key  position 
in  the  science  of  biology,  since  everything  the  organism  does  has  a  part  of 
its  cause  in  protoplasmic  activity.  This,  however,  does  not  mean  that  all 
biology  must  be  regarded  as  an  extension  of  cytology :  it  means  rather  that 
all  biological  problems  have  a  cytological  element  in  them.  Cytology  is 
therefore  an  integral  part  of  biology,  and  the  future  progress  of  the  science 
will  depend  very  largely  upon  how  well  such  integration  is  maintained. 


CHAPTER  II  ^^'VM0%^ 

THE  ORGANISM  AND  THE  CELL  ^~ 

The  bodies  of  animals  and  plants  are  composed  of  protoplasm  and  its 
products;  an  organism  is,  in  fact,  an  organized  mass  of  protoplasm 
interacting  with  its  environment.  The  protoplasm  constituting  the 
protoplast,  or  cell,  is  almost  universality  differentiated  into  two  unlike 
portions,  cytoplasm  and  nucleus,  each  of  these  being  bounded  by  a 
definite  membrane.  In  this  chapter  we  shall  examine  the  various  ways 
in  which  this  dual  organization  is  manifested  in  the  bodies  of  organisms. 
As  we  do  so  we  shall  point  out  certain  general  inferences  which  it  is  well 
to  have  in  mind  before  narrowing  the  scope  of  our  topics  in  subsequent 
chapters. 

The  Typical  Protoplast. — The  smallest  complete  example  or  expression 
of  the  fundamental  organization  of  most  living  things — nucleated  cyto- 
plasm— is  seen  in  the  cell.  This  cell  may  exist  singly  or  appear  as  one  of 
a  multitude  of  such  units  in  a  large  body.  Cells  differ  greatly  in  their 
internal  organization  and  in  the  envelopes  that  may  surround  them, 
but  for  the  present  we  shall  limit  our  attention  mainly  to  certain  com- 
ponents common  to  nearly  all  of  them,  leaving  further  details  to  be  dealt 
with  in  the  succeeding  chapter. 

The  cytoplasm  is  a  mass  of  colorless  protoplasm  bounded  externally 
by  its  specialized  plasma  membrane.  It  may  appear  as  a  clear  viscous 
fluid,  but  often  its  many  small  inclusions  give  it  a  granular  or  an  alveolar 
appearance.  In  plant  cells  it  commonly  encloses  a  considerable  mass  of 
cell  sap  against  which  it  forms  a  limiting  vacuolar  membrane,  and  also 
one  or  more  plastids  in  which  carbohydrates  are  formed. 

The  nucleus  is  enclosed  within  the  cytoplasm  and  is  seldom  normally 
in  direct  contact  with  any  other  component  of  the  cell.  It  is  bounded  by 
a  membrane.  Usually  it  appears  as  a  globule  of  clear  matter  in  which 
numerous  fine  threads  and  one  or  more  nucleoli  are  embedded. 

The  foregoing  provisional  description,  very  meager  though  it  is, 
will  serve  the  purposes  of  this  chapter.  The  importance  of  structural 
and  functional  organization  within  the  typical  protoplast  should  be 
stressed  at  once.  The  cytoplasm,  the  nucleus,  and  the  plastids  are 
regions  in  which  the  protoplasm  differs  structurally  and  carries  on  dif- 
ferent functional  activities:  they  are  to  be  regarded  therefore  as  organs 
of  the  protoplast.  The  membranes,  too,  are  such  specialized  regions  and 
should  be  thought  of  not  merely  as  barriers,  but  as  organs  of  interaction 

11 


12  FUNDAMENTALS  OF  CYTOLOGY 

between  C3rtoplasm,  nucleus,  and  environment.  Within  the  ty])ical  pro- 
toplast the  localization  of  certain  reactions  in  particular  regions  goes  hand 
in  hand  with  structural  differentiations,  much  as  it  does  at  a  higher  level 
of  organization  in  the  various  regions  of  a  large  and  complex  multi- 
cellular body. 

As  will  be  realized  more  fully  later  on,  the  concept  of  the  cell  as  a 
fundamental  organic  unit  has  occupied  a  large  place  in  biological  thought. 
(1)  It  has  afforded  the  student  of  organic  structure  a  convenient  con- 
crete unit  for  descriptions  of  the  minute  anatomy  and  development  of 
plants  and  animals.  (2)  The  physiologist  has  often  been  able  to  gain 
insight  into  the  functional  activities  of  complex  organisms  by  studying 
the  activities  of  single  cells.  (3)  The  fact  that  many  minute  organisms 
have  the  structure  of  a  single  cell  has  stimulated  speculation,  much  of  it 
doubtless  profitable,  on  the  problem  of  the  evolution  of  larger  organisms. 
We  therefore  begin  our  cytological  studies  with  the  typical  cell  before  us, 
even  though  our  conceptions  of  its  role  may  become  modified  as  we 
proceed. 

It  is  a  habit  of  long  standing  to  interpret  a  large  organism  in  terms 
of  its  cell  units.  We  cannot  emphasize  too  strongly  the  importance  of 
interpreting  the  behavior  of  the  cell  in  terms  of  the  complete  organism 
of  which  it  may  be  a  small  part.  The  relationship  existing  between  the 
two  individualities — the  multicellular  organism  and  the  unit  cell — can 
be  made  clearer  through  a  consideration  of  the  various  arrangements 
assumed  by  cytoplasm,  nuclei,  and  limiting  membranes  in  particular 
tissues  and  complete  organisms  and  by  observing  how  these  arrange- 
ments may  change  into  one  another  as  the  development  of  the  organism 
progresses. 

Ontogeny,  the  development  of  an  elaborate  individual  animal  or 
plant  from  a  fertilized  egg,  a  spore,  or  any  other  small  and  relatively 
simple  initial  mass  of  protoplasm,  is  surely  one  of  the  most  amazing 
phenomena  on  earth.  The  seemingly  simple  initial  protoplast  has, 
however,  its  own  peculiar  microscopic  and  submicroscopic  organization. 
It  is  a  major  task  of  biologists  to  describe  this  organization  and  to  show 
how  it  functions  in  the  gradual  transformation  of  the  initial  protoplast 
into  a  mature  organism  with  its  higher  degrees  of  organization.  This 
transformation  involves  almost  innumerable  structural  changes  and 
functional  reactions,  known  and  unknown.  Our  immediate  purpose  is 
not  to  describe  any  of  these  in  detail,  but  rather  to  deal  in  a  more  general 
way  with  certain  fundamental  aspects  of  ontogeny,  viz.,  growth,  differen- 
tiation, and  correlation.  We  shall  then  be  in  a  better  position  to  appre- 
ciate the  relationship  between  the  organism  as  a  whole  and  the  cell. 

Growth. — Growth  consists  primarily  in  the  synthesis  of  new  proto- 
plasm through  the  activity  of  that  already  present.     It  is  ordinarily 


THE  ORGANISM  AND  THE  CELL  13 

accompanied  by  an  increase  in  size,  although  a  cell  or  other  mass  of 
protoplasm  may  at  times  become  much  larger  because  of  the  accumula- 
tion of  some  nonprotoplasmic  product,  such  as  cell  sap.  Certain  effects 
of  growth  upon  the  form  assumed  by  protoplasts  will  now  be  examined. 

For  the  sake  of  simplicity  we  shall  begin  with  a  typical  cell  consisting 
of  a  spherical  mass  of  cytoplasm  enclosing  a  spherical  nucleus,  these  two 
portions  being  bounded  by  a  plasma  membrane  and  a  nuclear  membrane, 
respectively.  Synthesis  of  new  protoplasm  involves  interchanges  of 
materials  between  cytoplasm  and  nucleus,  the  part  that  the  nucleus  can 
play  being  determined  quantitatively  by  the  area  of  its  membrane. 
As  the  cell  and  nucleus  increase  in  volume,  the  area  of  the  membrane 
through  which  the  interchanges  must  occur  does  not  increase  at  the  sam(^ 
rate ;  hence  a  point  may  be  reached  beyond  which  the  interaction  between 
nucleus  and  cytoplasm  would  be  insufficient  to  support  further  growth 
of  the  whole  protoplast.  This  difficulty  is  usually  overcome  in  nuclear 
division,  whereby  the  nuclear  surface  present  in  the  protoplast  is  increased 
without  a  corresponding  increase  in  the  volume  of  any  individual  nucleus. 
Further  growth  can  then  proceed  until  the  critical  nucleocytoplasmic 
ratio  is  again  reached.  Sometimes  a  nucleus  may  increase  its  surface- 
volimie  ratio  by  altering  its  shape,  the  relative  amount  of  surface 
being  greater  in  nonspherical  objects. 

A  similar  limitation  on  the  growth  of  a  spherical  cell  is  imposed  by  the 
area  of  plasma  memljrane  through  which  the  protoplasm  interacts  with 
the  external  environment.  Here  again  the  block  to  further  growth  is 
overcome  by  a  division  of  the  protoplast,  or  often  by  the  assumption  of  a 
flattened  or  a  filamentous  form,  as  in  so  many  algae;  either  of  these 
methods  leads  to  an  increase  of  surface  per  unit  volume.  In  large  organ- 
isms like  ourselves  there  are,  of  course,  elaborate  structural  features,  such 
as  the  respiratory  and  circulatory  systems,  that  make  possible  interactions 
between  the  environment  and  the  innermost  regions  of  the  body. 

As  the  protoplasm  continues  to  grow  and  the  nuclei  to  multiply,  the 
patterns  assumed  by  the  cytoplasm,  nuclei,  and  membranes  may  come  to 
differ  widely  in  various  tissues  and  organisms.  For  convenience  one  may 
refer  to  such  patterns  as  protoplasmic  growth  patterns,  remembering,  how- 
ever, that  these  are  not  always  sharply  distinct.  In  Fig.  5  there  is 
represented  diagrammatically  the  development  of  six  such  patterns  from  a 
typical  protoplast. 

1.  The  growth  of  the  protoplasm  and  the  multiplication  of  its  nuclei 
may  not  be  accompanied  by  the  formation  of  partition  membranes,  the 
result  being  a  'multinucleate  mass.  Such  a  coenocytic  condition  is  seen 
throughout  the  vegetative  bodies  of  some  algae  and  fungi  and  as  a 
temporary  or  permanent  feature  of  certain  tissues  of  other  plants  and 
animals. 


14 


FUNDAMENTALS  OF  CYTOLOGY 


2.  The  nuclear  multiplication  may  be  accompanied  regularly  by  the 
development  of  partition  membranes,  the  result  here  being  the  multi- 
cellular mass  so  familiar  in  the  tissues  of  most  animals  and  plants. 

3.  As  the  nuclei  multiply,  the  cytoplasm  may  not  subdivide  com- 
pletely but  gradually  forms  a  network,  commonly  with  its  larger  portions 
containing  the  nuclei  and  remaining  continuous  with  one  another  through 
narrower  strands.     Such  a  pattern  is  found  in  some  animal  connective 


Multinucleate  Man 


Multicellular  Mass 


3 

Network 


Cell  Cluster 


Cells  in  Matrix 


Free  Cells 


Fig.  5. — Diagram  illustrating  development  of  six  different  protoplasmic  growth  patterns 
from  a  typical  protoplast.      Explanation  in  text. 

tissues  (Fig.  6).     The  network  lies  embedded  in  a  substance  that  may 
contain  cells  of  additional  types. 

4.  Each  nuclear  division  may  be  followed  by  a  cytoplasmic  division, 
the  resulting  cells  rounding  up  from  one  another  but  remaining  in  contact 
as  a  cell  cluster.  This  pattern  characterizes  a  certain  stage  (morula)  of 
certain  animal  embryos  (Fig.  94),  and  it  is  also  seen  in  "colonial"  algae 

(Fig.  7). 

5.  After  nuclear  and  cell  division  the  individual  cells  may  become 
separated  by  the  accumulation  of  an  intercellular  substance  (matrix) 
secreted  by  the  protoplasm  during  their  formation  and  growth.     The 


THE  ORGANISM  AND  THE  CELL 


15 


isecreted  substance  may  have  a  firm  consistency,  forming  with  the  included 
cells  a  definite  tissue.  Such  a  pattei-n,  which  is  exemplified  by  cartilage 
(Fig.  8),  may  be  termed  cells  in  matrix. 

6.  After  the  individual  cells  are  formed,  the}'  may  become  entirely 
independent  free  cells,  as  in  the  case  of  unicellular  organism.s  reproducing 
by  simple  fission. 

The  above  protoplasmic  growth  patterns  may  become  transformed  one 
into  another  as  the  structural  development  of  the  organism  or  tissue 
proceeds.  The  following  examples  will  serve  as  illustrations.  Pattern 
1  changes  into  pattern  2  in  the  developing  endosperm  of  most  flowering 
})lants  and  the  embryos  of  certain  gymnosperms,  the  appearance  of  walls 
throughout  a  multinucleate  mass  resulting  in  a  multicellular  tissue  (Fig. 


Fig.  6. — Reticular  connective  tissue  from 
lymph  node  of  cat.  In  the  continuous  cytoplasm 
is  a  network  of  nonelastic  fibers.  {After  M. 
Heidenhain.) 


Fig.  7. — A  colonial  green  alga 
(Eudorina).  The  cells  are  contained 
in  a  homogeneous  envelope.  (After 
G.  M.  Smith.) 


108).  In  developing  connective  tissues,  pattern  3  may  be  derived  from 
pattern  1  by  the  formation  of  large  masses  of  fluid  in  the  cytoplasm  or 
from  pattern  2  by  a  loosening  up  of  the  cells.  Pattern  2  may  give  rise  to 
l)attern  4  when  the  cells  of  undifferentiated  plant  tissue  round  up  from  one 
another  and  form  loose  parenchyma.  The  formation  of  matrix  character- 
izing cartilage  (pattern  5)  may  begin  in  a  multinucleate  mass  (pattern  1 ) 
or  a  multicellular  mass  (pattern  2).  Pattern  6  develops  from  pattern  1 
when  a  myxomycete  plasmodium  divides  up  into  individual  spores,  or 
from  pattern  2  when  spores  are  produced  in  an  anther  or  fcin  sporangium. 
In  the  foregoing  examples  the  several  patterns  hav(;  been  described  as 
arising  either  by  a  subdivision  of  the  initial  typical  protoplast  or  by  further 
subdivision  in  a  pattern  already  formed.  Similar  patterns  sometimes 
arise  by  an  aggregation  of  free  cell  units  or  a  vmion  of  tissue  cells  into  a 
continuous  mass  of  protoplasm.  Thus  free  cells  (pattern  ())  may  associate 
and  form  a  colonial  cluster  (pattern  4)  in  certain  algae.  Among  the  slime 
molds  there  is  a  group  of  species  in  which  free  cells  (pattern  ())  unite  in 


16 


FUNDAMENTALS  OF  CYTOLOGY 


great  numbers  and  form  large  pseudoplasmodia  in  which  the  original  cell 
boundaries  remain  evident  (pattern  2),  while  in  another  group  the  uniting 
cells  lose  their  identity  completely  and  form  a  true  Plasmodium  (pattern 
1).  In  the  anthers  of  many  flowering  plants  the  tapetum  is  at  first  a 
multicellular  tissue  (pattern  2),  but  while  the  microspores  complete  their 
development  the  walls  in  this  tissue  disintegrate  and  allow  the  protoplasts 
to  flow  together  as  a  tapetal  Plasmodium  (pattern  1). 

Much  interest  attaches  to  patterns  characterized  by  continuous  cyto- 
plasm because  of  the  light  they  shed  on  the  causes  of  ontogenetic  develop- 


-i**--.-' 

"    ^^'-i^ 
>%^* 


\.» 


»;     »- 


V«> 


Fig.  8. — Hyaline  cartilage  from  sternum  of  rabbit.      (CuurU^u  of  General  l-iifiaijicdl  Supply 

House,  Inc.,  Chicago.) 

ment  in  organisms  with  distinct  cells.  When  the  condition  has  arisen  as  a 
result  of  nuclear  division  without  cj^toplasmic  division,  the  resulting  body 
or  tissue  is  best  called  a  coenocyte,  whereas  such  a  body  or  tissue  produced 
by  a  union  of  previously  distinct  cells  is  known  as  a  syncytium.  Evidently 
there  are  tissues,  such  as  muscle  or  connective  tissue,  that  may  develop  a 
given  pattern  in  either  of  these  ways.  When  the  mode  of  development  in 
a  tissue  under  observation  is  unknown,  the  nontechnical  pattern  names 
used  in  the  foregoing  paragraphs  should  suffice. 

The  subdivision  and  the  aggregation  of  protoplasts  are  of  interest  not 
only  from  the  standpoint  of  ontogenetic  development:  their  relative 
importance  in  the  evolution  of  animals  and  plants  in  general  has  also  been 
a  much  debated  point.  The  true  historical  relationship  of  unicellular  and 
multicellular  oi-ganisms  is  at  best  uncertain,  but  that  the  facts  set  forth 


THE  OUGANISM  AND  THE  CELL 


17 


Mac. 


Mic- 


above  have  an  important  bearing  on  the  ciuestion  should  lie  evident  in 
what  follows. 

Differentiation. — ^By  differentiation  is  meant  a  progressive  change  from 
a  generalized  and  uniform  condition  to  a  more  specialized  and  hetero- 
geneous condition  in  the  protoplasmic  system.  It  results  in  the  structural 
alteration  of  regions  in  which  certain  functions  come  to  be  localized,  so 
that  what  is  at  first  a  mass  in  which  all  portions  appear  and  act  alike 
becomes  a  system  of  unlike  but  correlated  parts,  each  performing  one  or 
more  special  functions.  In  differentiation  "the  level  of  organization  is 
raised." 

Differentiation  can  occur  in  any  of  the  proto- 
plasmic growth  patterns  reviewed  above.  Even 
the  structure  of  the  typical  protoplast  is  reason- 
ably (though  not  necessarily)  considered  to  be 
the  result  of  an  ancient  differentiation  in  primitive 
living  svibstance,  the  c^^toplasm,  nucleus,  and 
membranes  representing  specialized  portions  act- 
ing in  harmony.  The  astonishing  extent  to  which 
differentiation  can  be  carried  in  a  unicellular 
organism  is  illustrated  by  those  protozoa  which 
have  clearly  specialized  locomotor,  digestive, 
excretory,  and  neuromotor  regions  (Fig.  9).  After 
examining  such  organisms  as  this,  one  can  no 
longer  accept  the  statement  that  "protozoa  are 
simple  organisms."  They  are  small  and  unicellu- 
lar, but  considering  their  size  and  mode  of  life 
they  are  perhaps  as  well  differentiated  as  we  are. 
No  other  single  cells  are  quite  so  intricately 
organized. 

A  growing  mass  of  protoplasm  having  any  one 
of  the  "patterns,"  although  it  retains  the  primi- 
tive cUfterentiation  into  cytoplasm,  nucleus,  and  membranes,  may  still  be 
regarded  as  generalized  -vvith  respect  to  other  expressions  of  differentiation 
that  are  yet  to  appear  within  it.  A  mass  of  embryonic  tissue,  for 
example,  has  many  nuclei,  memliranes,  and  other  components,  but  if  this 
same  type  of  structure  and  the  same  general  functions  pervade  the  whole 
mass  uniformly,  it  is  said  to  be  imdifferentiated  so  far  as  the  development 
of  further  specialized  organs  is  concerned. 

That  differentiation  resulting  in  the  same  general  form  of  body  or  in 
organs  having  the  same  function  may  occur  in  protoplasmic  systems  of 
unlike  pattern  is  shown  by  the  following  examples.  The  three  green 
algae  Stigeoclonium,  Cladophor-a,  and  Vaucheria  all  develop  branching, 
filamentous  bodies,  although  the  first  has  numerous  uninucleate  cells  and 


Fig.  9. — A   protozoan 
(Diplodinium).  M, 

mouth;  iV,  neuromotoi- 
apparatus;  Mac,  macro- 
nucleus;  Mic,  micro- 
nucleus;  V.V.,  contractile 
vacuole;  A,  anal  canal; 
C,  contractile  region;  S, 
skeletal  plates.  {After 
R.  G.  Sharp.) 


18 


FUNDAMENTALS  OF  CYTOLOGY 


~the  second  fewer  and  larger  coenocytic  compartments,  while  the  third  is 
completely  coenocytic,  having  no  cellular  subdivisions  whatever  in  the 
vegetative  body.  Their  common  body  form  must  be  developed  by  what 
is  common  to  all  of  them — nucleated  c^^toplasm. 

Even  more  striking  are  the  spore-bearing  organs  of  various  plants 
(Fig.  10).  In  each  of  these  selected  cases  the  organ  is  composed  of  a 
supporting  stalk  and  an  enlarged  terminal  portion  in  w^hich  the  spores  are 
formed.  In  the  first,  a  fruiting  body  of  a  slime  mold,  a  multinucleate 
Plasmodium  grows  upward  into  a  column  which  enlarges  at  the  top, 
becomes  surrounded  by  a  resistant  outer  wall,  and  develops  an  internal 
system  of  capillitium  filaments,  and  then,  only  when  these  differentiations 
are  practically  completed,  does  the  multinucleate  protoplasm  in  the  up- 
per portion  subdivide  into  spores.     In  the  second,  a  fruiting  body  of 


©J0%G©©^. 


\\2 


Fig.  10. — Differentiation  in  spore-bearing  portions  of  various  plants.      Semidiargainmatic. 
Explanation  in  text.      (A'o.  4  redrawn  from  J .  L.  Williams.) 

another  type  of  slime  mold,  a  large  number  of  separate  cells  become  closely 
aggregated  without  actually  fusing  and  build  up  a  stalked  structure  wdth  a 
slimy  sheath  and  spore-like  cysts.  In  the  third,  a  sporophj^te  of  a  liver- 
wort, a  mass  of  multicellular  tissue  develops  the  stalked  form,  a  sporan- 
gium wall,  a  system  of  internal  filaments  (elaters),  and  eventually  spores. 
In  the  fourth,  a  sporangium  of  an  alga,  a  single  uninucleate  cell  elongates, 
projects  from  the  body,  and  forms  a  distal  globular  enlargement  which 
subdivides  into  spores.  In  the  fifth  example,  the  fruiting  body  of  a 
mushroom,  the  organ  is  developed  by  a  mass  of  interwoven  filamentous 
hyphae. 

Of  equal  interest  in  connection  with  the  role  of  cells  in  structural  and 
functional  differentiation  are  the  following  observations  on  the  develop- 
ment of  animal  eggs.  The  larva  of  a  certain  annelid  w^orm  develops 
much  of  its  characteristic  form  and  internal  differentiation  up  to  a  certain 
stage  even  when  the  normal  subdivision  into  cells  is  suppressed  by  adding 
KCl  to  the  medium.  In  some  marine  animals  a  complete  embryo  may  be 
developed  by  a  single  one  of  the  8  or  16  cells  formed  by  the  first  3  or  4 
divisions  of  the  fertilized  egg,  whereas  in  others  the  embryo  lacks  certain 


THE  ORGANISM  AND  THE  CELL  19 

parts  even  after  a  portion  of  the  undivided  egg  is  removed.  This  indi- 
cates that  subdivision  into  cells  and  differentiation  are  two  processes  that 
may  be  variously  correlated  in  time,  differentiation  beginning  relativelj'' 
late  in  the  first  instance  and  relatively  early  in  the  second.  In  Amphibia 
it  has  been  shown  that,  if  a  group  of  cells  from  one  region  of  a  sufficiently 
>oung  lar\'a  be  transplanted  to  another  region,  the  structure  into  which  it 
develops  will  be  appropriate  to  the  new  position  rather  than  the  original 
one. 

It  appears,  then,  that  when  differentiation  of  regions  takes  place  in 
growing  protoplasm  these  regions  need  not  be  delimited  by  cell  l)()undai-ies. 
Cellular  structure  is  accessory,  and  cell  division  is  an  incident  of  growth 
rather  than  a  cause  of  differentiation.  The  kinds  of  subsidiary  units, 
])resent — cells,  hyphae,  nuclei,  plastids — do,  of  course,  share  in  deter- 
mining the  kinds  of  specialization  that  can  occur,  but  which  of  these  do 
take  place  in  the  various  regions  is  determined  by  their  positions  in  the 
whole  system.  The  region  behaves  as  it  does,  not  l)ecause  it  is  cells,  but 
because  it  is  protoplasm  with  a  certain  physicochemical  constitution 
responding  to  conditions  that  are  in  some  degree  peculiar  to  that  portion 
of  the  growing  mass.  This  is  strikingly  illustrated  by  the  coenocytic  alga 
Bryopsis,  which  develops  a  regular  and  characteristic  body  form  while  the 
streaming  cytoplasm  carries  the  nuclei  about  from  region  to  region  within 
it.  In  a  word,  ontogenetic  differentiation  is  to  be  regarded  as  an  act  of 
the  developing  system  as  a  whole,  whatever  its  protoplasmic  growth  type. 

The  controlling  influence  of  the  whole  upon  the  activities  of  its  parts  in 
multicellular  tissues  has  been  emphasized  anew  in  recent  studies  of  cell 
behavior  during  the  development  of  plant  organs.  In  the  transforma- 
tion of  the  small  ovary  into  the  large  fruit  of  the  squash  plant,  it  is 
found  that  growth  in  early  stages  is  chiefl}^  by  cell  multiplication,  whereas 
in  later  stages  it  is  entirely  by  cell  enlargement.  Meanwhile  the  fruit 
increases  in  size  and  in  dry  weight  at  constant  rates,  and  its  parts  differ- 
entiate and  change  in  relative  proportions;  in  other  words,  growth  and 
differentiation  of  the  whole  proceed  uniformly  whether  the  constituent 
cells  are  multiplying  rapidly,  slowly,  or  not  at  all  and  whether  they  are 
becoming  progressively  smaller  (by  divisions)  or  larger.  Moreover,  the 
rate  and  the  time  of  cessation  of  cell  division  in  fruits  of  this  kind  are  the 
same  regardless  of  cell  size.  In  long  gourds  the  nuclear  division  figures 
when  first  formed  lie  at  various  angles  with  respect  to  the  long  axis  of  the 
fruit,  but  at  later  stages  they  turn  more  nearly  parallel  to  it  as  though  in 
response  to  some  polarizing  force  acting  along  the  axis;  hence  the  new 
partitition  walls  develop  at  right  angles  to  this  axis  and  the  number  of 
cells  along  it  is  increased. 

Similar  studies  of  growing  grass  roots  likewise  indicate  a  dependence  of 
the  cell  upon  forces  acting  in  the  organ  as  a  whole.     In  regions  where  the 


20  FUNDAMENTALS  OF  CYTOLOGY 

cells  are  elongating,  it  can  be  seen  that  a  wave  of  elongation  joasses  from 
the  base  toward  the  apex  of  the  root,  this  wave  at  a  given  moment  affect- 
ing alike  whatever  portions  of  neighboring  cells  occupy  the  same  level, 
whether  these  portions  are  basal,  median,  or  apical.  The  wave  is  some- 
thing not  dependent  upon  boundaries  of  the  unit  cells.  Here,  as  in  the 
cucurbit  fruits  and  coenocytes,  the  unity  of  behavior  and  of  organization 
inheres  primarily  in  the  whole  rather  than  in  the  elements  composing  it. 

Correlation. — In  every  normal  mass  of  protoplasm,  whatever  its 
growth  pattern  or  degree  of  differentiation,  the  many  diversified  activities 
are  so  coordinated  that  it  behaves  as  a  consistent  whole,  or  individual, 
from  the  beginning  of  development  onward ;  without  such  harmony  there 
obviously  could  be  no  organism.  How  this  harmony  is  maintained  has 
never  been  fuU}^  explained.  Recent  work  on  higher  plants  and  animals 
has  shown  that  diffusible  substances  play  an  important  role  in  this  con- 
nection, and  something  of  the  sort  may  well  occur  within  the  limits  of 
single  cells.  In  multicellular  tissues  the  fine  protoplasmic  strands 
(plasmodesms)  connecting  neighboring  protoplasts  in  all  probability 
facilitate  correlation  (Fig.  56).  In  animals  the  nervous  system  functions 
as  a  specialized  correlating  mechanism.  It  has  also  been  shown  that 
gradients  in  the  rate  of  metabolism  and  in  electric  potential  along  the 
various  axes  of  symmetry  are  correlated  with  differentiation  with  respect 
to  these  axes.  Reversal  of  the  electrical  polarity  ma}^  result  in  a  reversal 
of  morphological  pattern.  Moreover,  there  is  some  evidence  that  in  and 
about  an  organ  or  developing  embryo  there  is  a  characteristic  pattern  of 
potential — a  "field" — that  exercises  some  measure  of  control  over  what 
occurs  within  it.  "  Differentiation,  upon  such  a  view,  is  to  be  looked  upon 
as  a  setting  up  of  new  fields,  each  resulting  from  changes  in  size  or  position 
during  ontogeny  or  phylogeny."  All  this  suggests  that  a  physicochemical 
explanation  of  correlation  msLy  be  hoped  for  and  that  a  more  satisfactory 
conception  of  organic  "wholeness"  may  some  day  be  attained. 

Tissues  and  Organs. — Since  the  fundamental  processes  of  develop- 
ment— growth,  differentiation,  and  correlation — may  occur  normally  in 
the  several  protoplasmic  growth  patterns,  it  is  scarcely  adequate  to  define 
a  tissue  as  a  group  of  cells  and  their  products.  Many  tissues  are  just 
this,  but  a  better  definition  would  be  one  applicable  also  to  other  patterns, 
including  the  plasmodial  state. 

Although  we  commonl}^  think  of  an  organ  as  a  specialized  multicel- 
lular structure  because  of  familiar  examples,  the  more  comprehensive 
definition,  "any  part  of  an  organism  performing  some  definite  function," 
is  more  nearl}'  adequate  if  one  remembers  that  a  protozoan  as  well  as  a 
metazoan  may  have  distinct  regional  differentiations  associated  with 
special  localized  functions.  On  the  basis  of  function,  which  gives  organs 
their  significance,  the  contractile  region  within  a  protozoan  cell  is  as  much 


THE  ORGANISM  AND  THE  CELL  21 

an  organ  as  the  muscle  of  a  frog.  The  flat,  green  expansions  of  curtain 
green  algae  are  functionally  "leaves"  even  though  they  are  coenocytic 
instead  of  cellular  like  the  leaves  of  vascular  plants. 

These  points  are  perhaps  sufficient  to  emphasize  the  thought  that  in 
our  cytological  study  of  animals  and  plants  we  shall  miss  much  that  is  of 
first  importance  if  we  consider  cells,  their  components,  and  their  products 
only  in  terms  convenient  for  the  description  of  structure  and  neglect  their 
relation  to  the  active  life  of  the  organism  of  which  they  are  parts. 

Conclusions. — In  this  chapter  we  have  dealt  at  some  length  with  the 
lelationship  between  two  units  or  individualities  present  in  large  organ- 
isms— the  organism  as  a  whole  and  the  cell.  Obviouslj^  there  is  a  recip- 
local  action  between  them  when  both  are  present,  the  part  affecting  the 
whole  while  the  whole  affects  the  part.  The  relative  importance  of  the 
two  has  been  variously  conceived.  The  proponents  of  the  cell  theory 
stressed  the  cell  as  the  primarj'-  agent  of  organization,  while  adherents  of 
the  organismal  theory  insist  upon  the  primacj^  of  the  whole,  cells  when 
present  being  important  but  subsidiarj^  parts.  The  former  regarded 
multicellular  organisms  as  having  arisen  phylogenetically  as  aggregates 
of  unicellular  individuals,  whereas  the  latter  hold  it  to  be  more  probable 
that  single  unicellular  individuals  became  internally  sul^divided  as  they 
became  larger.  The  probabilitj^  of  an  "evolution  from  Protozoa  to 
sponges  and  coelenterates  by  multiplication  of  nuclei  in  an  already  dif- 
ferentiated cytoplasm"  has  recently  been  emphasized  anew  (Kofoid). 

This  subject  can  be  followed  further  in  other  works  on  biology,  but  it 
has  been  thought  well  to  set  forth  early  in  the  present  book  some  of  the 
basic  facts  of  structural  development  insofar  as  the}'  concern  cells  as  units, 
together  ^\dth  a  suggestion  of  certain  theoretical  interpretations.  It 
should  then  be  easier  to  appreciate  the  significance  to  the  organism  of 
those  cytological  details  to  which  we  shall  soon  restrict  our  attention. 
The  prevalent  multicellular  state  is  doubtless  the  most  efficient  basis  for 
differentiation  in  all  but  very  small  organisms  and  has  conditioned  much 
evolutionary  progress,  but  we  h&ve  seen  that  the  essential  features  of 
development  can  occur  without  it.  "The  principle  of  protoplasmic 
differentiation  is  more  general  and  fundamental  than  that  of  cells  as 
units"  (Heidenhain). 


CHAPTER  III 
THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 

In  the  previous  chapter  the  fact  that  the  cell  is  not  always  a  distinct 
element  in  tissues  was  emphasized  as  one  that  should  aid  in  clarifying  our 
conceptions  of  factors  responsible  for  the  action  of  the  complex  organism 
as  a  whole,  especially  during  its  ontogeny.  Nevertheless  in  most  organ- 
isms and  tissues  the  cell  does  appear  clearly  as  a  more  or  less  standardized 
unit  having  cytoplasm,  nucleus,  and  membranes.  In  addition  to  these 
there  may  be  other  diverse  functional  and  structural  differentiations  that 
cause  some  cells  to  look  very  unlike  "typical "  protoplasts.  We  shall  now 
extend  our  description  of  the  protoplast  by  reviewing  in  more  detail  its 
principal  internal  features. 

Cytoplasm. — The  cytoplasm  consists  ordinarily  of  an  optically  clear, 
somewhat  viscous  fluid  {hyaloplasm)  in  which  there  may  be  embedded  a 
multitude  of  small  droplets  and  granules  that  give  it  a  visible  structure. 
The  fundamental  structure  necessary  to  the  performance  of  vital  activi- 
ties is  known  to  lie  in  the  hyaloplasm  beyond  the  limit  of  visibility  with 
the  photomicroscope;  this  matter  will  be  considered  further  in  the  next 
chapter.     The  cytoplasmic  portion  of  the  protoplast  is  called  the  cytosome. 

The  cytosome  is  often  differentiated  into  fairly  distinct  regions  differ- 
ing in  viscosity.  In  many  cells  the  more  viscous  portion,  the  plasmagel, 
forms  a  cortical  layer  of  variable  thickness  just  beneath  the  plasma  mem- 
])rane,  while  the  more  fluid  portion,  the  plasmasol,  lies  farther  in.  The 
cytoplasm  may  change  rapidly  and  locally  from  one  state  to  the  other, 
and  it  is  evident  that  such  alterations  play  a  role  in  ameboid  locomotion 
and  the  streaming  observed  in  tissue  cells. 

In  embryonic  or  meristematic  tissues  the  cytoplasm  seems  to  be  less 
differentiated  than  the  other  cell  components  enclosed  within  it  and  more 
like  what  we  may  imagine  primitive  protoplasm  to  have  been.  It  is, 
however,  capable  of  differentiation  in  many  ways.  In  muscle  tissue,  for 
example,  it  may  be  almost  completely  transformed  into  fine  longitudinal 
myofibrils  that  function  somehow  in  the  act  of  contraction  (Fig.  11). 
The  cytoplasm  of  nerve  cells  has  delicate  neurofibrils  probably  concerned 
in  the  conduction  of  stimuli  (Figs.  12,  13).  In  the  protozoa  the  cytoplasm, 
or  at  least  its  external  hyaline  portion  (ectoplasm),  may  develop  loco- 
motor extensions  in  the  form  of  undulating  membranes,  cilia,  and  flagella 
of  many  types.     The  pseudopodia  formed  by  amebas  and  mj^xomycetes 

22 


THE  STin'CTVRAL  COMMON KNTS  OF  I'ROTOI'LASTS 


23 


consist  of  both  h,yaline  ectoplasm  and  granular  cndoplasni.  Since  most 
of  the  visible  changes  in  differentiating  cells  occur  in  the  cytoplasm,  the 
latter  has  sometimes  been  regarded  as  an  organ  of  differentiation ,  some- 
what as  the  nucleus  has  been  called  an  organ  of  heredity. 

At  the  outer  surface  of  the  cytoplasm,  whether  it  is  surrounded  b}'-  a 
thick  cell  wall  or  not,  there  is  a  film  of  ultramicroscopic  thinness,  the 
plasma  membrane.  In  case  a  layer  of  ectoplasm  is  present,  as  in  the 
ameba,  the  plasma  membrane  is  at  its  outer  boundary.     Such  a  membrane 


Fii;.  11. —  Portioii.s  of  two  huiiiuii  niu^;cle  fil)eis.  The  myofibrils  vuu  Icusthwi-^e. 
Modifications  in  the  associated  fibrils  at  regular  intervals  are  responsible  for  the  transverse 
striations.      {Courtesy  of  General  Biological  Supply  House,  Inc.,  Chicago.) 

evidently  develops  by  the  accumulation  of  certain  protoplasmic  constit- 
uents to  the  exclusion  of  others  and  the  arrangement  of  these  constituents 
into  a  layer  having  a  special  type  of  structure.  It  seems  to  resemble  in 
some  measure  the  surface  film  on  a  pond  or  drop  of  water,  Avhere  the 
molecules  have  been  shown  to  be  arranged  regularh'^  and  closel}'  in  a 
pavement-like  layer.  The  elastic  and  other  physical  properties  of  tliis 
external  membrane  have  been  studied  with  the  aid  of  tlie  micromanipu- 
lator (page  45).  Such  studies  have  shown  that  when  the  membrane  is 
torn  (if  not  too  greatly)  it  is  quickly  renewed  by  the  protoplast. 

The  physical  and  chemical  properties  of  the  plasma  membrane  are 
largely  responsible  for  the  physiological  behavior  of  the  protoplast,  in 
particular  its  interaction  with  its  environment.     What  substances  shall 


24 


FUNDAMENTALS  OF  CYTOLOGY 


enter  or  leave  the  protoplast  and  the  rate  of  their  movement  are  deter- 
mined not  only  by  the  nature  of  the  substances,  but  also  by  the  character 
of  the  membrane.  The  membrane  is  semipermeable,  i.e.,  it  may  allow 
the  solvent  but  not  the  solute  to  pass,  and  physiologists  are  attempting 
to  account  for  this  significant  property  and  its  fluctuations  in  terms  of 
physic ochemical  constitution. 

At  the  boundary  between  the  cytoplasm  and  each  of  the  other  main 
parts  of  the  protoplast  to  be  described  below  there  is  a  special  membrane 
of  some  kind,  so  that  interchanges  there,  too,  are  determined  b}^  regional 


.*. 


The  cell  has  numerous  prolongations, 


^,/_:?.#:^  /  #■ 

Fig.  12. —  Motor  nerve  cells  in  spinal  cord  of  ox. 
the  nerve  fibers,  which  are  embedded  in  a  tissue  known  as  neuroglia.  Fine  striations  in 
the  cytoplasm  are  neurofibrils  (compare  Fig.  13).  {Courtesy  of  General  Biological  Supply 
House,  Inc.,  Chicago.) 

differentiations  involving  the  cytoplasm.  To  what  extent  the  special 
alterations  at  such  an  interface  involve  changes  in  the  materials  on  either 
side  of  it  is  not  W'cll  known.  There  are  reasons,  both  experimental  and 
theoretical,  for  the  view  that  a  membrane  formed  where  two  different 
fluids  meet  arises  b}^  a  local  modification  of  both  fluids.  The  membranes 
within  the  protoplast,  notably  that  around  the  nucleus,  would  in  this 
sense  be  double  structures.  Precipitation  membranes  composed  of  new 
components  formed  by  chemical  interaction  of  two  fluids  probably 
play  a  minor  role.  It  should  always  be  borne  in  mind  that  the  behavior 
of  the  protoplast  depends  not  onlj^  upon  the  general  composition  of  its 
principal  parts,  but  also  upon  further  special  modifications  in  areas  where 
two  unlike  components  meet. 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS  25 

The  problem  of  membranes  involves  a  further  special  differentiation 
of  the  cytoplasm  known  as  kinoplasm.  This  has  been  studied  chiefly 
ill  plant  cells.  It  appears  typically  in  the  form  of  strands  or  channels  of 
fluid  streaming  with  included  granules  through  the  unmodified  cytoplasm, 
or  frophoplas7n.  It  seems  to  form  a  more  or  less  continuous  system  with 
the  plasma  membrane,  the  vacuole  membrane,  and  sheaths  of  similar 
material  about  the  plastids  and  nucleus.  Like  the  membranes,  the  kino- 
plasmic  strands  appear  to  have  a  high  lipide  content.  With  a  few  impor- 
tant exceptions,  cytologists  have  neglected  the  kinoplasm,  and  it  is  much 
in  need  of  further  study,  especially  in  view  of  its  apparent  relation  to  the 
i:)rotoplasmic  surface  membranes. 

Nucleus. — The  nucleus  has  claimed  a  large  share  of  the  attention  of 
cytologists  ever  since  its  discovery  more  than  a  century  ago.  There  arc 
numerous  reasons  for  this.  Excepting  chloroplasts  of  most  plants,  the 
nucleus  is  the  most  conspicuous  organ  of  a  protoplast  under  the  microscope, 
especially  in  stained  preparations.  By  observing  the  effects  of  its  removal 
from  certain  cells,  it  has  been  shown  to  be  necessary  for  synthetic  metab- 
olism in  the  protoplast.  At  the  time  of  division  it  passes  through  an 
amazingly  complicated  but  very  orderly  series  of  changes  that  never  fail 
to  fascinate  the  observer.  C>i:ogenetic  studies  have  shown  that  the 
mode  of  ontogenetic  development  and,  hence,  the  particular  characters 
exhibited  by  the  organism  are  related  to  the  constitution  of  the  nucleus 
and,  furthermore,  that  the  inheritance  or  noninheritance  of  certain 
parental  characters  is  due  to  the  behavior  of  the  chief  nuclear  components, 
the  chi'omosomes,  during  the  reproductive  stages  of  the  life  cycle.  Pre- 
occupation with  nuclear  behavior  has  doubtless  been  too  great  at  times, 
but  fortunately  there  have  been  other  workers  who  have  stressed  the 
importance  of  membranes,  plastids,  and  other  components  of  the  cyto- 
some,  so  that  altogether  a  fairly  well  balanced  conception  of  the  proto- 
plast's activity  is  being  built  up. 

It  cannot  yet  be  said  that  nuclei  are  present  in  all  animals  and  plants. 
In  some  minute  organisms,  particularly  certain  bacteria,  there  are  bodies 
whose  reactions  and  behavior  at  least  suggest  their  nuclear  nature,  but 
their  minute  size  and  various  uncertainties  regarding  the  value  of  the 
criteria  used  to  distinguish  them  from  other  minute  bodies  leave  the  ques- 
tion quite  open.  In  higher  organisms  it  is  not  so  difficult  to  characterize 
the  nucleus,  for  in  spite  of  many  variations,  some  of  them  rather  extreme, 
the  same  general  type  of  fundamental  structure  seems  to  be  present  in 
practically  all  groups.  Such  typical  nuclei  will  serve  as  the  basis  of  the 
following  description. 

The  nucleus,  when  not  undergoing  division,  is  said  to  be  in  the  meta- 
bolic or  energic  stage  because  many  of  its  most  important  functions  are 
exercised  at  this  time.     Unfortunately  it  has  long  been  called  the  "  resting 


26 


FUNDAMENTALS  OF  CYTOLOGY 


stage,"  an  obviously  inappropriate  term.  At  this  stage  the  nucleus  is 
bounded  by  a  membrane.  The  permeability  of  this  membrane  is  known 
to  differ  in  certain  respects  from  that  of  the  plasma  membrane:  the  two 
are  not  formed  between  the  same  pair  of  substances.  Although  various 
substances  must  be  interchanged  through  the  membrane,  the  cytolysis 
following  its  tearing  by  a  needle  shows  that  it  separates  substances  capa- 
ble of  strong  interaction. 

Within  the  membrane  is  a  mass  of  clear  nuclear  sap,  or  karyolymph ,  in 
which  the  remaining  nuclear  constituents  are  embedded.  The  most 
important  of  these  constituents  is  evidently  a  substance  in  the  form  of 
numerous  crooked  threads,  the  chromoncmafa  (=  color  threads).     These 


Fig.  13. — Nerve  cell  from  earth- 
worm, showing  the  fine  neurofibrils  within 
the  cytoplasm.      {After  J.  Kowalski.) 


Fig.  14. — Nuclei  in  young  floral  axis 
of  Maianthemum.  In  the  narrow  cells  of 
the  developing  vascular  bundle  (right)  the 
nuclei  become  greatly  elongated.  Young 
plastids  are  present  in  the  cytoplasm. 


are  so  named  because  they  contain  a  substance  {chromatin)  which  is 
strongly  stainable  with  certain  dyes  and  can  thus  be  made  to  stand  out 
plainly  in  the  clear,  unstained  karyolymph.  In  living  nuclei  they  are 
only  faintly  seen  or  even  may  be  invisible  because  their  refractive  index  in 
ordinary  light  is  so  nearly  like  that  of  the  karyolymph,  but  suitable  meth- 
ods reveal  their  presence  (Fig.  15).  Their  visibility  va&y  vary  with 
alterations  in  the  degree  of  hydration  during  the  nuclear  cycle.  They 
frequently  appear,  especially  in  fixed  material,  to  be  connected  by  fine 
strands  (anastomoses),  forming  thus  a  network  (reticulum).  The  extent 
to  which  anastomoses  are  normal  structures  is  uncertain.  The  chro- 
monemata  are  of  special  interest  because  they  represent  the  chromosomes 
at  this  stage.  In  stages  of  nuclear  division  an  additional  stainable  sub- 
stance (matrix)  is  combined  with  the  chromonemata,  giving  the  chromo- 
somes the  compact  form  characteristic  of  those  stages  (Chap.  VII). 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


27 


In  some  nuclei  there  are  several  dense,  highly  stainable  lumps  in  the 
midst  of  the  chromonemata  (Fig.  16).  These  are  chromocenters  and  seem 
to  represent  regions  where  the  chromonemata  are  more  closely  packed  and 
have  retained  more  of  the  matrix.  Such  regions  occur  in  definite  positions 
in  certain  chromosomes.  When  each  chromosome  has  a  single  one,  the 
number  of  chromosomes  can  sometimes  be  determined  by  coimting  the 
deeply  stained  spots  in  the  metabolic  nucleus.  Ordinarily  chromosomes 
can  be  counted  only  during  nuclear  division. 

Each  typical  nucleus  has  one  or  more  nucleoli  lying  in  the  karyolymph. 
These  differ  chemically  from  the  material  of  the  chromocenters  and  can 


^i^.:':-m 


^-iS-      •         •.♦ 


-.%:'-' 


Fig.  15. — Living  nucleus  in  Tradescantia 
.stamen  hair  mounted  in  paraffin  oil.  The 
mottled  appearance  is  due  to  the  numerous 
fontorted  cliromonemata.  (Photograph  hy 
H.  Telezynski.) 


B 
Fig.  16. — A,  nucleus  of  bean  (Phase- 
olus),  with  chromocenters.  {After  E. 
Kuhn.)  B,  cell  of  touch-me-not  (Impa- 
tiens),  with  nucleus  in  prophase.  Eu- 
chromocentera  are  present  (see  page  87). 
(After  V.  Gregoire.) 


easily  be  distinguished  from  them  by  suitable  staining  methods.  They 
develop  in  close  union  with  certain  chromosomes  in  the  newly  formed 
nuclei  at  the  close  of  division;  this  is  why  they  usually  lie  against  one  or 
more  of  the  chromonemata  in  the  metabolic  nucleus.  Their  number  also 
is  related  to  their  connection  with  chromosomes.  These  points  will  be 
discussed  in  Chap.  YH. 

The  physical  consistency  of  the  nucleus  as  a  whole  obviously  depends 
upon  the  consistencies  and  relative  amounts  of  its  several  components — 
membrane,  karyolymph,  and  chromonemata.  In  certain  animal  eggs  it 
behaves  under  the  mici'omanipulator  like  a  very  fluid  droplet  with  a 
firmer  membrane.  In  cells  of  several  other  kinds  it  seems  to  be  more 
viscous  throughout  than  the  cytoplasm  and  can  be  moved  about  bodily 


28 


FUNDAMENTALS  OF  CYTOLOGY 


Avithout  visible  injury.  The  relative  specific  gravities  of  the  nuclear  com- 
ponents and  other  portions  of  the  protoplast  can  be  determined  by  the  use 
of  the  centrifuge  (Fig.  30).  In  an  electrical  field,  free  nuclei  or  cells  very 
rich  in  nuclear  material  tend  to  pass  toward  the  anode,  showing  that  they 
carrj^  a  negative  charge,  whereas  cytoplasm  or  cells  with  little  chromatin 
tend  to  go  in  the  opposite  direction.  In  the  nucleus  it  is  the  chromatin 
that  carries  the  negative  charge,  the  karyolymph  and  usually  the  nucle- 
olus being  positive. 

The  nucleus,  like  the  rest  of  the  protoplast,  consists  of  chemical  sub- 
stances of  several  classes,  among  which  proteins,  lipides,  and  water  play 
the  major  roles.  Of  special  importance  is  the  chemical  nature  of  the 
chromatic  threads  and  the  chromosomes  of  which  the}^  are  the  principal 
constituents,  for  it  is  largely  upon  their  composition  that  the  peculiar 
powers  of  the  nucleus  in  determining  the  course  of  development  and  the 


Fig.   17. — Three  stages  in  mitosis  in  einl)ryo  of  Ascaris,  showing  centrioles  and  asters. 


phenomena  of  heredity  seem  to  depend.  Anal3^ses  have  shown  that  the 
chromatin  is  a  nucleoprotein  composed  of  nucleic  acid  and  certain  basic 
proteins.  Further  discussion  of  the  chemical  nature  of  nuclei  w\\\ 
follow  in  the  next  chapter  (page  51). 

The  Centrosome.— In  the  cytoplasm  of  most  animal  cells  and  of 
certain  lower  plant  cells  a  centrosome  is  commonly  present.  It  is  not 
found  in  seed  plants.  Typically  it  consists  of  a  minute,  deeply  staining 
granule,  the  centriole,  or  often  a  pair  of  these,  surrounded  bj^  a  mass  of 
less  stainable  substance,  the  centrosphere.  In  some  cells  only  a  centriole 
is  visible,  while  in  others  nothing  but  centrosphere  substance  seems  to  be 
present.  The  aspect  of  the  centrosome  varies  widely  in  cells  of  different 
kinds  and  especially  in  different  stages  of  nuclear  division,  when  one  of  its 
chief  functions  is  evidently  performed  (Fig.  17).  It  also  plays  a  con- 
spicuous role  dining  the  development  of  certain  motile  cells,  notably  male 
gametes,  where  it  is  concerned  in  the  formation  of  the  motor  apparatus. 
Further  discussion  of  centrosomes  is  therefore  deferred  to  chapters  dealing 
with  these  topics. 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


29 


Plastids. — Plastids  are  protoplasmic  bodies  characteristic  of  the 
vegetable  kingdom.  They  are  present  in  nearl}'  all  plant  cells  and  play 
an  important  role  in  metabolism.  Plastids  confront  the  biologist  with 
problems  of  three  kinds:  cj'tological,  biochemical,  and  phylogenetic. 
Cytologists  have  already  learned  much  about  plastids,  but  some  of  the 
most  important  points  regarding  their  structure,  visible  alterations,  and 


Fig.  is.  ('lil(ii(ii.l:i~t,-<  ill  «ametophytes  of  fern  {Polypodium).  a.  luinual  gameto- 
phyte.  6,  c,  <l,  persi.^^tont  modifications  (chain-like,  plate-like,  and  budding  types)  induced 
in  chloroplasts  by  X-ray  treatment  of  .spores.      (.After  L.  Knudson.) 


relation  to  other  cytoplasmic  differentiations  remain  obscure.  Bio- 
chemists are  gradually  approaching  a  better  understanding  of  the  exact 
chemical  changes  that  take  place  in  plastids  during  the  all  important 
process  of  photosynthesis.  They  are  also  studying  the  significant  rela- 
tion of  certain  plastid  pigments  to  vitamins.  Biologists  in  gencMal  would 
like  to  know  more  about  the  histoi'ical  origin  of  i)l;istids  and  their  role  in 
the  divergent  evolution  of  organisms  with  different  tyi)es  of  nutrition. 
The  familiar  green  chloroplasts  (Figs.  18,  19)  occupy  a  i)e('uliarl\' 
strategic  position  in  the  living  world,  for  within  them  carbon  dioxide 


30 


FUNDAMENTALS  OF  CYTOLOGY 


and  water  react  in  such  a  manner  as  to  yield  a  sugar.  The  energy  for 
the  reaction  is  obtained  from  visible  light.  The  green  pigment  chloro- 
phyll absorbs  this  energy  and  transfers  it  to  the  reacting  substances,  and 
oxygen  is  liberated  as  a  by-product.  This  process  of  photosynthesis  is 
the  primary  source  of  the  world's  organic  food  supply.  The  reaction 
resulting  in  the  production  of  a  gram  molecule  of  hexose  may  be  repre- 
sented in  a  convenient  though  misleadingly  simple  manner  as  follows: 
6CO2  +  6H2O  +  673  kg.  cal.  =  CeHisOe  +  6O2.  The  energy  of  sun- 
light, after  being  thus  captured  and  stored,  is  released  to  the  organism 
through  the  process  of  respiration,  which  may  be  represented  as  follows: 
CeHioOe  +  6O2  =  6CO2  +  6H2O  +  673  kg.  cal. 

Chlorophyll  is  not  the  only  pigment  in  the  chloroplast.     Chlorophyll 
itself  exists  in  two  slightly  different  forms  designated  as  a  and  h.     Present 


Gelativona  Sheath 


Cell  Wall 
-_J- 


Nucleus 


Dense  Cytoplasm. 


Chloroplast 


Pyrenoid 


Central  Vacuole 


Fig.   19. — A  cell  of  a  green  alga  (Spriogyra).      {From  Smith,  Overton  et  al.,  A  Textbook  of 
General  Botany,  Ath  ed..  New  Yoric,  The  Macmillan  Company.) 

with  these  are  the  yellow  carotenoid  pigments  xanthophyll  and  carotene, 
whose  color  is  evident  after  the  chlorophyll  disintegrates  in  drying  autumn 
leaves.  Chemical  analysis  of  tobacco  leaves  has  revealed  the  following 
amounts  of  these  pigments  in  milligrams  per  square  meter  of  leaf  surface : 
chlorophyll  a,  29.30;  chlorophyll  b,  10.38;  xanthophyll,  10.63;  carotene, 
3.52.  Four  of  the  many  known  carotenoids  show  a  pronounced  vitamin 
A  activity,  although  their  value  to  the  plant  is  obscure.  There  is  also 
evidence  that  vitamin  C  (ascorbic  acid)  is  present  in  the  chloroplast. 
In  the  algae  other  pigments  accompany  the  chlorophyll.  Familiar 
examples  are  fucoxanthin,  a  carotenoid,  in  the  brown  algae  and  phy- 
coerythrin,  a  chromoprotein,  in  the  red  algae.  Other  carotenoids  such  as 
riboviolascin  occur  in  the  purple  bacteria.  Bearing  directly  upon  the 
problem  of  the  early  stages  of  organic  evolution  is  the  striking  chemical 
resemblance  of  the  complex  chlorophyll  molecule  to  that  of  the  red 
hemin  in  animal  hemaglobin.  The  place  of  the  magnesium  atom  in 
chlorophyll  is  occupied  by  iron  in  hemin,  thereby  rendering  hemoglobin 
an  efficient  oxygen  carrier. 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


31 


Plastids  with  no  color  are  known  as  leucoplasts.  Ordinarily  they  are 
small  and  occur  in  considerable  numbers  in  meristematic  plant  cells  and 
in  parts  not  exposed  to  light.  Under  appropriate  conditions  some  of 
them  enlarge  and  develop  into  green  chloroplasts  or  plastids  of  other 
colors.  Under  other  conditions,  notably  in  roots  and  other  storage 
organs,  they  remain  colorless  but  become  active  in  the  conversion  of 
soluble  carbohydrates  into  granules  of  storage  starch; 
such  leucoplasts  are  therefore  known  as  aniyloplasts 
(Figs.  20,  31).  Hence  the  carbohydrate  appearing  as 
starch  in  a  potato  tuber  has  been  through  two  plastids. 
It  was  first  elaborated  in  chloroplasts  in  the  leaf  as  a 
soluble  sugar;  if  not  removed  at  once,  it  was  converted 
by  enzymes  into  visible  starch  granules.  Later  this 
starch  was  reconverted  to  sugar  and  transported  to  the 
tuber,  whore,  within  the  amyloplasts,  it  was  again  trans- 
formed into  granules  of  starch.  In  some  plants,  e.g., 
the  yellow-green  algae,  diatoms,  and  the  onion  plant, 
the  visible  products  of  synthetic  activity  are  fats. 

Of  the  greatest  interest  is  the  recent  announcement 
that  the  cellulose  of  plant  cell  w-alls  is  elaborated  in 
minute  colorless  plastids  in  higher  plants  and  in 
chloroplasts  in  an  alga.  We  shall  revert  to  this  impor- 
tant subject  in  Chap.  VI. 

Plastids  of  higher  plants  are  often  csiWed  chromoplnsts 
when  they  show  some  color  other  than  green;  in  literal 
terms,  however,  a  chloroplast  is  also  a  chromoplast,  or 
chromato}:)hore.  The  ordinary  tomato  fruit  is  red 
l)ecause  lycopene,  related  to  carotene,  appears  in  the 
chloroplasts  during  ripening.  Nasturtiums  owe  their 
yellow  color,  though  not  their  red,  to  their  chromo- 
plasts.  Such  special  pigments  may  develop  in  leuco- 
plasts or  in  chloroplasts.  The  red,  light-sensitive 
eyespots  of  certain  flagellate  and  algal  cells  seem  to  be 
plastid-like  differentiations.  Other  eyespots  have  a 
different  origin. 

Structure  of  the  CJdoroplast. — The  fact  that  a  chloroplast  sw"ells  in 
distilled  water  or  a  hypotonic  solution  and  the  manner  in  which  its 
boundary  often  appears  to  separate  from  the  green  substance  under  such 
conditions  indicate  the  presence  of  a  limiting  membrane  with  osmotic 
properties.  The  ground  substance  (stroma)  of  the  chloroplast  appears  as 
colorless  cytoplasm.  B}^  grinding  and  centrifugation  it  is  possible  to 
separate  the  plastids  from  other  cell  components  and  to  show  that  50 
l)er  cent  of  the  i)rotein  in  tobacco  leaves  is  in  the  plastids.     The  (;hloro- 


FiG.  20.— Cells 
of  pea  root  tip. 
The  elongate  leu- 
coplasts contain 
starch  grains. 
Note  their  ar- 
rangement near 
poles  of  dividing 
nuclei.  (After  R. 
H.  Bowcn.) 


32 


FUNDAMENTALS  OF  CYTOLOGY 


phyll  appears  to  be  confined  to  numerous  small  platelets,  or  grana, 
embedded  in  the  stroma.  These  grana  are  sometimes  few  and  large 
enough  to  be  easily  seen,  but  under  some  physiological  conditions  the}" 
are  so  small,  numerous,  and  closely  packed  that  the  chloroplast  appears 
very  finely  granular  or  even  homogeneous.  Even  in  these  latter  cases 
the  stroma,  free  of  grana,  may  be  observed  in  pseudopodium-like  exten- 
sions or  in  torn  specimens.  The  fibrous  appearance  often  exhibited  by 
chloroplasts  is  probably  due  to  a  linear  arrangement  of  the  grana  and  to 
variations  in  the  submicroscopic  structure  of  the  stroma  itself. 


Fig.  21. — Chloroplasts  with  pyrenoids.  a,  in  a  green  alga  (Draparnaldia).  b,  in  a 
liverwort  (Anthoceros);  pyrenoid  consists  of  numerous  small  bodies,  c,  in  a  green  alga 
(Oedogonium);  starch  granules  are  near  pyrenoids  and  elsewhere  in  the  plastid.  (c,  after 
F.  Schmitz.) 

Chemical  tests  show  that  the  granum,  after  the  extraction  of  its 
chlorophyll,  consists  mainly  of  protein  and  lipide.  Attempts  have  been 
made  to  ascertain  the  exact  form  of  association  between  these  materials 
and  the  chlorophyll  Avdth  the  aid  of  polarized  light  and  fluorescence  in 
ultraviolet  light.  The  results,  taken  together  with  observations  on  the 
chemical  and  physical  behavior  of  proteins  and  lipides,  have  led  to  a 
current  hypothesis  of  chloroplast  structure.  On  this  hypothesis  the 
chlorophyll  forms  a  series  of  monomolecular  films  on  the  surfaces  of 
numerous  protein  layers  lying  more  or  less  parallel  throughout  the 
granum.  The  chlorophyll  molecules  have  their  hydrophile  ends  asso- 
ciated with  the  protein  and  their  lipophile  ends  with  lipide  molecules. 


Tllh:  STRiJCTUUAL  COMPONENTS  OF  PROTOPLASTS  33 

The  arrangement  is  such  that  between  each  two  protein  layers  there  are 
two  films  of  chlorophyll  molecules,  a  double  layer  of  lipide  molecules,  a 
few  xanthophyll  molecules,  and  water.  The  distance  between  two  of  the 
protein  layers  is  about  0.005/u.  To  what  extent  this  represents  the  true 
structure  no  one  can  say  at  present,  but  hypotheses,  right  or  wrong,  are 
valuable  as  long  as  they  are  subject  to  experimental  test. 

In  some  algae  the  chloroplast  contains  one  or  more  peculiar  bodies 
known  as  pyrenoids  (Fig.  21).  These  appear  like  small  masses  of  protein 
in  the  midst  of  the  stroma  and  evidently  play  some  definite  role  in  the 
elaboration  or  deposition  of  carbohydrates,  for  starch  grains  develop  in 
their  immediate  vicinity  and  commonly  form  a  dense  mass  about  each 
of  them.  Such  behavior  strongly  suggests  localized  enzymatic  activity. 
In  the  liverwort  Anthoceros  and  the  pteridophyte  Selaginella  there  are 
compound  pyrenoids,  the  parts  of  which  give  the  appearance  of  trans- 
formation into  starch  granules.  There  are  no  known  pyrenoids  in  seed 
plants. 

Development  and  Multiplication  of  Plastids. — When  the  meristematic 
tissues  in  a  young,  actively  growing  bud  of  a  seed  plant  are  examined,  it 
is  found  that  the  plastids  there  do  not  have  their  mature  characters,  but 
appear  rather  as  very  small  globules  or  rodlets;  these  are  plastid  pri- 
mordia,  or  proplastids.  As  the  young  stem  tip  and  leaves  grow  and  the 
cells  multiply,  the  proplastids  also  grow  and  multiply  by  division.  This 
is  apparently  their  only  mode  of  multiplication,  for  there  is  as  yet  no 
conclusive  evidence  that  they  ever  arise  anew  in  the  cytoplasm.  Sooner 
or  later,  when  the  leaf  tissues  become  further  differentiated,  the  enlarged 
proplastids  become  transformed  into  chloroplasts.  Division  may  occur 
even  after  they  are  fully  differentiated.  In  root  meristems  the  story  is 
essentially  the  same,  except  that  leucoplasts  rather  than  chloroplasts  are 
formed. 

The  division  of  the  young  plastids  during  the  development  of  most 
tissues  is  not  definitely  correlated  in  time  with  that  of  the  nuclei  and  cells. 
However  in  some  plants,  such  as  the  liverwort  Anthoceros  and  various 
algae  having  only  one  plastid  per  cell,  the  plastid  divides  regularly  just 
before  or  during  the  division  of  the  other  cell  elements,  thus  preserving 
the  one-plastid  condition.  It  should  be  added  that  many  plastids 
undergo  striking  alterations  in  shape  and  that  these  should  not  be 
interpreted  too  hastil}^  as  stages  of  division. 

The  bud  tissues  described  above  are  derived  from  embryonic  cells  and 
ultimately  from  the  fertilized  egg.  This  raises  the  question  of  the  con- 
tinuity of  plastids  as  individuals,  multipl^dng  only  by  division,  through 
successive  life  cycles.  In  the  angiosperms,  proplastids  appear  to  be 
present  at  all  stages  in  the  formation  of  spores  and  gametes,  the  plastids 
thus  being  continuous  from  one  generation  to  the  next.     The  inheritance 


34  FUNDAMENTALS  OF  CYTOLOGY 

of  certain  characters  involving  plastids  suggests,  however,  that  in  some 
but  not  all  of  these  plants  the  male  gamete  contributes  no  functional 
cytoplasm  or  plastids  to  the  offspring,  inheritance  of  such  characters 
being  purely  maternal  in  such  cases.  It  seems  probable  that  in  ferns  and 
brj^ophytes,  too,  there  is  such  a  continuity  of  plastids.  In  these  groups  it 
is  plain  that  the  motile  male  gamete  loses  most  if  not  all  of  its  cytoplasm 
wdth  any  contained  plastids  before  it  unites  with  the  egg.  In  Anthoceros, 
mentioned  in  the  preceding  paragraph,  the  single  plastid  characterizes 
all  the  cells  in  the  life  cycle  with  the  exception  of  the  male  gamete.  Again 
in  certain  algae  (Spirogyra,  Zygnema) ,  whose  gametes  have  essentially  the 
structure  of  vegetative  cells,  the  plastids  can  be  observed  as  distinct  indi- 
viduals through  all  the  reproductive  phases  (Figs.  117,  118). 

In  the  absence  of  adequate  evidence  to  the  contrary,  observations  such 
as  the  foregoing  speak  for  the  probable  validity  of  the  theory,  long  held  by 
many  cytologists,  that  plastids  are  permanent  protoplasmic  organs  always 
derived  from  their  predecessors  by  division.  An  element  of  doubt  still 
remains,  however,  for  in  certain  instances  it  is  reported  that  the  proplastids 
grade  off  to  the  lower  limit  of  visibility,  suggesting  the  possible  presence 
of  newly  formed  ones  as  yet  too  small  to  be  seen.  Furthermore,  it  is 
difficult  to  distinguish  with  certainty  minute  proplastids  from  other 
equally  small  bodies  in  the  cytoplasm.  Origin  anew  might  be  suspected 
in  view  of  other  cytoplasmic  specializations  during  development.  The 
observation  that  in  certain  algal  cells  the  chlorophyll  is  diffused  through- 
out the  cytoplasm  recalls  the  speculation  that  plastids  as  organs  arose 
historically  when  an  important  function  performed  throughout  the  cyto- 
plasm became  localized  along  with  corresponding  structural  alterations  in 
the  regions  concerned.  At  present,  however,  there  are  not  sufficient 
grounds  for  stating  that  anything  of  this  kind  occurs  in  the  development 
of  plastids  by  each  individual  plant. 

This  section  on  plastids  may  be  concluded  with  the  reminder  that  it  is 
chloroplasts  that  make  available  to  organisms  the  remarkable  properties 
of  carbon  with  its  capacity  for  forming  the  complex  compounds  required 
as  building  materials  and  sources  of  energy.  It  is  no  wonder  that  a 
famous  botanist  once  said  that  he  always  felt  like  taking  off  his  hat  to  the 
chloroplast. 

Chondriosomes. — The  presence  of  small  bodies  known  as  chondrio- 
somes,  or  mitochondria,  is  all  but  universal  in  the  cytoplasm  of  animals 
and  plants  (Fig.  22).  They  appear  in  living  cells  as  minute  granules, 
vesicles,  rodlets,  threads,  and  strings  of  beads,  and  they  often  vary  in 
abundance  and  form  in  different  phases  of  cellular  activity.  Thej^  are 
reported  to  arise  anew  in  the  cytoplasm,  and  they  can  be  seen  to  divide; 
in  special  cases  only  (e.g.,  certain  animal  spermatocytes)  does  the  division 
coincide  with  that  of  the  cell.     In  many  types  of  tissue  they  tend  gradu- 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


35 


ally  to  disappear  as  differentiation  approaches  completion.  Special 
techniques  are  required  for  their  study  in  fixed  preparations.  They 
can  be  well  fixed  with  fluids  containing  formalin  and  potassium  dichro- 
mate,  but  both  they  and  the  proplastids  are  dissolved  by  some  of  the  more 
highly  acid  fluids,  especially  those  containing  acetic  acid,  commonly  used 
for  the  preservation  of  nuclei. 

There  is  a  verj^  large  bodj'  of  literature  on  the  subject  of  chondrio- 
somes,  but  they  are  still  an  enigma  to  the  cytologist.  Naturally  many 
suggestions  have  been  made  regarding  their  significance.  For  the  most 
part  these  fall  under  two  general  heads:  (1)  the  view  that  they  are  reserve 
products  or  by-products  of  some  form  of  cytoplasmic  activity  common  to 
nearly  all  organisms,  these  prodvicts  being  utilized  as  energy  sources  or  in 
other  ways ;  (2)  the  view  that  they  are  organs  or  organ-like  bodies  playing 
a  definite  role  in  the  elaboration  of  metabolic  products  or  in  differentiation. 


A 


.       '      c  *  d 

Fig.  22. — Choiidiiosoiae?.  in  liver  cells  of  fishes,  a  ncHinal  cell  from  Fundulus  kept  at 
temperature  of  21°C.;  b,  cell  from  Fundulus  kept  at  temperature  (37.5  to  40°C.)  inducing 
heat  rigor;  c,  cell  from  Fundulus  in  extreme  heat  rigor  induced  at  45  to  50°C.;  d,  normal 
cell  from  goldfish.      {After  R.  C.  MacCardlc.) 

The  first  of  these  views  was  suggested  by  the  decrease  in  abundance 
and  frequent  disappearance  of  the  chondriosomes  as  tissues  mature  and 
by  their  apparent  use  in  the  elaboration  of  secretion  products  in  certain 
gland  cells.  The  second  view  was  suggested  by  what  was  interpreted  as 
the  direct  transformation  of  chondriosomes  into  various  intracellular 
specializations,  such  as  myofibrils  and  neurofibrils,  the  chondriosome 
being  looked  upon  as  a  sort  of  organ  of  differentiation.  Organ-like  roles 
in  connection  with  enzj'me  activity,  secretion,  respiration,  and  adsorption 
catalysis  have  also  been  regarded  as  possibilities.  Of  particular  interest 
was  the  observation  that  the  chondriosomes  bear  a  striking  resemblance 
to  young  plastids  in  form,  chemical  composition  and  specific  gravity; 
in  fact,  many  cytologists  have  inclined  to  the  view  that  plastids  arise  in 
plant  cells  by  the  transformation  of  chondriosomes  of  a  certain  type. 

The  problem  of  chondriosome-proplastid  relationship  has  been  a  par- 
ticularly vexing  one  for  a  number  of  years.  Recent  work  on  the  shapes, 
color  reactions,  and  pigmentation  of  these  bodies  has  emphasized  anew 
their  apparent  distinctness  (Figs.  23,  24),  whereas  some  observers  are 


36 


FUNDAMENTALS  OF  CYTOLOGY 


unable  to  accept  this  view.  If  this  problem  could  be  definitely  solved 
with  the  discovery  of  the  exact  reasons  why  some  of  the  minute  bodies 
develop  into  plastids  while  others  in  the  same  cell  or  in  other  organisms  do 
not,  we  should  be  nearer  to  a  solution  of  the  more  general  problem  of  the 
real  significance  of  chondriosomes  in  protoplasmic  activity.  Researches 
to  date  have  at  least  served  to  foster  a  more  critical  interpretation  of  the 
effects  of  fixation. 


Fig.  23. — Cell  from  inner  side  of  bean 
pod  after  treatment  with  Janus  green  B. 
m,  mitochondria;  O,  oil  globules;  P,  poly- 
morphic plastids,  colorless  and  partially 
impregnated  with  chlorophyll  (shaded). 
(After  H.  Sorokin.) 


Fig.  24. — Cells  from  young  shoot  of  rye. 
In  the  cytoplasm  are  small  granular 
mitochondria,  large,  deeply  staining  plastid 
primordia,  and  plastids  containing  starch. 
{After  J.  A.  O'Brien,  Jr.) 


Vacuoles. — Vacuoles  are  liquid-filled  cavities  in  the  cytoplasm  or,  very 
rarely,  in  other  portions  of  the  protoplast  (Figs.  25,  26).  They  are  char- 
acteristic chiefly  of  plants,  where  they  are  conspicuously  developed  in  cells 
of  nearly  all  kinds.  They  are  also  prominent  in  protozoan  cells,  while 
smaller  globules  staining  with  neutral  red  in  the  tissues  of  higher  animals 
seem  to  be  of  the  same  general  nature.  By  virtue  of  their  osmotic  proper- 
ties, plant  vacuoles  function  in  the  maintenance  of  turgor,  which  is  of 
importance  to  metabolic  acti\-ity  and  contributes  to  the  support  of 
herbaceous  bodies.  Fiu'thermore,  they  serve  as  repositories  for  certain, 
classes  of  reserve  products  and  by-products.  To  what  extent  such 
secretory  activity  involves  reactions  within  the  vacuoles  rather  than  in  the 
cytoplasm  near  by  is  not  well  known.  The  vacuoles  in  animal  tissues  play 
problematic  roles.  The  rhythmic  filling  and  discharge  of  contractile 
vacuoles  in  lower  animals  and  plants  appears  to  be  an  excretory  process, 
and  it  is  further  thought  probable  that  it  aids  in  the  regulation  of  hydro- 


THE  STRLCrURAL  (JOMFONENTS  OF  RROTOl'LAST.S 


3: 


static  pressure.     The  movement  of  the  gametes  in  Spirogyra  is  (lep(>ndent 
in  part  upon  the  activity  of  such  vacuoles. 

In  the  meristematic  cells  of  plants  the  vacuoles  are  usually  much 
smaller  and  more  numerous  than  in  differentiated  cells.     As  the  cells 


Fm.    25. — Cell    from    onion    root,    showing  Fig.    26. — Various    forms    assumed    by 

vacuoles.  vacuoles    in     fusiform     cambium     cells    of 

locust  tree.      (After  I.  W.  Bailey.) 

derived  from  the  meristem  enlarge,  multiply,  and  differentiate,  their 
vacuoles  increase  in  size,  undergo  internal  chemical  changes,  and  gradu- 
ally unite,  thus  forming  one  or  more  vacuoles  of  large  size  (Fig.  27). 
Often  vacuoles  in  living  cells  can  be  seen  to  become  fragmented  as  a 


Fig.  27. — Vacuoles  (stippled)       a-d,  successive  stages  in  bud  of  a  conifer  (Abies);  e,  pollen 
grain  of  a  conifer  (Cephaloiaxus).      (After  P.  Dangeard.) 

result  of  protoplasmic  streaming;  also  at  the  time  of  cell  division  they 
may  be  passively  divided. 

The  origin  of  the  small  vacuoles  in  the  meristem  has  been  variously 
conceived.  It  was  at  one  time  thought  that  the  cytoplasm  contained 
individualized  bodies  (tonoplasts)  derived  only  from  previous  ones  by 


38  FUNDAMENTALS  OF  CYTOLOGY 

division  throughout  the  Hfe  cycle  and  that  with  the  accumulation  of  cell 
sap  in  the  tonoplasts  these  extended  to  form  the  membranes  of  vacuoles. 
The  vacuole  membrane  is  still  called  the  iono-plasi.  It  is  now  more  gen- 
erally believed  that  vacuoles  arise  bj^  a  hydration  and  an  accumulation 
of  certain  cytoplasmic  colloids,  the  membrane  being  formed  by  the  cyto- 
plasm at  the  interface.  It  is  being  found  very  difficult,  however,  to 
demonstrate  that  vacuoles  do  arise  anew  in  such  a  manner. 

The  tonoplast  resembles  the  plasma  membrane  in  physical  consistency 
and  to  some  degree  in  its  semipermeability.  It  appears,  however,  to  be 
more  resistant  to  injur}'  and  may  manifest  semipermeabilit}'^  after  the  cell 
is  killed.  Its  permeability  to  certain  substances  may  be  remarkably  low, 
the  concentration  of  acids  in  the  vacuoles  of  some  cells  being  greater  than 
enough  to  kill  the  cell  if  they  were  applied  to  it  externally.  This  is 
notably  true  of  food  vacuoles  of  protozoa  during  digestion.  The  results 
of  permeability  studies  are  thought  to  indicate  that  the  tonoplast  contains 
more  lipide  than  the  plasma  membrane. 

The  cell  sap  in  plant  vacuoles  of  the  common  type  is  a  slightly  viscous 
fluid  composed  of  water  and  substances  of  many  kinds  in  molecular  or 
colloidal  solution.  Salts,  sugars,  organic  acids  (oxalic,  malic,  citric,  etc.), 
glucosides,  alkaloids,  amides,  proteins,  enzymes,  tannins,  and  other 
compounds  can  be  identified  in  different  cases.  Some  cells  secrete  visible 
globules  of  protein,  gums,  resins,  and  other  materials  in  such  amounts  that 
the  sap  has  a  milky  appearance.  In  extreme  cases  such  cells  form  exten- 
sive systems  of  latex  tubes  ramifying  through  the  other  tissues  of  the  plant ; 
familiar  examples  are  the  dandelion  and  the  rubber  tree.  Globules  of 
rubber  are  elaborated  in  the  cytoplasm  and  secreted  into  the  sap  in 
Ficus.     Crystals  of  various  compositions  also  may  occur  in  the  cell  sap. 

The  reactions  of  cell  sap  to  chemical  tests  and  in  staining  procedures 
may  vary  widely  in  different  plants  and  at  different  stages  of  development 
in  a  given  tissue.  This  is  due  both  to  differences  in  pH  and  to  the  pres- 
ence or  absence  of  certain  particular  compounds.  In  meristematic  tissues 
the  cell  sap  ordinarilj'-  has  a  slightly  alkaline  or  a  neutral  (pH  7)  reaction, 
but  as  the  vacuoles  enlarge  in  the  differentiating  cells  it  soon  becomes 
decidedly  acid,  the  pH  falling  to  5  or  even  lower.  Phenolic  compounds 
such  as  tannin  also  have  a  profound  effect  upon  stainabilit.y,  the  same  cell 
sometimes  showing  vacuoles  of  two  colors  correlated  with  differences  in 
tannin  content.  Of  several  vital  stains  commonly  used  for  vacuoles,  the 
best  is  neutral  red.  It  quickly  accumulates  in  the  vacuoles  of  living  cells, 
leaving  the  cytoplasm  and  nucleus  colorless,  and  it  remains  there  until 
death  occurs. 

Many  vacuoles  contain  natural  pigments  in  their  sap.  The  most 
prevalent  of  these  are  the  anthocyanins,  which  are  commonly  reddish  in 
very  acid  saps  and  blue  or  purple  in  more  alkaline  mediums.     Such 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


31) 


l)igmonts  are  mostly  resjionsiljle  for  these  colors  in  flowers  and  fruits. 
Yellow  flavone  pigments  rarelj^  arc  evident  in  the  sap  of  j^etals  (e.g., 
snapdragons),  although  they  may  be  made  to  appear  in  some  white  flowers 
upon  hj'drolysis  of  the  glucosides  of  which  they  are  constituents.  It  is 
rather  the  plastid  pigments  that  give  so  many  plants  their  yellow  colors. 

Golgi  Material. — The  Golgi  material,  named  after  its  discoverer, 
appears  in  the  cytoplasm  of  nearly  all  animal  cells  prepared  with  certain 
special  techniques  designed  to  deposit  silver  or  osmium 
as  a  dark  precipitate.  Under  such  conditions  the  ma- 
terial appears  in  the  form  of  separate  small  bodies  or  as 
a  more  or  less  continuous  system  of  strands,  these  forms 
being  fairly  constant  in  certain  types  of  cell.  Only 
rarel}^  can  it  be  distinguished  from  the  c,ytoplasm  in 
living  cells.  Its  composition  is  not  well  known,  but 
lipides  are  evidently  present.  Its  tendency  to  show 
blackened  and  nonblackened  portions  in  silver-impreg- 
nated material  suggests  the  occurrence  of  two  main  con- 
stituents which  may  not,  however,  have  such  an 
arrangement  in  the'  living  cell.  Its  behavior  in  cen- 
trifuged  cells  indicates  its  physical  distinctness  from 
the  chondriosomes  and  also  that  its  viscosity  in  different 
cells  varies  with  respect  to  that  of  the  cytoplasm. 
At  the  time  of  cell  division  it  is  distributed  passively 
and  more  or  less  equally  to  the  daughter  cells  (Fig.  28). 

The  function  of  the  Golgi  material  is  most  evident 
in  gland  cells  (Fig.  29).  During  secretory  activity'  in 
these  cells,  it  becomes  more  plentiful  and  droplets  of 
the  secretion  product  make  their  appearance  in  contact 
with  it.  The  droplets  accumulate  near  the  surface  of 
the  cell  and  are  eventually  excreted.  It  is  not  yet  clear 
whether  this  should  be  interpreted  as  a  synthesis  or  a 
condensation  of  the  secretion  globules  by  the  Golgi 
material,  or  w^hether  the  globules  themselves,  which 
stain  with  neutral  red,  are  active  vacuoles  in  which  the 
secretion  is  produced  from  materials  in  the  cytoplasm  near  l)y.  On  the 
latter  theory  the  Golgi  networks  seen  in  silver  preparations  are  inter- 
preted as  an  alteration  of  dense  chondriosome-containing  cytoplasm 
between  the  vacuoles  in  this  region  of  the  cell.  The  rok^  of  the  Golgi 
material  in  nonglandular  cells  is  problematical,  though  secretory  or  other 
elaborative  activity  on  a  smaller  scale  is  suggested. 

Attempts  to  "homologize"  the  Golgi  material  with  some  constituent 
of  plant  cells  have  not  met  with  definite  success.  When  meristematic 
plant  cells  are  treated  with  the  special  methods  mentioned  above,  the 


Fig.  28.— Moi- 
aphase  of  first  divi- 
sion in  spermato- 
cytes of  a  bug 
(Euschistus)  pre- 
pared by  dififerent 
methods  to  show 
filamentous  chon- 
driosomes (above) 
and  rounded  Golgi 
bodies  (below) . 
Chromosomes  at 
center.  (After  R. 
H.  Bowen.) 


40 


FUNDAMENTALS  OF  CYTOLOGY 


aspects  presented  by  vacuoles  in  their  various  stages  of  development  ma}- 
be  strikingly  like  those  of  the  Golgi  material  in  animal  cells.  That  both 
are  involved  in  secretory  activity  is  also  suggestive.  It  is  true  that 
plastids,  too,  when  similarly  treated,  often  show  a  strong  resemblance 
to  the  Golgi  material.  In  the  sperm-forming  cells  of  mosses  there  is  not 
only  a  likeness  in  form  and  stainability,  but  a  mass  of  material  arising 
from  the  plastid  performs  the  same  peculiar  function  as  does  a  product  of 
the  Golgi  material  in  animal  spermatogenesis  (page  121).  This  would 
seem  to  be  a  stronger  argument  for  homology.     A  third  constituent  of 


Fig.  29. — The  relation  of  the  Golgi  apparatus  to  secretion.  1—5,  formation  of  secretory 
droplets  in  goblet  cell  in  intestine;  6,  secretory  globules  with  attached  bits  of  Golgi  material 
from  pancreas.      {After  R.  H.  Bowen.) 

plant  cells,  the  so-called  osynio-pkilic  'platelets,  has  been  brought  into  the 
controversy. 

An  interesting  light  on  this  question  has  come  from  bean  root  cells 
subjected  to  very  high  centrifugal  forces — 400,000  times  gravity  for  15  to 
20  minutes.  In  cells  thus  treated  the  cell  constituents  become  arranged 
in  order  of  their  relative  specific  gravity  as  shown  in  Fig.  30.  It  is  to  be 
noted  that  the  plastids  and  chondriosomes  are  relatively  heavy,  whereas 
the  osmiophilic  platelets  are  relatively  light,  just  as  the  Golgi  material  is 
shown  to  be  in  animal  cells  similarly  treated. 

Animals  and  plants  have  been  going  their  separate  ways  in  evolu- 
tionary specialization  for  a  very  long  time,  the  differences  in  nutrition 
and  cytological  structure  having  become  great  enough  to  enjoin  caution 
in  the  drawing  of  homologies.     We  shall  nevertheless  continue  to  be 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS 


41 


l^-rWf- 

.•<■  -  .  -V 

'.©.     .J          o 

^  o./^^-^r 

o    °'.    .:%'r 

>/^     #  fit: 

■' vv^^y  .  > 

Vo'r «     >o   o 

l°\V-:-,®^^?J 

O  v°>  o«^ 


m^ 


impressed  in  later  chapters  by  similarities  rather  than  by  (lif'ferences  in 
the  fundamental  cytological  features  of  the  two  organic  kingdoms. 

Ergastic  Matter. — Accumulations  of  nonproto- 
plasmic  materials  in  or  on  the  protoplast  are  called 
ergastic  suhsfances  (ergon  =  work).  They  are  for  the 
most  part  products  of  the  protoplasm's  metabolic 
work.  They  may  represent  reserve  matcnials  later 
used  as  sources  of  energy  in  further  work,  or  the 
useless  by-products  of  such  activity,  or  supporting 
structures  that  render  bodies  of  certain  t,ypes  pos- 
sible. Such  ergastic  matter  may  occur  in  an^^  part 
of  the  protoplast,  although  it  is  rarely  observed  in 
nuclei.  The  cell  sap,  described  in  a  previous  section, 
may  be  regarded  as  a  mass  of  ergastic  materials  in 
an  aqueous  medium. 

The  most  conspicuous  ergastic  substances  in 
plants  are  carbohydrates,  starch  and  cellulose  being 
the  representatives  of  this  group  most  often  observed 
in  tissues.  The  starch  elaborated  in  the  chloroplast 
appears  as  visible  granules,  and  when  deposited  by 
amyloplasts  in  storage  organs  the  granules  may 
become  very  large.  Researches  on  the  structure  of 
such  granules  have  shown  them  to  consist  of 
numerous  concentric  layers  which  have  been  de- 
posited successively  about  a  hilum,  the  point  at  which 
deposition  began  (Fig.  31).  Unequal  deposition  on 
the  various  sides  results  in  a  granule  of  eccentric  form 
and  structure.  Compound  grains  with  more  than 
one  hilum  are  plentiful  in  some  tissues. 

The  layering  in  the  starch  granule  has  been  coi- 
related  with  periodic  activity  of  the  plastic!  caused 
by  the  alternation  of  day  and  night.  Each  laj-er 
consists  of  CeHioOs  units  arranged  in  a  regular  "space 
lattice":  the  granule  is  a  spherocrystal.  When  such 
granules  are  examined  in  polarized  light  between 
crossed  Nicol  prisms,  they  present  a  characteristic 
and  beautiful  appearance,  each  of  them  being  a  bright 
body  traversed  by  a  dark  cross  with  its  arms  meeting 
at  the  hilum  (Fig.  32).  It  is  sometimes  possible  to 
identify  small  granules  in  this  way  when  other  tests  fail. 

Cellulose  is  the  chief  material  in  plant  cell  walls.  Like  starch,  it 
consists  of  CeHioOa  units  (anhydrous  glucose  residues)  arranged  in  the 
form  of  a  space  lattice,  the  layers  here  being  i-elatively  flat.     Associated 


^^l^^^f§)OOoS 


Vic.  :M).  Xolinal 
and  centrifuged  eells 
of  V)ean  root.  In  the 
centrifuged  cell  (be- 
low) the  centrifugal 
end  is  directed  down- 
ward. In  order  of 
relative  and  decI■ea^^- 
ing  .specific  gravity 
the  nonnuclear  com- 
ponents are  (1)  .starch 
granules  and  pla.«tids 
(when  present),  (2) 
chondriosomes  and 
proplastids,  (.3)  cyto- 
plasm, (4)  osmiophilic 
platelets,  (5)  cell  sap, 
(())  lipide  material. 
{After  H.  W.  Beams 
and  R.  L.  Kirifj.) 


42 


FUNDAMENTALS  OF  CYTOLOGY 


with  it  are  other  materials;  these  will  be  discussed  in  Chap.  VI.  Cellulose 
and  hemicelluloses  frequently  serve  as  reserve  products,  notably  in  the 
endosperm  of  certain  seeds. 


Fig.  31. — Cell  from  stem  of  an  angio- 
sperm  {Pellionia)  with  large  starch  gran- 
ules. Most  of  the  plastid  substance  forms 
a  thick  cap  at  one  end  of  the  granule. 


Fig.  32. — Potato-starch  granules  photo- 
graphed through  a  polarizing  microscope 
with  crossed  Nicol  prisms. 


Glycogen  is  an  important  reserve  carbohydrate  formed  in  animal 
cells;  it  occurs  also  in  blue-green  algae  and  various  fungi.  Mucilages 
and  gums  are  further  examples  of  ergastic  carbohydrates. 


Fig.  33. — Large  oil  drop- 
let in  cell  of  young  root  of 
heliotrope  ( Valeriana) . 
{After  A.  Meyer.) 


Fig.  34. — Development  of  crystal  aggregate  ("druse") 
in  castor  bean  plant.  1-5,  single  crystals  and  aggregates; 
6,  7,  dendritic  growth;  8-10,  developing  crystals  within 
cell;  11,  druse  in  mature  parenchyma  cell  at  petiole  base. 
(After  F.  M.  Scott.) 


Proteins  as  ergastic  products  occur  in  crystalline  and  noncrystalline 
masses.  Perhaps  the  most  familiar  protein  reserves  in  plants  are  the 
aleurone  granules  of  certain  seeds,  notably  those  of  cereals,  legumes, 
walnuts,   and  the  castor  bean.     Such  granules  may  have  amorphous 


THE  STRUCTURAL  COMPONENTS  OF  PROTOPLASTS  13 

protein  only,  or  both  amorphous  antl  crvstalhno  components;  other 
substances  may  be  present  also.  Protein  res(n'V(\s  are  w<'ll  known  in  the 
yolk  of  animal  eggs  where  they  commonl.\-  occur  in  associnlion  with  l';itty 
yolk  globules. 

Plants  infected  with  some  viruses  show  characteristic  intracellular 
bodies  not  present  in  normal  tissues.  These  are  amorjjhous  in  some 
cases,  while  certain  other  viruses  result  in  the  formation  of  l)otii  am(n-- 
phous  and  crystalline  inclusions.  Insoluble  complexes  reseml)ling  the.se 
can  be  produced  artificially  by  combining  purified  viruses  with  proteins 
of  certain  kinds,  which  suggests  that  such  bodies  in  infected  tissues  may 
be  similar  combinations  of  the  virus  with  normal  or  abnormal  materials 
of  the  host  plant.  This  single  example  may  serve  as  a  remindei-  of  the 
mutual  assistance  rendered  by  cytology  and  pathology. 

Fats  and  oils  occur  as  reserves  in  the  form  of  globules  in  the  cj'to- 
plasm  of  many  plant  and  animal  cells,  particularly  in  seeds,  spores,  and 
eggs.  Minute  lipide  globules  are  of  common  occurrence  in  the  cytoplasm 
of  active  cells.  Ver}^  large  oil  globules  are  sometimes  encountered  (Fig. 
33).     Waxes  are  ergastic  products  of  importance  in  many  plants. 

Inorganic  crj^stals  form  another  class  of  ergastic  substances  (Fig.  34). 
These  occur  in  great  variety  in  plant  tissues,  the  needle-shaped  "raph- 
ides"  composed  of  calcium  oxalate  being  very  frequently  encountered 
in  cytological  work  on  living  tissues. 

Ergastic  substances,  then,  are  of  man}-  kinds.  The  same  chemical 
compound  maj^  occur  at  any  one  moment  as  a  relatively  inert  mass  in 
the  protoplast;  at  another  moment  it  may  be  in  solution  and  participat- 
ing actively  in  the  work  of  the  protoplasm.  Ergastic  matter  is  therefore 
to  be  characterized  by  its  relative  inactivit,y  rather  than  its  composition. 
This  is  in  harmony  with  the  view  adopted  in  the  follo^^^ng  chapter,  viz., 
that  protoplasm  is  an  organized  living  system  of  substances  that  by 
themselves  are  not  li\ing. 


CHAPTER  TV 
PROTOPLASM 

Few  scientific  achievements  rank  in  importance  with  the  discovery 
that  the  phenomena  of  hfe  occur  in  a  colorless,  somewhat  viscous  fluid- 
like material  having  certain  properties  common  to  all  organisms.  Every 
plant  and  animal  type,  and  probably  every  individual,  has  its  own  charac- 
teristic type  of  protoplasm,  but  the  fundamental  features  of  this  sub- 
stance are  strikingly  the  same  everywhere.  This  highly  significant  fact 
was  given  eloquent  expression  by  Thomas  Huxley  (1868)  in  his  classic 
essay  on  "The  Physical  Basis  of  Life,"  which  ranks  as  a  masterpiece  of 
popular  scientific  exposition. 

Physical  Characters  of  Protoplasm. — By  direct  observation  with  the 
aid  of  the  ordinary  microscope,  protoplasm  is  revealed  as  a  clear  fluid, 
called  hyaloplasm,  in  which  there  usually  are  distributed  globules, 
granules,  and  various  special  differentiations.  Many  activities,  such  as 
the  characteristic  streaming  in  vacuolate  plant  cells  and  the  responses  to 
certain  experimental  treatments,  may  be  studied  in  this  way,  but  the 
investigator  must  employ  additional  special  techniques.  Among  these 
aids  are  dark-field  illumination,  which  reveals  the  presence  of  very 
minute  particles;  ultraviolet  photography,  which  yields  images  showing 
fine  structural  detail;  fluorescence  in  ultraviolet  light,  which  gives  evi- 
dence of  composition;  polarized  light,  by  which  it  is  possible  to  learn  much 
concerning  ultramicroscopic  structure  and  chemical  composition;  the 
high-speed  centrifuge,  which  yields  data  on  specific  gravity  and  viscosity; 
the  micromanipulator,  wnth  which  it  is  possible  to  operate  on  living  cells 
under  high  magnifications;  and  the  electron  microscope. 

The  results  of  such  investigations  have  been  numerous  and  of  excep- 
tional value.  For  example,  it  has  been  possible  to  measure  with  con- 
siderable accuracy  the  viscosity  of  protoplasm  in  different  cells,  in 
localized  regions  of  the  same  cell,  and  in  the  same  region  at  different 
stages  of  functional  activity.  The  values  obtained  range  from  only  two 
or  three  times  that  of  water  in  the  granule-free  hyaloplasm  of  certain 
eggs  to  hundreds  of  times  this  value  in  some  other  types  of  protoplasm. 
Commonly  the  viscosity  of  protoplasm  in  active  cells  is  about  that  of 
glycerin  or  light  machine  oil.  Exceedingly  high  values  sometimes 
reported  are  probably  due  to  secondary  differentiations  in  the  protoplast. 
The  average  viscosity  tends  to  be  lower  in  plants  than  in  animals. 

44 


PROTOPLASM  -15 

Although  nerve  cells  and  epithelial  cells  show  a  relativelj^  high  viscosity, 
there  appear  to  be  less  viscous  channels  within  them. 

The  viscosity  of  protoplasm  may  be  experimentally  decreased  by 
hydrostatic  pressure  or  by  stirring;  it  may  be  increased  by  anesthetics, 
heat,  electric  currents,  ultraviolet  light,  and  X  rays.  These  changes  are 
reversible,  but  extreme  treatments  may  result  in  irreversible  coagulation 
or  complete  cytolysis.  Experiments  of  this  nature  have  demonstrated 
the  dependence  of  certain  protoplasmic  processes,  notably  cell  cleavage 
and  the  fascinating  phenomenon  of  streaming  with  its  various  results, 
upon  orderly  alterations  in  viscosity.  One  method  of  measuring  ^-iscosity 
is  that  of  observing  the  rate  of  movement  of  nickel  particles  placed 
within  the  protoplasm  and  then  subjected  to  centrifugal  or  electromag- 
netic force. 

Elasticit}',  a  characteristic  of  special  importance 
in  view  of  the  hints  it  affords  regarding  the  ultrami-  -»-=— 

croscopic  structure  of  protoplasm,  can  be  demonstrated 
wdth  the  micromanipulator  and  by  observing  the  tend- 
ency of  introduced  nickel  particles  displaced  by  the 
electromagnet  to  return  to  their  original  position  when 
the  current  is  cut  off.  Red  blood  cells  and  certain 
nuclei  have  been  stretched  between  two  micromanipu- 
lator needles  until  they  were,  respectively,  4  and  25  j^-j^^  ;35— 8iir- 
times  their  original  diameter  (Fig.  4),  and  upon  release  face  layer  of  starfish 
they  returned  to  nearly  their  normal  shape.  Such  ^th^  needle  ^mT  mi- 
behavior  may  be  due  in  part  to  the  bounding  mem-  cro manipulator, 
branes,  but  the  nickel-particle  technique  shows  clearly  /  er  .  m  ers.) 
that  protoplasm  itself,  and  not  merely  its  membranes,  is  elastic  as  well 
as  ductile. 

Refined  mechanical  aids  have  made  it  possible  to  learn  a  great  deal 
about  the  phj^sical  nature  of  the  external  membranes  of  cells.  Echino- 
derm  eggs,  because  of  their  large  size  and  other  desirable  characters, 
have  been  used  for  this  and  many  other  types  of  cytological  study.  After 
the  removal  of  certain  external  coats,  the  protoplasmic  surface  film  of 
the  egg  lies  exposed.  The  physical  characteristics  of  this  film  can  be 
demonstrated  by  drawing  it  out  in  the  form  of  a  slender  strand  from  the 
egg  surface  and  then  allowing  it  to  return  to  its  original  position  (Fig.  35). 
If  not  broken  the  material  coalesces  perfectly  with  the  egg,  while  small 
bits  if  broken  away  round  up  into  droplets.  This  and  a  variety  of  other 
treatments  have  shown  that  the  surface  membrane  is  elastic  and  water- 
immiscible,  though  permeable;  it  shrinks  without  wrinkling,  extends 
without  increase  of  surface  tension,  easily  engulfs  droplets  of  paraffin 
oil,  and  undergoes  rapid  renewal  fiom  within  when  not  too  greatly 
torn. 


46  FUNDAMENTALS  OF  CYTOLOGY 

Streaming  movements  of  various  kinds  appear  to  be  of  general  occur- 
rence in  protoplasm.  They  have  been  studied  chiefly  in  amebas,  Plas- 
modia of  slime  molds,  dividing  animal  eggs,  and  the  highly  vacuolate 
cells  of  plants.  The  movement  may  involve  the  entire  protoplast,  the 
cytoplasm  streaming  as  one  mass  and  carrying  the  various  inclusions 
with  it,  or  only  localized  portions  may  be  concerned,  the  other  regions 
showing  no  visible  change.  No  complete  explanation  of  this  fascinating 
phenomenon  has  yet  been  given.  With  the  aid  of  the  motion-picture 
camera  it  has  been  found  that  the  streaming  observed  in  the  slime  mold 
results  from  a  rhythmic  contraction  and  relaxation  of  the  protoplasm, 
and  the  force  involved  has  been  measured.  Contraction  and  relaxation 
in  protoplasm  are  now  attributed  mainly  to  a  folding  and  unfolding  of 
linear  protein  molecules  (see  page  48).  The  energy  necessary  to  proto- 
plasmic streaming  is  evidently  derived  from  respiration,  but  the  manner 
in  which  this  energy  is  utilized  in  producing  the  movements  is  unknown. 

Protoplasm  as  a  Colloidal  System. — The  physical  properties  of  proto- 
plasm are  largely  dependent  upon  the  fact  that  it  is  a  complex  colloidal 
system.  Matter  is  in  the  colloidal  state  w^hen  it  has  the  form  of  numerous 
small  particles,  the  resulting  properties  being  most  characteristically 
displayed  when  the  particles  are  between  about  0.1  and  0.001^  in  at  least 
one  dimension.  This  is  below  the  reach  of  the  ordinary  microscope. 
Such  particles  are  molecular  aggregates  except  perhaps  in  the  case  of 
extremely  large  molecules.  In  a  colloidal  system  at  least  two  phases  are 
essential :  a  medium  which  constitutes  the  continuous  phase  and  a  second 
substance  dispersed  as  particles  within  it.  The  phases  may  be  liquid, 
solid,  or  gaseous,  and  they  may  have  any  chemical  composition  so  long  as 
they  are  dissimilar  enough  to  remain  physically  distinct.  The  chemical 
constituents  of  a  given  phase  are  called  com-ponents.  The  important 
feature  of  all  colloidal  systems,  which  vary  greatly  in  minute  structure, 
is  that  the  phases  lie  in  contact  with  each  other  over  a  surface  of  enormous 
extent,  even  in  a  minute  cell.  This  fact  means  much  when  it  is  remem- 
bered that  many  reactions  are  promoted  b}^  forces  acting  at  surfaces. 

It  is,  of  course,  the  fluid  colloidal  systems  that  are  of  particular 
significance  in  biology.  Such  a  system  is  known  as  a  sol  if  it  flows  readily 
and  as  a  gel  if  it  does  not.  It  may  be  made  to  pass  from  one  state  to  the 
other,  often  by  relatively  small  alterations  in  temperature,  electrical 
charge,  or  the  degree  of  hydration.  Thus  a  sol  may  become  a  gel  (gela- 
tion, pectization),  and  the  gel  may  again  become  a  sol  (solation,  peptiza- 
tion). Irreversible  coagulation  may  also  occur.  In  a  fluid  colloidal 
system  most  of  the  continuous  phase  is  free  and  easily  removable,  but 
some  of  it  may  constitute  a  denser  solvation  layer  at  the  surface  of  the 
other  jjhase  and  strongly  resist  forces  tending  to  remove  it.  When  the 
layer  consists  of  water,  the  colloidal  particle  is  said  to  be  hydrated. 


PROTOPLASM  47 

That  protoplasm  is  a  colloidal  system  is  indicated  by  many  of  its 
characteristics.  Like  other  colloids  it  differs  from  true  solutions  in  its 
manner  of  flow,  in  the  relation  of  its  viscosity  to  stress,  and  in  behavior 
involving  surface  tension,  adsorption,  and  permeability.  Its  physical 
consistency  varies  widely  during  certain  processes  such  as  cell  division 
and  is  strongly  affected  b.v  stimuli  of  various  kinds.  It  even  exceeds 
some  other  colloidal  systems  in  its  resistance  to  separation  of  phases  by 
centrifugal  forces:  after  being  centrifuged  for  1  hour  at  400,000  times 
gravity,  Ascaris  eggs  recovered  and  divided,  and  cleavage  actually 
occurred  during  centrifugation  at  100,000  times  gravity. 

It  is  not  known  at  present  what  type  or  tj'pes  of  colloidal  structure  are 
characteristic  or  essential  in  protoplasm.  Although  protoplasm  fre- 
quently shows  within  the  visible  range  a  structure  like  that  of  an  emulsion, 
it  is  uncertain  how  far  such  a  structure  continues  into  the  submicroscopic 
range.  As  a  matter  of  fact  there  are  rather  definite  indications  that  the 
structure  is  not  primarily  of  the  emulsoid  type:  protoplasm  is  elastic, 
emulsions  are  not;  protoplasm  has  a  limited,  though  high,  imbibition 
limit,  whereas  emulsions  do  not;  protoplasm  coagulates,  emulsions  do  not 
(coagulation  of  milk  involves  the  protein,  casein,  not  the  fat  forming  the 
visible  emulsion) ;  across  the  plasma  membrane  is  a  continuous  water 
path,  and  if  the  other  phase  or  phases  were  discontinuous  the  membrane 
would  disintegrate  in  water.  Such  considerations  point  to  the  existence 
of  some  sort  of  structural  framework  not  capable  of  indefinite  dispersion 
like  an  emulsion. 

The  view  that  protoplasm  has  an  important  fibrous  element  in  its- 
structure  along  with  nonfibrous  constituents  has  recently  gained  strong 
support.  The  "fibrillar  theorj^"  of  many  years  ago  was  based  largely 
upon  what  was  directly  seen  in  living  and  fixed  cells.  Our  modern 
interpretation  has  come  not  only  from  investigations  of  protoplasm  with  a 
variety  of  new  techniques,  but  also  from  physical  and  chemical  studies  on 
inorganic  colloidal  systems,  on  certain  products  of  biological  activity,  and 
especially  on  the  proteins.  The  "fibers"  that  now  concern  us  are  mainly 
something  far  smaller  than  the  workers  of  half  a  century  ago  had  in 
mind. 

Especially  instructive  are  the  results  of  researches  on  the  structure  of 
inorganic  systems,  notably  those  formed  by  vanadium  pentoxide,  zinc 
oxide,  and  silicon  hydroxide  in  water.  In  polarized  light  between  crossed 
nicols,  these  su))stances  wken  flowing  in  narrow  channels  show  double 
refraction  of  a  type  that  indicates  the  presence  of  minute  linear  elements 
lying  parallel;  moreover,  such  an  arrangement  is  sometimes  assumed 
spontaneously  without  flow.  The  linear  elements  here  are  evidently 
chains  of  elongate  molecules:  in  the  silica  gel,  for  example,  the  Si(OH).j 
molecules  join  end-to-end,  losing  HoO  at  each  junction,  to  form  long 


48 


FUNDAMENTALS  OF  CYTOLOGY 


chains.  When  such  long  molecules  or  molecular  chains  have  a  random 
arrangement,  the  system  is  isotropic.  When  they  lie  closely  parallel 
and  are  free  to  move  upon  each  other,  they  are  in  the  paracrystalline,  or 
"liquid-crystal,"  state,  and  if  they  lie  in  a  medium  of  different  refrac- 
tive index,  the  system  exhibits  double  refraction.  In  the  gel  state, 
characterized  by  considerable  firmness  and  elasticity,  the  chains  evidently 
tend  to  associate  in  a  sort  of  network  or  to  unite  closely  here  and  there  into 
bundles,  or  micelles.  Such  elastic  gels  may  quickly  liquefy  when  jarred 
or  stirred  owing  to  a  property  known  as  thixotropy. 

In  such  phenomena  the  cytologist  finds  welcome  clues  to  the  sub- 
microscopic  structure  of  protoplasm.  For  example,  double  refraction 
can  be  detected  in  muscle  cells,  plastids,  chromosomes,  and  mitotic 
spindles;  furthermore,  ice  crystals  formed  \\'ithin  these  structures  tend  to 


NH. 


COOH  NH 


RCH  NHH  COOH 

Union    of   two    amino    ocids    with  loss    of    HiO 


RCH 


Polypeptide  chain  of   indefinite  lengtti 

Fig.  36. — The  constitution  of  the  polypeptide  chain.     The  symbol  R  stands  for  side  chains 

of  several  kinds:  NH2,  COOH,  SH,  OH,  etc.      {Adapted  from  Frey-WyssHng.) 

be  oriented  accordingly.  Gelled  regions  in  protoplasm  frequently  pass 
into  the  sol  state  when  stirred  with  the  micromanipulator  needle.  Proto- 
plasm is  elastic.  A  certain  myxomycete  Plasmodium  can  pass  through 
pores  Iju  in  diameter  slowly  and  by  itself,  but  it  is  destroyed  if  pressed 
through  gauze  with  openings  200^1  in  diameter.  All  such  observations, 
together  with  the  visible  fibrous  differentiations  more  directl}^  observable, 
point  to  the  conclusion  that  protoplasm  has  a  fibrous  constituent  in  its 
fundamental  structure. 

It  is  now  thought  probable  that  the  submicroscopic  fibrous  constituent 
of  protoplasmic  structure  consists  primarily  of  proteins.  Researches  on 
proteins  show  that  their  molecules  consist  of  amino  acids  arranged  in  the 
form  of  long  polypeptide  chains  (Fig.  36).  Such  a  chain  may  be  extended 
as  a  "fibrous  molecule"  which  could,  with  a  molecular  weight  of  35,000, 
reach  a  length  of  0.1  m-  The  results  of  studies  with  X  rays  and  polarizefl 
light  show  that  molecules  in  this  extended  form,  singly  or  in  bundl(\s 
(micelles),  are  present  in  tissues  with  mechanical  functions   (muscles, 


PROTOPLASM  49 

tendons)  and  in  certain  ]irodiicts  of  protoplasmic  activity  (hair,  siliv). 
The  molecules  in  silk,  which  is  inelastic,  are  fully  extended,  whereas  those 
in  wool,  which  is  elastic,  are  wavy  or  folded.  In  other  proteins  the  chain 
may  be  very  closely  folded,  forming  a  "globular  molecule."  With  a 
molecular  weight  of  36,000  such  a  molecule  would  have  a  diameter  of 
about  0.005m.  Such  molecules  constitute  the  so-called  mobile  proteins, 
while  the  fibrous  molecules  form  the  structure  proteins.  It  has  been  possi- 
ble to  separate  the  two  kinds  in  cells:  about  an  eighth  of  the  proteins  in  an 
echinoderm  egg  and  about  two-thirds  of  those  in  kidney  cells  are  of  the 
structural  type.  There  is  controversy  over  the  question  of  the  degree  of 
distinctness  and  the  relative  importance  of  the  two  types  in  determining 
the  physical  characters  of  protoplasm,  but  the  extremely  suggestive  nature 
of  the  above  findings  is  evident. 

Chemical  Nature  of  Protoplasm. — The  immense  difficulty  of  ascertain- 
ing the  chemical  composition  of  protoplasm  with  any  degree  of  accuracy 
scarcely  needs  to  be  pointed  out.  With  protoplasm  are  always  associated 
some  of  its  products ;  relative  amounts  of  the  constituents  vary  in  different 
tissues  and  at  different  phases  of  activity;  the  high  sensitivity  to  reagents 
and  the  alterations  occurring  at  death  greatly  complicate  the  problem  of 
analysis.  It  is  nevertheless  possible  to  form  a  general  idea  of  its  composi- 
tion, and  with  further  improvements  in  method  our  knowledge  of  it  will 
doubtless  gain  in  definiteness.  In  general  it  is  found  that  protoplasm  in 
the  active  state  consists  of  more  than  75  per  cent  water  and  less  than  25 
per  cent  materials  representing  the  dry  weight.  The  dry  matter  is 
roughly  90  per  cent  organic  (proteins,  fats,  carbohydrates)  and  10  per  cent 
inorganic. 

Water,  one  of  the  commonest  substances  in  nature,  is  of  the  utmost 
importance  to  organisms.  Without  water  or  something  like  it — and  there 
is  nothing  like  it — life  as  we  know  it  is  inconceivable.  Water  acts  as  a 
solvent  and  conveyor  of  reacting  materials,  is  a  medium  of  reaction,  and 
participates  in  reactions  through  hydrolysis  and  dehydration.  Because 
of  its  unique  properties  it  very  largely  determines  the  character  of  the 
colloidal  system  of  which  it  is  a  part  as  well  as  the  types  of  reaction  that 
occur.  In  inactive  protoplasm,  such  as  that  in  dry  spores,  the  percentage 
of  water  may  fall  to  a  very  low  value.  The  protoplast  contains  free 
water  and  water  bound  at  the  surface  of  the  colloidal  particles.  The 
bound  water  is  difficult  to  remove  by  heat,  and  it  also  resists  the  effects 
of  very  low  temperatures,  remaining  unfrozen  after  the  free  water  has 
crystallized.  It  is  probably  this  crystallization  of  free  water  that  kills 
protoplasm  at  low  temperatures:  spores  that  have  been  deprived  of  their 
free  water  may  survive  the  temperature  of  liquid  air.  It  has  recently 
been  found  that  undehydrated  cells  may  not  be  killed  by  intense  cold  if  the 
temperature  is  lowered  very  rapidly  through  the  freezing  range:  the  water 


50  FUNDAMENTALS  OF  CYTOLOGY 

then  vitrifies  instead  of  crystallizing,  and  the  cells  survive  and  resume 
activity  after  the  temperature  is  raised  very  rapidly  through  the  range 
where  crystallization  would  otherwise  occur.  This  has  an  interesting 
bearing  on  the  problem  of  storing  living  material  at  low  temperatures. 

The  inorganic  salts  in  protoplasm  are  fairly  numerous  though  small  in 
amount.  They  occur  in  part  in  the  free  water  and  in  part  as  ions  bound 
by  the  organic  constituents.  They  incorporate  many  of  the  40  or  more 
essential  elements,  some  of  which  are  present  in  extremely  small  amounts 
and  can  be  detected  only  by  extremely  sensitive  methods.  The  amounts 
present  often  do  not  indicate  the  amounts  necessar}^,  but  in  some  instances 
their  ratio  of  concentration  is  very  significant.  For  example,  sodium 
lowers  viscosit}^  and  increases  the  permeability  of  membranes,  whereas 
calcium,  necessary  to  membrane  formation,  has  the  opposite  effects;  in 
combination  the  two  are  antagonistic.  The  ratio  of  calcium  to  sodium 
salts  is  about  the  same  in  sea  water,  blood,  and  balanced  protein-lipide 
emulsions,  a  fact  that  surely  has  interesting  theoretical  implications  for 
the  student  of  evolution. 

Among  the  carbohydrates  the  pentoses,  hexoses,  and  their  condensation 
products  (polysaccharides)  are  of  special  importance  in  the  constitution 
and  activity  of  protoplasts.  The  pentoses,  C5H10O5,  are  one  of  the  main 
components  of  nuclear  chromatin  (page  51),  while  various  pentosans, 
(CsHioOs)^,  are  the  principal  constituents  of  many  plant  mucilages  and 
gums  and  are  components  of  pectins.  Among  stored  foods  are  hexoses, 
C6H12O6,  including  glucose,  levulose,  mannose,  and  galactose,  as  well  as 
hexosans,  (CeHioOs)^,  notably  starch  in  plants  and  glycogen  in  animals. 
Cellulose,  a  hexosan,  is  the  main  constituent  of  most  plant  cell  walls. 
Carbohydrates  other  than  the  pentoses  do  not  enter  directly  into  the 
actual  constitution  of  protoplasm  but  serve  as  sources  of  energy  and  build- 
ing materials. 

Fats  and  oils  occur  in  great  variety  in  protoplasts.  Although  it  is  often 
impossible  to  tell  in  what  degree  a  given  kind  is  a  true  constituent  or  only 
a  product  of  protoplasm,  there  can  be  no  doubt  that  some  of  them,  notably 
the  phospholipides,  are  among  the  fundamental  constituents.  The  fragrant 
essential  oils  of  plants  are  not  fats  but  belong  to  other  chemical  classes. 
Although  of  great  commercial  imi^ortance,  their  value  to  the  plant  is 
questionable. 

True  fats,  which  contain  only  carbon,  hydrogen,  and  oxygen,  are  salt- 
like combinations  (esters)  of  fatty  acids  and  glycerol  (the  glycerides)  or  of 
fatty  acids  and  other  alcohols  (the  sterols  and  most  waxes) .  The  fats  are 
of  importance  as  reserve  food  and  together  with  sterols  and  waxes  function 
in  retarding  loss  of  water  at  surface  membranes.  Ergosterol,  a  sterol 
found  in  plants,  becomes  the  antirachitic  vitamin  D  upon  irradiation  with 
ultraviolet  light.     Vitamins  A  and  E  are  commonly  found  in  tissues  high 


PROTOPLASM  51 

in  fat.  Variable  amounts  of  free  fat  occur  in  cells,  but  much  of  the  fatty 
material  exists  in  some  form  of  combination  with  the  proteins. 

The  compound  fats  are  combinations  of  fatty  acids,  nitrogen-contain- 
ing bases,  and  either  phosphorus  or  carbohydrates.  Such  fat -like  com- 
pounds containing  nitrogen  \\\t\\  or  without  phosphorus  are  called  lipides. 
The  phospholipides  are  of  special  interest,  for  they  appear  to  perform  a 
major  role  in  the  formation  and  activit}^  of  protoplasmic  surface  mem- 
branes, thus  sharing  largely  in  determining  permeability  and  water- 
immiscibility.  Lecithin,  a  prominent  member  of  this  class,  is  abundant 
in  all  cells.  It  occurs  in  a  finely  divided  state  in  the  cytoplasm,  and  by 
virtue  of  its  possession  of  hydrophile  and  lipophile  groups  it  probably 
functions  in  maintaining  the  colloidal  state. 

The  proteins,  which  with  lipides  and  water  represent  the  main  con- 
stituents of  protoplasm,  are  elaborate  compounds  containing  carbon, 
hydrogen,  oxygen,  nitrogen,  often  sulphur,  and  sometimes  phosphorus. 
They  are  built  up  from  amino  acids  with  XHo  substituted  for  H  in  the 
group  attached  to  the  COOH  group.  The  protein  casein  has  about  20 
amino  acids  in  its  molecule.  Every  kind  of  organism  evidentl}^  differs 
in  some  measure  from  every  other  in  its  proteins,  a  fact  that  is  of  impor- 
tance with  respect  to  such  matters  as  immunity,  allergy,  and  the  differ- 
entiation of  species. 

The  simple  natural  proteins,  which  yield  only  amino  acids  when 
hydrolyzed  by  enzymes  or  acids,  are  present  in  great  variet}^  in  protoplasts. 
Albumins  and  globulins  are  important  constituents  of  cytoplasm  and  are 
often  present  in  large  quantities  in  eggs  and  seeds.  The  enzyme  urease 
is  a  globulin,  and  other  enzymes  also  are  proteins.  Other  simple  proteins 
characterize  the  cereals  {e.g.,  glutenin,  oryzein,  zein,  gliadin,  hordein) 
and  animal  tissues  (e.g.,  keratin,  elastin,  gelatin,  collagen).  The  histones 
are  relatively  simple,  while  the  simplest  and  most  basic  of  all  natural 
proteins  are  the  protamines.  The  best  known  protamine  (salmin)  from 
fish  sperm  has  only  four  amino  acids,  and  its  formula  is  C81H115N45O18. 

The  conjugated  proteins  in  nature  are  simple  proteins  in  combination 
with  other  organic  groups;  they  yield  amino  acids  and  nonproteins  when 
hydrolyzed.  As  examples  may  be  mentioned  the  glycoproteins  in  mucus 
and  "tissue  cements,"  the  chromoproteins  in  certain  plant  and  animal 
pigments,  the  lecithoproteins  probably  present  in  all  cytoplasm  and  its 
membranes,  and  the  nucleoproteins  found  in  various  parts  of  the  proto- 
plast and  of  special  importance  in  nuclei. 

Special  attention  should  be  given  to  the  proteins  of  nuclei.  As  pointed 
out  previously  (page  50),  the  material  composing  the  chromonemata, 
which  are  significant  constituents  of  the  chromosomes,  is  mainly  a  nucleo- 
protein  made  up  of  proteins  and  nucleic  acid.  This  highly  stainable 
material  has  been  called  nuclein  or,  more  commonly,  chromatin.     The 


52  FUNDAMENTALS  OF  CYTOLOGY 

proteins  concerned  are  relatively  simple  ones  such  as  protamines  (in  fish 
sperm)  or,  more  commonly  in  animals,  the  somewhat  more  complex  and 
less  basic  histones.  In  plants  the  nuclear  proteins  are  less  well  known, 
but  they  appear  to  resemble  histones  in  composition.  Of  great  signifi- 
cance is  the  previously  cited  fact  that  each  type  of  organism  studied  seems 
to  have  its  own  peculiar  kind  of  protein.  So  far  as  nuclear  materials  are 
concerned,  differences  between  species  appear  to  reside  largely  in  the 
protein  portion  of  the  nucleoprotein  molecule  and  probably  to  a  lesser 
degree  in  the  nucleic  acid  portion  responsible  for  the  chromatin's  high 
stainability  with  basic  dyes.  Nucleic  acid  is  composed  of  chemical 
groups  of  three  main  types:  (1)  phosphoric  acid  groups,  (2)  pentose 
carbohydrate  groups  (sometimes  hexoses?),  and  (3)  purine  and  pyrimi- 
dine  bases.  Nucleic  acid,  like  the  proteins  with  which  it  is  associated,  has 
the  remarkable  ability  to  form  long  chains.  Of  extraordinary^  significance 
is  the  recent  discovery  that  the  tobacco  mosaic  virus,  which  like  other 
viruses  has  the  power  of  increasing  its  substance  when  in  a  protoplasmic 
medium,  is  a  nucleoprotein. 

The  karyolymph  consists  mainly  of  proteins  less  highly  polymerized 
than  those  of  the  chromosomes.  The  nucleolus  has  at  least  two  main 
constituents:  (1)  a  protein  that  does  not  stain  with  iron-hematoxylin  and 
(2)  a  stainable  sulphuric  ester  of  a  polysaccharide.  At  certain  stages 
a  form  of  nucleic  acid  can  be  detected  in  the  nucleolus  and  in  some  cases 
in  the  cytoplasm.  The  small  amount  of  mineral  matter  in  nuclei  lies 
in  the  chromatic  elements  rather  than  in  the  karyolymph,  to  judge  from 
the  location  of  ash  in  incinerated  tissues.  The  enormous  chemical  com- 
plexity of  the  nucleus  is  evident  when  one  considers  that  in  a  sperm  cell 
of  ordinary  size  the  nuclear  portion  has  a  dry  weight  of  scarcely  a  billionth 
of  a  milligram;  yet  this  minute  mass  of  material,  which  constitutes  about 
3  per  cent  of  the  weight  of  the  living  sperm  head,  carries  the  physical 
basis  of  the  paternal  hereditarj^  contribution  to  the  next  generation. 

The  Staining  of  Protoplasm. — The  staining  reactions  of  protoplasm 
depend  upon  its  chemical  composition,  its  colloidal  state,  and  certain 
characteristics  of  the  dye  solutions.  It  is  mainly  the  proteins  that  take 
up  the  stains,  but  certain  products  of  other  kinds,  such  as  minute  fat 
droplets,  may  be  so  abundant  and  stainable  as  to  obscure  the  effects  of 
the  stains  on  the  protoplasm  itself.  The  dyes  employed,  aside  from 
valuable  natural  ones  like  hematoxylin  and  carmine,  are  for  the  most  part 
coal-tar  products.  These  dyes  are  commonly  employed  as  salts  and 
fall  into  two  main  classes:  basic  dye  solutions  carry  the  color  in  the 
cations,  whereas  acid  dye  solutions  carry  it  in  the  anions.  Familiar 
examples  of  the  former  group  are  safranine  (red),  crystal  violet  (blue  or 
violet),  and  methyl  green;  members  of  the  latter  group  are  eosin  (red), 
methyl  blue,  and  fast  green. 


PROTOPLASM  53 

The  successful  staining  of  living  protoplasm  with  "vital"  dyes,  which 
are  nontoxic  in  dilute  solution,  requires  considerable  skill.  Some  of  these 
dyes  are  indicators  of  the  degree  of  acidity  or  alkalinity,  since  the}^  alter 
their  color  in  passing  through  characteristic  regions  of  the  pH  scale.  In 
this  way  it  has  been  found  in  cells  of  various  kinds  that  the  living  nucleus 
is  slightly  alkaline,  with  a  pH  of  about  7.5  to  7.6,  whereas  the  cytoplasm 
is  usuall}^  at  about  6.7  to  6.9,  or  slightly  acid.  Injury  causes  the  acidity 
to  increase,  the  pH  of  the  cytoplasm  falling  to  5.2  to  5.5.  The  cell  may 
later  recover,  but  not  if  the  hydrogen-ion  concentration  is  maintained  too 
long  at  this  level.  When  an  ameba  is  placed  in  a  dilute  solution  of 
methyl  red,  it  becomes  pale  yellow  throughout,  showing  that  the  pH 
in  all  parts  is  well  above  5.2.  If  a  slight  amount  of  acid  is  then  injected 
into  the  cytoplasm  near  the  nucleus,  a  local  reddening  of  the  cytoplasm 
and  then  of  the  nucleus  indicates  a  lowering  of  the  pH  to  some  point 
below  5.2.  Both  regions  soon  recover  their  yellow  color,  showing  that 
the  protoplasm  contains  or  produces  buffering  substances  tending  to 
maintain  its  normal  reaction  in  the  vicinitj^  of  pH  7,  the  neutral  point. 

Living  nuclei  can  be  stained  with  dilute  solutions  of  weakly  basic 
dyes,  which  enter  cells  freely,  or  by  acid  ones  when  injected.  Protozoa 
ma}^  live  with  nuclei  and  chromosomes  stained  with  neutral  red,  and 
certain  stages  of  mitosis  in  plants  can  occur  with  chromosomes  colored 
by  Hoffmann's  violet  or  malachite  green.  Cytoplasm  ordinarily  does  not 
take  the  stains  markedly,  much  of  the  color  observed  being  rather  in 
vacuoles  and  inclusions.  Lipide-soluble  dyes  appear  to  stain  the  cyto- 
plasm itself  in  some  degree. 

The  fixation  of  tissues  with  special  fluids  designed  to  render  their 
components  firm  and  more  resistant  to  reagents  employed  in  sectioning 
techniques  also  has  effects  on  staining.  The  staining  may  be  greatly 
improved  by  previous  fixation,  but  one  must  always  be  on  guard  against 
interpreting  fixation  artefacts  as  natural  appearances.  After  fixation 
the  nucleus  acts  as  an  acid  and  stains  markedly  with  basic  coal-tar  dyes. 
This  is  because  the  nucleic  acid,  although  combined  with  other  substances 
in  such  a  way  as  to  render  the  living  nucleus  actively  alkaline,  neverthe- 
less gives  the  nucleus  a  strong  potential  acidity.  Hence  when  fixed 
tissues  are  placed  in  properly  prepared  solutions  of  basic  dyes,  the  nega- 
tive bonds  of  the  phosphoric  acid  groups  in  the  nucleic  acid  unite  with  the 
colored  cations  of  the  solution.  The  cytoplasm,  on  the  contrary,  com- 
monly acts  as  a  base  in  fixed  tissues  and  unites  with  the  colored  anions  in 
solutions  of  acid  coal-tar  dyes.  It  is  by  manipulating  a  pair  of  dyes 
differing  in  both  color  and  reacting  power  that  double-staining  effects  are 
achieved. 

The  proteins  react  as  they  do  in  such  procedures  largely  because  they 
are  amphoteric,  i.e.,  they  have  the  properties  of  both  bases  and  acids 


54  FUNDAMENTALS  OF  CYTOLOGY 

because  of  the  uncombined  NH2  and  COOH  groups  in  the  amino  acids 
of  which  they  are  composed.  The  reaction  of  a  given  cell  protein 
depends  upon  the  relation  existing  between  its  isoelectric  point  and  the 
pH  of  the  medium:  it  acts  as  an  acid  and  stains  with  a  basic  dye  if  its 
isoelectric  point  is  below  the  pH  of  the  solution.  When  the  relation  of 
these  two  factors  is  reversed,  the  protein  acts  as  a  base  and  takes  the  acid 
dye.  Hence  when  it  is  desired  to  stain  the  nucleus  with  one  dye  and 
the  cytoplasm  with  another,  success  may  depend  upon  adjustments  of 
the  pH  of  the  solutions  with  respect  to  the  somewhat  different  isoelectric 
points  of  the  two  regions.  Exact  values  are  difficult  to  determine  because 
of  the  chemical  complexity  of  the  protoplasmic  system  and  the  fvu'ther 
complications  introduced  by  other  variable  factors. 

One  of  the  most  useful  staining  techniques  now  used  in  cytology  is  the 
Feulgen  reaction,  which  consists  in  the  restoration  of  color  to  decolorized 
basic  fuchsin  by  aldehyde  groups  in  the  pentose  component  of  thy- 
monucleic  acid.  It  is  thus  rather  highly  specific  for  chromatin  and  can 
be  used  to  distinguish  chromosomes  from  other  bodies.  Ribonucleic 
acid,  present  in  nucleoli  and  sometimes  in  cytoplasm,  gives  a  negative 
Feulgen  test,  but  it  can  be  detected  through  the  absorption  of  ultraviolet 
light  which  it  shows  in  common  with  tl\vmonucleic  acid  of  the  chromatin. 

Conclusions.- — The  matters  discussed  in  this  chapter  all  have  a  more 
or  less  direct  bearing  on  the  work  of  the  cytologist,  who  is  aware  that  his 
own  understanding  of  every  cytological  object  and  process  will  be 
deepened  bj'  what  the  physicist  and  chemist  can  help  him  to  learn  about 
protoplasm.  Complete  comprehension  of  protoplasmic  activity  is  a  goal 
that  cannot  be  approached  rapidly  and  perhaps  can  never  be  reached,  yet 
it  is  helpful  to  have  in  mind  a  provisional  picture  of  protoplasm  as  a 
physicochemical  system. 

Pix)toplasm  is  an  extraordinarily  complex  mixture  of  materials  of 
manj^  kinds,  each  of  which  has  some  share  in  determining  the  nature  of  its 
activities.  It  may  be  thought  of  as  a  vast  array  of  ions,  molecules,  and 
molecular  aggregates,  some  of  them  large  enough  to  be  visible,  forming  a 
colloidal  system  of  numerous  phases.  Certain  proteins,  because  of  their 
linear  molecules  and  chain-forming  ability,  seem  to  constitute  a  sort  of 
loose  submicroscopic  framework  to  which  some  of  the  lipides,  phos- 
pholipides,  and  other  materials  are  attached.  Lecithin,  with  its  hj^dro- 
phile  and  lipophile  groups,  acts  as  a  link  between  proteins  and  fats. 
Water  molecules  in  great  numbers,  together  with  inorganic  ions  and 
molecules,  occur  in  the  interstices  of  the  framework.  The  whole  mass  is 
capable  of  streaming  because  the  unions  between  the  various  substances 
in  the  framework  are  readily  broken  and  reestablished  in  new  waj^s. 
Local  variations  of  this  structure  occur  in  the  membranes,  plastids, 


PROTOI'LASM  55 

nuclei,    and    (jtlici-    microscopically    visible    specializations    witiiin    the 
protoplast. 

Protoplasm  is  therefore  more  than  a  mere  mixture:  it  is  a  delicately 
balanced  organized  system  of  substances  combined  in  certain  proportions 
and  patterns  and  interacting  harmoniously  in  a  consistent  manner  through 
long  and  varied  life  cycles.  Life  is  the  resultant  of  all  these  amazingly 
well-correlated  activities:  it  is  a  property  not  of  this  or  that  component, 
but  of  the  system  as  a  whole.  The  subject  of  this  chapter  is  one  that 
should  interest  not  merely  the  cytologist,  but  every  person  interested  in 
his  relation  to  the  rest  of  nature,  for  protoplasm  is  the  physical  basis  of 
his  being  as  well  as  of  every  other  living  thing. 


CHAPTER  V 
THE  DIVISION  OF  THE  PROTOPLAST 

The  division  of  one  protoplast  into  two  can  be  seen  with  Uttle  diffi- 
culty under  a  microscope,  yet  the  process  is  one  that  investigators  armed 
with  many  techniques  have  only  begun  to  understand.  The  significance 
of  much  that  is  seen  occurring  is  evident,  but  precisely  how  the  various 
changes  are  accomplished  remains  to  be  discovered.  Ordinarily  the 
division  of  a  free  cell  or  a  tissue  cell  results  in  two  cells  that  have  the  same 
structure  and  capacities  as  the  cell  that  produced  them.  In  the  develop- 
ment of  the  body  (soma)  of  a  large  organism,  a  long  series  of  such  divisions 
occurs,  the  many  resulting  cells  eventually  becoming  unlike  in  appearance 
as  the  soma  differentiates.  When  a  reproductive  cell — a  spore  or  an  egg 
— is  produced,  it  has  all  the  capacities  essential  to  the  development  of  a 
complete  individual.  We  are  therefore  faced  with  the  problem  of  deter- 
mining how  the  highly  complex  organization  of  the  protoplast  can  be 
duplicated  w^hen  division  occurs  and,  further,  just  what  it  is  in  this  organi- 
zation that  enables  a  spore  or  an  egg  to  become  an  adult  organism 
manifesting  both  general  and  particular  characters  of  the  previous 
generation.     It  is  the  first  of  these  cjuestions  that  now  concerns  us. 

For  the  study  of  somatic  cell  division  in  plants,  one  may  employ  large 
cells  that  can  be  kept  living  in  aqueous  mediums  or  paraffin  oil  while 
being  examined  wdth  the  microscope.  The  cells  of  certain  filamentous 
algae  {Zygnema,  Sphacelaria),  the  marginal  cells  of  very  young  leaves 
(Tradescantia),  and  the  hairs  on  certain  stamens  (Tradescantia)  and 
grass  stigmas  (Arrhenathcrum)  have  been  used  very  successfully  in  this 
way.  Young  root  tips  have  long  been  favorite  material  for  somatic- 
division  studies  in  higher  plants,  for  the  regular  arrangement  of  the  cells 
and  the  large  number  of  divisions  visible  in  one  stained  section  rendei- 
them  almost  perfect  objects  for  the  purpose  (Fig.  37).  In  animals  the 
dividing  eggs  of  echinoderms  and  fishes  are  particularly  good  (Fig.  38). 
In  later  stages  of  somatic  development  the  embryonic  membranes  of 
mammals  and  the  tail  fins  of  tadpoles  yield  excellent  division  figures 
(Fig.  157).  The  somatic  type  of  division  is  also  well  displayed  by 
spermatogonia  (but  not  by  spermatocytes). 

It  should  be  reahzed  that  details  of  the  division  process  vary  wddely 
in  different  organisms  and  tissues.  In  this  chapter  we  shall  confine 
attention  to  typical  examples. 

56 


THE  DIVISION  OF  THE  PROTOPLAST 


57 


SOMATIC  CELL  DIVISION  IN  PLANTS 

Cell  division  includes  both  the  division  of  the  nucleus  by  a  process 
known  as  mitosis,  or  karyokinesis,  and  the  di\ision  of  the  cytosome,  or 


-f^i^ 


Fig.   37. — Portion   of  longitudinal   section   of   root   tip   of   onion.     Prophase,    nietapha.sc, 
anaphase,  and  telophase  stages  of  mitosis  are  visible.      {Courtesy  of  G.  H.  C'onant.) 

cytokinesis.  Mitosis  often  occurs  without  cytokinesis,  as  in  coenocytes, 
and  sometimes  cytokinesis  takes  place  without  nuclear  division.  Mitosis 
and  cell  division  are  therefore  not  svnonvmous  terms. 


58 


FUNDAMENTALS  OF  CYTOLOGY 


Comparatively  little  is  well  known  concerning  the  particular  factors 
responsible  for  the  onset  of  cell  division.  Since  the  mitotic  changes  are 
so  conspicuous  and  precede  cytokinesis,  it  is  often  assumed  that  cell 
division  begins  with  the  nucleus,  all  othei-  changes  being  a  consequence  of 


Fig.  38. —  Mitosis  in  embryonic  cells  of  whitefish:  prophase,  metaphase,  and  two  stages  of 
anaphase.      {Courtesy  of  General  Biological  Supply  House,  Inc.,  Chicago.) 

its  behavior.  In  certain  meristematic  cells  with  large  vacuoles  it  has 
been  shown,  however,  that  the  cytoplasm  forms  a  sort  of  diaphragm,  or 
phragmosome,  across  the  cell  at  the  plane  of  future  cytokinesis  before  the 
nucleus,  about  to  divide,  becomes  oriented  with  respect  to  this  plane 


THE  DIVISION  OF  THE  PROTOPLAST 


59 


(Fig.  39).  This  indicates  that  the  plane  of  cell  division  is  determined  by 
factors  acting  at  an  early  stage  throughout  the  cell  and  not  by  the  nucleus 
alone. 

Mitosis. — Mitosis  is  a  process  in  which  each  of  the  chromosomes,  the 
principal  constituents  of  the  nucleus,  undergoes  a  longitudinal  doubling, 
the  halves  of  all  the  chromosomes  then  separating  into  two  similar  groups 
which  reconstitute  two  new  nuclei  (daughter  nuclei).  Only  rarely  or 
under  very  exceptional  circumstances  does  a  nucleus,  without  respect  to 
the  chromosomes  as  individuals,  divide  b}^  simple  constriction  {amitosis); 
mitosis  is  the  almost  universal  method  of  nuclear  division. 

In  the  root  tip  of  a  plant  with  large  chromosomes  the  course  of  mitosis 
is  essentially  as  follows  (Fig.  40).     The  nucleus  in  the  metabolic  stage 


^ 


*:^%?- 


■"tl^T" 

I 

.    -s^l 

'       ■^-• 

y 

c 

r 

'■"i 

___&^ 

4'.i 

"•'W 

..•;'.  i.4jjjgjfe»' •  ■-i.aa 


E  F  G  H 

Fig.  39. — Division  of  vacuolate  pith  cell,  showing  the  cytoplasmic  diaphragm  (phrag- 
mosome)  present  before  mitosis.  Semidiagrammatic.  {After  E.  W.  Sinnott  and  R. 
Block.) 

preceding  a  division  contains  numerous  chromonemata  which,  because  of 
their  number,  length,  and  coiled  or  contorted  condition,  can  seldom  be 
traced  far  as  individuals.  The  prophase  comprises  all  the  changes  that 
transform  the  chromosomes  from  this  metabolic  condition,  in  which  their 
chromonemata  have  little  or  no  matrix  about  them  and  are  all  uniformly 
dispersed  in  the  nucleus,  into  the  compact  separate  individuals  seen  at  the 
mid-point  of  mitosis.  In  the  early  part  of  the  prophase  the  mass  of 
chromonemata  becomes  less  uniform,  so  that  the  threads  belonging  to 
different  chromosomes  stand  apart  more  clearly  as  indi^•iduals,  though 
their  length  at  this  stage  usually  precludes  following  them  from  end  to 
end.  They  are  more  or  less  spirally  coiled,  and  close  examination  shows 
them  to  be  longitudinally  double ;  hence  at  this  stage  each  chromosome  is 
actually  represented  by  two  chromonemata  running  closely  parallel. 
The  two  longitudinal  halves  of  a  chromosomt^  at  this  stage  or  at  any  other 
are  known  as  chromatids.  As  the  prophase  ad^'ances  to  its  middle  stage, 
the  chromonemata  tend  to  relax  their  coils  and  thicken  somewhat,  so  that 
the  doubleness  appears  more  plainly.  In  the  later  prophase  the  second 
chromosomal  constituent,  the  matrix,  accumulates  about  each  chromo- 


GO 


FUNDAMENTALS  OF  CYTOLOGY 


J    V 


MID- PROPHASE 


^   r 


&fj'^   ^ 


%  ©^  ^#   p 


ANAPHASE 


y      V 


EARLY    TELOPHASE  MID- TELOPHASE  LATE    TELOPHASE 

Fig.  40. — Diagram  of  somatic  cell  division  based  on  studies  of  plants  with  large  chromo- 
somes. The  relation  of  one  chromosome  pair  to  the  nucleolus  is  indicated.  Three  stages 
of  cytokinesis  by  cell-plate  development  are  shown  in  the  last  row.  Further  explanation 
in  text. 


THE  DIVISION  OF  THE  PROTOPLAST 


61 


nema  which  again  becomes  more  closely  coiled;  thus  the  chromosome  soon 
becomes  a  thicker,  smoother,  double  body  comprising  two  chromatids,  each 
composed  of  chromonema  and  matrix.  In  ordinary  preparations  the 
deeply  stained  matrix  renders  the  chromonema  invisible,  but  suitable 
methods  reveal  it.  In  some  cases  there  is  visible  evidence  that  the 
chromonema  in  each  chromatid  is  in  turn  longitudinally  double,  the  whole 
chromosome  by  the  end  of  the  prophase  therefore  having  four  half- 
chromatich.  The  nucleolus  commonly  disappears  late  in  the  prophase  as 
the  matrix  becomes  abundant  and  stainable. 

The  nucleus  next  passes  rapidly  through  a  stage  known  as  the  pro- 
vietaphasc  into  the  mctaphasc.  This  involves  a  complicated  series  of 
changes  in  which  the  karyolymph,  probably  with  the  cooperation  of  some 
cytoplasmic  substance,  liecomcs  transformed  into  the  achromaiic  figure,  or 


I 


_f3 

Fiu.  41. — Stages  in  mitosis  in  root  tips,     a,  anaphase;  b,  telophase;  c,  (/,  early  prophase; 
e,  late  prophase.      {After  L.  W.  Sharp.) 


spindle.  That  this  change  consists  primarily  in  a  definite  rearrangement 
of  materials,  presumably  protein  chains,  into  positions  parallel  with  the 
longitudinal  axis  of  the  spindle,  and  a  differentiation  into  two  components, 
one  relatively  firm  and  the  other  more  fluid,  is  indicated  by  several  lines 
of  evidence:  the  spindle,  unlike  the  material  previously  present,  is  aniso- 
tropic; it  offers  axial  resistance  to  swelling  or  shrinking  agents;  it  splits 
longitudinally  in  shrunken  cells;  Brownian  movement  of  occasional  parti- 
cles in  the  more  fluid  regions  is  greatest  parallel  to  the  longitudinal  axis ; 
fixation  usually  gives  the  spindle  a  longitudinally  striated  or  fibrillar 
aspect. 

The  spindle  in  root  tips  commonly  begins  its  development  at  two 
opposed  poles  of  the  nucleus,  apparently  outside  the  nuclear  membrane 
shrinking  inward  in  these  regions  (Fig.  42).  Sometimes  it  develops  more 
or  less  simultaneously  throughout  the  nucleus  with  no  membrane  shrink- 
age. In  either  case  the  membrane  (eventually  disappears,  leaving  the 
chromosomes,  which  have  meanwhile  moved  toward  the  equatorial  plane 
of  the  nucleus,  in  the  midst  of  the  spindle.     The  double  chromosomes, 


62 


FUNDAMENTALS  OF  CYTOLOGY 


normally  constant  in  number  in  a  given  kind  of  plant,  quickly  become 
arranged  in  such  a  way  that  a  certain  specialized  portion  of  each  of  them 
occupies  a  position  in  the  equatorial  plane.  This  portion  consists  of  the 
spindle-attachment  regions,  or  kinetochores,  of  the  two  chromatids.  The 
two  kinetochores  face  opposite  spindle  poles,  while  other  portions  of 
the  chromosome  maj^  lie  in  any  position.  When  this  stage  is  reached,  the 
nucleus  is  in  the  metaphase  of  division. 

As  the  kinetochores  take  up  their  positions  at  the  equator,  a  new  ele- 
ment appears  in  the  mitotic  figure.  At  the  kinetochore  of  every  chromatid 
there  appears  a  small  mass  of  material  which  gradually  extends  poleward 
through  the  spindle  substance  as  a  so-called  tractile  Jibe?-.  Whether  this 
represents  a  local  modification  of  the  spindle  substance,  a  fluid  extruded 


i^K^Kl.--    -1^1^^   '<:?^Jv\    ''irr  'u^ 


1  2  3  4 

Fig.    42. — Spindle   development   in   root    tip   of   hyacinth.      Explanation   in    text.      {After 

W.  Robyns.) 

from  the  chromatid,  or  an  actual  pseudopodium-like  extension  of  the 
chromatid  is  not  yet  agreed  upon  b,y  cytologists.  The  fact  that  it  some- 
times contains  a  Feulgen-positive  material  strongly  suggests  its  chromo- 
somal origin.  That  it  actually  exerts  a  tractile  force  is  seriously  doubted. 
In  the  anaphase  the  two  chromatids  of  each  chromosome  separate  and 
pass  toward  opposite  poles,  the  kinetochores  moving  ahead  along  the 
course  of  the  tractile  fibers.  After  each  chromatid  becomes  free  from  the 
other  and  goes  its  independent  way,  it  should  be  referred  to  as  a  chromo- 
some, the  two  half-chromatids  being  advanced  accordingly  to  the  rank 
of  chromatid.  In  the  anaphase,  as  in  the  metaphase,  the  general  mor- 
phology of  the  chomosomes  is  usually  well  displayed,  for  they  tend  to 
lie  well  separated  from  one  another  and  show  the  location  of  their  kine- 
tochores clearly  (Fig.  43).  Long  chromosomes  may  present  a  very 
confusing  appearance  during  the  earlier  portion  of  the  anaphase,  for 
even  though  the  kinetochores  pass  poleward  regularly,  the  other  portions 


rilE  DIVISION  OF  THE  PROTOPLAST  03 

which  have  been  lying  in  various  positions  are  drawn  into  many  odd 
shapes.  By  the  close  of  the  anaphase  the  tractile  fibers  ha\e  disap- 
peared, and  the  chromosomes  at  each  pole  form  a  close  group.  Hotwecii 
the  two  groups  lies  the  spindle  through  which  they  have  recently  passed. 
The  mechanism  of  their  anaphasic  movement  will  be  discussed  in  a  later 
section. 

The  telophase  is  the  stage  during  which  the  two  groups  of  chromo- 
somes, after  completing  their  anaphasic  movement,  reorganize  as  the 
two  new  nuclei.  Some  of  the  alterations  undergone  by  the  chromosomes 
in  the  prophase  are  now  reversed:  the  matrix  loses  its  stainability  or 
disappears,  leaving  the  chromonemata  visible,  while  the  latter  associate 
more    closely    with    their   neighbors    and    form    a    uniform    threadwork 


fk 


'F, 


^ 


Fig.  43. — Chromosomes  at  late  prophase,  metaphase  (polar  view),  and  anapha.se  of 
mitosis  in  microspore  of  Trillium.  The  spindle  is  not  well  shown  in  smear  preparations  of 
this  kind.      (After  H.  E.  Warmke.) 

dispersed  throughout  the  enlarging  nucleus.  The  presence  of  two 
chromonemata  in  each  anaphase  chromosome  may  account  for  the 
fact  that  in  the  completed  telophase  nucleus  there  often  appear  to  be 
more  chromonemata  than  the  known  number  of  chromosomes. 

While  the  chromosomes  are  undergoing  their  transformation,  other 
telophasic  changes  take  place.  Nucleoli  appear  among  the  chromone- 
mata as  the  matrix  disappears,  and  it  is  known  that  they  arise  at  definite 
points  on  certain  chromosomes.  If  there  are  two  or  more  nucleoli,  they 
may  fuse  or  remain  separate,  depending  upon  their  relative  positions. 
The  nuclear  membrane  arises  about  the  group  of  chromosomes  as  the 
telophase  begins.  The  karolymph  appears  and  increases  in  amount  as 
the  nucleus  enlarges,  but  its  origin  and  its  relation  to  the  disappearing 
matrix  are  not  understood. 

The  extent  to  which  the  telophasic  alterations  are  carried  varies  with 
the  type  of  tissue  and  rate  of  division.  In  older  regions  of  a  root  tip 
where  divisions  occur  slowly,  a  metabolic  stage  characterized  by  finely 
dispersed  chromonemata  is  develo])ed,  whereas  in  regions  where  mitoses 
occur  in  very  rapid  succession,  a  ])rophase  may  ])egin  l)eforc  the  preceding 


64 


FUNDAMENTALS  OF  CYTOLOGY 


telophase  has  advanced  so  far.     The  stage  between  two  mitoses  occumng 
in  rapid  succession  is  called  the  interphase. 

The  division  cycle  of  the  chromonemata  in  somatic  mitosis  may  be 
summarized  as  follows  for  a  single  chromosome.  In  the  metabolic  stage 
the  chromosome  is  represented  by  two  chromonemata  which  rank  as 
chromatids.  In  the  early  prophase  these  appear  as  a  double  spiral 
thread.  As  the  prophase  advances  the  two  become  less  closely  asso- 
ciated, and  by  the  time  the  metaphase  is  reached  each  of  them  has 
divided  into  two  half-chromatids,  making  four  chromonemata  in  the 
whole  metaphase  chromosome.  In  the  anaphase  the  two  chromatids 
move  apart  toward  opposite  poles.  Each  is  now  an  independent  daughter 
chromosome,  and  its  two  chromonemata  are  now  chromatids.  These 
chromonemata  represent  the  chromosome  through  the  telophase  and 
the   ensuing   metaboljc   stage.     Thus   a   chromonema   becomes   visibly 


ABC  abc 


ABCcibc 


Fig.  44. — Diagram  illustrating  the  equational  character  of  somatic  initosis. 

double  slightly  over  one  mitotic  cycle  in  advance  of  the  time  at  which 
the  halves  are  to  separate.  There  are  reasons  for  believing  that  the 
threads  are  doubled  submicroscopically  before  any  doubleness  is  seen 
and,  further,  that  the  chromosomes,  particularly  large  ones,  may  even 
be  more  highly  compound  in  terms  of  visible  chromonemata  than  indi- 
cated in  these  paragraphs.  The  foregoing  will  serve  as  a  convenient 
provisional  disposition  of  the  matter  until  some  alternative  interpretation 
has  become  better  established.  Chromosome  structure  will  be  discussed 
further  in  Chap.  VII. 

Finally,  the  significance  of  the  mitotic  form  of  nuclear  division  may 
be  emphasized.  At  the  close  of  a  typical  mitosis  there  are  two  nuclei 
that  are  quantitatively  and  qualitativelj^  similar  to  each  other  and  to 
the  nucleus  from  which  they  arose.  The  qualitative  aspect  is  of  special 
significance.  The  nucleus  is  not  merely  a  homogeneous  mass  of  some 
protein  or  other  substance,  but  an  intricately  organized  system  of  mate- 
rials of  many  kinds  with  definite  chemical  and  spatial  relations.  The 
chromonemata  contain  a  series  of  special  constituents  essential  to  normal 
development,   and  in  mitosis  these  constituents,   after  being  doubled, 


THE  DIVISION  OF  THE  PROTOPLAST  65 

are  equally  apportioned  to  the  two  daughter  nuclei  (Fig.  44).  As  a 
result,  the  organization  and  capacities  characteristic  of  the  original 
nucleus  are  exactly  reproduced  in  the  two  new  ones:  somatic  mitosis  is 
eqiiational.  From  this  it  follows  that  the  essential  organization  present 
in  the  nucleus  of  a  fertilized  egg  is  reproduced  in  all  the  nuclei  of  the  adult 
soma,  for  all  these  result  from  a  succession  of  equational  mitoses.  A 
simple  quantitative  mass  division  of  the  nucleus  without  respect  to  its 
differentiated  components  would  disrupt  the  system,  and  normal  develop- 
ment could  not  continue.  In  the  chapter  on  meiosis  we  shall  encounter  a 
form  of  nonequational  division,  but  it  is  an  orderly  process  of  such  nature 
that  a  complete  outfit  of  materials  is  still  maintained. 

Cytokinesis. — The  division  of  the  cytoplasmic  portion  of  the  pi-o- 
toplast  is  variously  correlated  in  time  with  mitosis.  In  some  tissues 
no  cytokinesis  follows,  in  others  it  follows  after  all  signs  of  recent  mitosis 
have  disappeared,  whereas  in  the  root  meristem  and  other  somatic 
tissues  of  higher  plants  it  commonly  begins  immediately,  even  before 
mitosis  has  been  completed.  In  this  last  case  mitosis  and  cytokinesis 
appear  like  two  parts  of  one  process,  for  the  region  of  the  cell  in  which 
cytokinesis  commences  is  still  occupied  by  the  remains  of  the  mitotic 
spindle.  As  a  result,  c^-tokinesis  in  these  tissues  is  of  a  type  characterized 
by  the  development  of  a  cell  plate.  Cytokinesis  in  many  other  plant 
cells  and  in  animals  is  accomplished  by  constriction  or  furrowing. 

Studies  on  living  cells,  notably  those  of  stamen  hairs,  show  that 
cytokinesis  by  cell-plate  formation  begins  as  follows.  The  spindle 
becomes  less  prominent  near  the  two  early  telophase  nuclei  and  widens 
at  the  equator  into  a  barrel-shaped  figure,  the  phragmoplast.  Some 
chemical  change  within  it  is  indicated  by  the  fact  that  it  now  stains 
like  the  cytoplasm  with  chrysoidine,  a  vital  dye,  whereas  during  meta- 
phase  and  anaphase  it  did  not.  Meanwhile,  even  before  widening  begins 
in  some  instances,  small  droplets  appear  near  the  equator  and  gradually 
unite  to  form  a  continuous  cell  plate  across  the  phragmoplast  (Figs. 
45,  46).  In  some  cells  the  cell  plate  appears  as  a  continuous  film  from 
the  start.  In  fixed  material  the  developing  cell  plate  commonly  appears 
at  first  like  a  series  of  granules  or  spindle-fiber  swellings  at  the  eciuator. 
The  phragmoplast  continues  to  fade  away  near  the  nuclei  and  to  widen 
at  the  equator,  while  the  cell  plate  extends  at  its  margins  until  the  latei-al 
walls  of  the  cell  are  reached.  The  remains  of  the  phragmoplast  then 
disappear. 

That  the  young  cell  plate  is  composed  of  fluid  is  shown  by  the  fact 
that  upon  plasmolysis  the  two  new  cells  easily  round  up  from  each  other, 
leaving  fluid  but  no  definite  membrane  between  them.  Veiy  soon,  how- 
ever, the  cell  plate  undergoes  both  physical  and  chemical  alterations, 
and  if  the  two  cells  are  then  separated  by  plasmolysis  a  firm  membi-ane 


66 


FUNDAMENTALS  OF  CYTOLOGY 


remains  in  the  intervening  fluid.  Strictly  .speaking,  cytokinesis,  the 
division  of  the  cytosome,  has  occurred  as  soon  as  the  halves  of  the  original 
protoplast  are  capable  of  rounding  up  as  two  inde]:)endent  protoplasts, 
for  to  do  this  each  of  them  must  have  completed  its  plasma  membrane 
on  the  side  next  to  the  cell  plate.  The  cell  plate,  with  certain  modifica- 
tions, remains  as  the  intercellular  substance,  or  middle  lamella,  upon 


1' urination  of  cull  ijlato  in   T radvscantia  stamen 
hair.     (After  W.  A.  Becker.) 


Fig.  46. — Phraginoplast 
with  cell  plate  in  iris  endo- 
sperm.    (After  V.  J  lingers.) 


which  the  cellulose  wall  layers  are  deposited,  thus  completing  the  parti- 
tion which  separates  the  new  protoplasts  in  plant  tissues  of  this  kind. 
The  development  and  nature  of  the  cell  wall  will  be  described  in  the 
next  chapter  (page  75). 

SOMATIC  CELL  DIVISION  IN  ANIMALS 

Somatic  division  in  animals  differs  in  many  cases  from  that  in  most 
plants  in  two  conspicuous  features:  (1)  the  achromatic  figure  is  often 
much  more  elaborate,  having  a  pair  of  asters  at  the  spindle  poles  and 
commonly  a  centrosome  at  the  focus  of  each  aster;  (2)  cytokinesis  is 
accomplished  by  a  furrow  which  progresses  inward  from  the  periphery 
of  the  cell,  rather  than  by  a  cell  plate  originating  in  the  middle  and 
extending  to  the  periphery. 

Mitosis. — Chromosome  behavior  during  mitosis  in  animals  is  essen- 
tially like  that  in  plants.  The  same  series  of  principal  phases  is  passed 
through,  and  the  main  significant  result  is  the  same:  the  division  of  the 
nucleus  is  equational,  the  original  nucleus  and  the  two  daughter  nuclei 
all  being  alike  in  chromosomal  composition  and  functional  capacity. 
In  recent  years  chromonemata  have  been  studied  less  in  animals  than 
in  plants.  Animal  and  plant  mitoses  may  differ  in  minor  ways,  but  it 
seems  likely  that  the  two  kingdoms  will  not  be  found  to  disagree  widely 
in  any  very  fundamental  feature  of  chromosome  behavior. 


THE  DIVISION  OF  THE  PROTOPLAST 


(17 


The  achromatic  figure  develops  in  typical  cases  as  follows  (Fig.  48). 
Lying  in  the  cj'toplasm  neai*  the  nucleus  is  a  centriole.  As  the  prophasic 
alterations  within  the  nucleus  begin,  the  centriole  divides  if  not  alroad.x- 
double,  and  the  daughter  centrioles  move  slowly  apart.     About  each 


Fig.  47. — iSection  of  spennary  of  crayfish  {Potamobius),  .showing  numerous  stages  of  mitosi.-s. 
{Courtesy  of  General  Biological  Supply  House,  Inc.,  Chicago.) 

of  them  there  appears  in  the  cytoplasm  a  system  of  radiations  known  as 
an  aster.  Between  the  two  may  be  seen  a  bundle  of  lines  called  the 
central  spindle,  all  three  parts  together  constituting  an  amphiaster.  The 
centrosomes  continue  to  diverge,  the  asters  increasing  in  prominence, 
until  they  reach  opposite  sides  of  the  nucleus.  By  the  time  they  reach 
these  positions,  and  often  before  this,  the  nuclear  materials  complete 


68 


FUNDAMENTALS  OF  CYTOLOGY 


their  prophasic  changes  and  the  nuclear  membrane  disappears,  all  the 
elements  concerned — amphiaster,  karyolymph,  chromosomes — establish- 
ing the  metaphase  figure.  In  many  cases  each  centriole  is  now  clearly 
double,  ha\'ing  divided  nearly  one  nuclear  cycle  in  advance  of  the  i^ro- 


FiG.  48. — Diagram  of  astral  mitosis  and  cytokinesis  in  an  animal  cell. 

phase  in  which  its  halves  are  to  separate.     Such  astral  figures  are  also 
found  in  certain  fungi  and  algae  (Fig.  49). 

The  asters,  in  the  possession  of  which  the  mitotic  figure  differs  so 
conspicuously  from  that  in  higher  plants,  are  evidently  developed  by 
the  formation  and  gradual  extension  of  centripetally  moving  streams, 
or  "astral  rays,"  in  the  cytoplasm  about  the  centrosomes.     The  regions 


X 


It 


''V/v^^T-^,.  ;h 


Fig.  49. — Astral  mitosis  in  brown  algae,  a,  centrioles  with  asters  moving  apart  along 
nuclear  membrane  in  apical  cell  of  Stypocauloii.  b,  metaphase  in  oogonium  of  Fucus;  the 
spindle  is  intranuclear,      (a,  after  W .  T.  Swingle;  b,  after  S.  Yamanouchi.) 

between  the  streams  are  gelled,  so  that  the  whole  aster,  in  spite  of  its 
fluid  streams,  has  a  relatively  firm  consistency  and  can  be  moved  about 
in  the  more  fluid  cytoplasm  with  a  micro-needle.  When  one  of  the 
minute  oil-like  droplets  occasionally  seen  in  the  sand-dollar  egg  is  pushed 
from  the  fluid  portion  of  the  cytoplasm  into  the  periphery  of  the  aster, 


THE  DIVISION  OF  THE  PROTOPLAST  (59 

it  is  (tarried  inward  toward  the  cciitcr.  The  centripetal  movement  of 
fluid  along  the  rays  is  somehow  compensated  by  a  gradual  outwaid 
movement  of  other  materials.  There  is  evidence  that  the  fluid  rays 
contain  oriented  ''structure  proteins."  The  precise  origin  of  the  spindle 
portion  of  the  achromatic  figure  is  more  difficult  to  determine.  Both 
the  karyolymph  and  some  cytoplasmic  component  between  the  diverging 
centrosomes  evidently  develop  an  orientation  revealed  b}^  the  appearance 
of  "  fibers  "  on  fixation,  but  the  manner  in  which  they  share  in  the  develop- 
ment of  the  spindle  is  not  yet  clear. 

In  the  anaphase  and  telophase  the  chromosomes  behave  as  already 
described  for  plants,  the  chromosomes  mo\'ing  to  the  poles  where  they 
reorganize  the  two  daughter  nuclei.  The  equator  which  they  have  left 
ordinarily  shows  no  conspicuous  change,  though  a  little  refractive  and 
stainable  material  may  often  accumulate  there.  The  asters,  near  which 
the  daughter  nuclei  lie,  remain  conspicuous  until  after  c,ytokinesis,  a 
process  in  which  they  appear  to  play  a  major  role. 

Cytokinesis. — Typical  cytokinesis  in  animals,  like  that  in  higher 
plants,  involves  the  achromatic  figure,  but  it  does  so  in  a  very  different 
manner.  It  invoh'es  also  a  special  series  of  changes  at  the  cell  membrane, 
these  acting  with  the  internal  forces  to  produce  the  cleavage  furrow 
which  divides  the  cytosome. 

The  large  eggs  of  echinoderms,  amphibians,  and  certain  other  animals 
are  particularly  well  suited  to  studies  of  the  factors  responsible  for  cell 
cleavage.  With  the  completion  of  the  achromatic  figure,  the  echinoderm 
egg  becomes  noticeably  elongated,  and  this  is  correlated  with  an  enlarge- 
ment of  the  two  elastic,  semisolid  asters.  It  is  also  found  that  the  cortical 
plasmagel  becomes  firmer  in  consistency  just  before  the  furrow  appears 
and  remains  so  during  its  inward  growth.  That  this  gelation  of  the 
protoplasm  is  a  major  factor  in  producing  the  furrow  is  indicated  by 
the  results  of  treatments  causing  a  return  to  the  sol  state.  Thus  if  the 
asters  in  a  cleaving  egg  are  liquefied  by  stirring  with  a  micro-needle, 
the  furrow  developing  between  them  disappears.  Similarly,  when  the 
rigidity  of  the  cortical  plasmagel  is  reduced  by  hydrostatic  pressure,  the 
furrow  ceases  to  grow  inward  or  even  recedes,  depending  upon  the  degree 
of  solation;  furthermore,  when  the  pressure  is  removed,  gelation  occurs 
once  more  and  the  inward  growth  of  the  furrow  is  resumed.  It  is  believed 
b\-  some  investigators  that  the  gelation  produces  its  cleaving  effect  by 
exerting  a  contractile  tension  in  the  equatorial  plane  of  the  cell,  since 
gelation  in  certain  other  colloidal  systems  is  known  to  produce  such 
forces. 

These  changes  AAdthin  the  cell  are  correlated  with  alterations  at  its 
surface.  By  observing  the  movements  of  small  particles  adhering  to  the 
surface  membrane  of  an  egg  beginning  its  cleavage,  it  can  be  established 


70  FUNDAMENTALS  OF  CYTOLOGY 

that  a  wave  of  local  stretching,  or  increase  in  area,  begins  at  the  polar 
regions  and  progresses  toward  the  site  of  the  furrow.  This  stretching 
continues  as  the  furrow  develops,  the  surface  actually  moving  inward 
along  the  walls  of  the  furrow  and  becoming  the  membranes  of  the  daugh- 
ter cells.  In  at  least  one  instance  it  has  been  reported  that  this  ingrowth 
stops  short  of  the  middle  of  the  egg,  the  membranes  in  the  central  area 
being  formed  anew  in  association  with  a  granular  substance  accumulated 
there.  The  physicochemical  problem  of  accounting  for  these  changes 
in  terms  of  alterations  in  viscosity,  surface  tension,  and  other  factors 
has  long  occupied  the  attention  of  biologists,  and  it  is  believed  that 
substantial  progress  is  being  made  toward  its  solution. 

Highly  interesting  contributions  to  the  eventual  solution  of  this 
problem  have  bepn  afforded  by  nucleate  and  nonnucleate  egg  fragments 
obtained  from  normal  eggs  by  shaking,  centrifugation,  or  constriction 
with  a  hair  noose.  When  nonnucleate  fragments  of  Arbacia  eggs  were 
treated  with  parthenogenetic  agents  (hypertonic  sea  water,  ultraviolet 
light),  asters  appeared  and  cytokinesis  frequently  followed.  In  some 
cases  numerous  successive  divisions  occurred,  giving  nonnucleate  blas- 
tulae,  one  of  which  had  as  many  as  500  cells  and  lived  a  month  or  more. 
In  an  amphibian  {Triton),  similar  phenomena  were  observed.  A  frag- 
ment with  a  nucleus  and  accessory  asters  formed  a  blastula  containing 
nuclei  and  asters  in  some  cells  but  only  asters  in  others.  Such  accessory 
asters  may  be  retentions  from  previous  mitoses,  or  they  may  be  cytasters, 
which  have  long  been  known  to  form  anew  in  cells  under  experimental 
treatment.  Such  facts  indicate  a  degree  of  independence  between  the 
nuclear  cycle  and  the  astral  cycle,  the  two  being  well  correlated  in  normal 
cells,  although  either  is  capable  of  continuing  alone  for  a  limited  time. 

Cytokinesis  by  furrowing  in  tissue  cells  presents  an  even  more  difficult 
problem  than  that  in  free  cells.  The  activities  of  any  one  of  the  cells  are 
determined  in  part  by  its  neighbors,  yet  it  seems  likel.y  that  the  principal 
forces  at  work  in  cleaving  eggs  are  paralleled  in  tissue  cells.  Asters  may 
be  less  prominent  in  the  latter,  but  such  cells  can  still  have  localized 
regions  of  high  viscositj^,  and  the  furrows  in  the  two  cases  are  often 
strikingly  similar.  There  is  also  the  further  problem  of  accounting  for 
the  furrows  that  cleave  large  multinucleate  plasmodia  into  cells  with 
single  nuclei. 

Between  the  limiting  membranes  of  adjacent  cells  of  a  tissue  is  a  layer 
of  material  known  as  intercellular  substance.  This  is  primaril}^  a  secretion 
of  the  protoplasm  and  varies  greatly  in  amount  in  different  tissues. 
Its  physicochemical  complexity  is  indicated  by  the  variety  of  fibrous  and 
other  modifications  that  may  appear  within  it,  and  these  may  have  a 
large  share  in  determining  the  functional  value  of  some  tissues,  such  as 
certain  connective  tissues  and  cartilage. 


THE  DIVISION  OF  THE  PliOTOPLAST  71 

FURTHER  ASPECTS  OF  CELL  DIVISION 

Causes  of  Anaphasic  Chromosome  Movement. — To  anyone  who 
follows  the  nucleus  through  all  the  visible  changes  comprising  one 
mitotic  cycle,  it  is  plain  that  the  problem  of  explaining  this  cycle  in  terms 
of  physics  and  chemistry  is  one  of  extraordinary  complexity.  It  has 
nevertheless  been  hoped  that  an  understanding  of  at  least  one  phase  of  the 
process,  anaphasic  chromosome  movement,  might  soon  be  reached.  A 
full  account  of  attempts  to  solve  this  part  of  the  problem  would  occupy 
many  pages,  but  the  conclusion  would  be  a  brief  one,  viz.,  that  no  satis- 
factory solution  has  yet  been  foimd.  At  the  same  time  it  should  be 
helpful  to  enumerate  some  of  the  principal  observations  and  hypotheses 
that  promise  to  contribute  to  an  eventual  explanation. 

The  early  theory  that  spindle  fibers  attached  to  the  chromosomes 
simply  contract  and  drag  the  chromosomes  apart  has  not  fared  well  in 
the  light  of  subsequent  work.  Recently,  however,  an  oscillatory  inde- 
pendent movement  of  the  several  chromosomes  at  metaphase  observed 
in  living  cells  has  brought  the  suggestion  that  localized  alterations  in 
viscosity  (gel-sol  changes)  in  the  immediate  neighborhood  of  the  chromo- 
somes play  a  role  in  their  later  movement,  for  gelation,  as  stated  in  the 
previous  section,  is  known  to  be  accompanied  by  contraction  in  many 
nonliving  colloidal  systems. 

Appearances  near  the  kinetochores,  in  particular  the  formation  of 
"tractile  fibers"  and  small  projections  on  the  chromosomes  w^here 
movement  begins,  strongly  suggest  a  slow  streaming  of  the  viscous 
materials;  moreover,  the  aster  when  present  is  known  to  have  streams 
flowing  toward  the  poles.  Despite  these  appearances  it  has  not  j'et  been 
possible  to  demonstrate  that  diffusion  streams  in  the  spindle  substance 
are  a  major  factor  in  chromosome  movement,  and  if  this  were  demon- 
strated the  streaming  would  still  have  to  be  explained. 

Elongation  of  the  spindle,  which  is  sometimes  observed  and  can  be 
experimentally  modified,  has  been  cited  as  a  factor  in  chromosome 
movement.  In  one  prominent  hypothesis  the  initial  separation  of  the 
chromatids  was  attributed  to  an  action  of  the  tractile  fiber  mechanism, 
subsequent  movement  poleward  being  due  to  spindle  elongation  in  the 
region  between  the  two  lots  of  chromatids  attached  to  it.  This  inter- 
pretation, too,  has  met  obstacles:  elongation  may  not  occur;  experimental 
alterations  of  spindle  viscosity  may  not  produce  the  expected  effects 
on  movement;  chromosomes  in  some  organisms,  notably  in  hybrids,  may 
not  all  move  poleward  even  though  they  occupy  the  equatorial  plane 
together  at  metaphase. 

Forces  of  electrical  repulsion  and  attraction  have  long  been  looked 
upon  as  factors  of  special  importance.     As  investigations  continue  it 


72  FUNDAMENTALS  OF  CYTOLOGY 

becomes  increasingly  probable  that  repulsions  of  some  sort  do  play  a  role, 
llioush  forces  of  attraction  are  more  difficult  to  demonstrate.  If  the 
chromosomes,  which  carry  a  negative  charge,  lie  in  a  spindle  with  positive 
poles,  the  combination  of  forces  could  result  in  movement.  The  same 
result  could  follow  if  between  all  parts  there  were  repulsions  and  these 
varied  in  relative  intensity  during  the  nuclear  cycle.  Attraction  of 
centrioles  for  chromosomes  is  sometimes  strongly  suggested  in  the  pro- 
phase when  they  move  along  together  on  opposite  sides  of  the  nuclear 
membrane,  but  its  importance  here  or  in  anaphase  is  still  uncentain. 

Anaphasic  movement  evidently  depends  not  only  upon  the  spindle 
mechanism,  but  also  upon  changes  going  on  in  the  chromosomes  them- 
selves. This  is  indicated  by  special  cases,  including  hybrids,  in  which 
metaphasic  arrangement  and  anaphasic  movement  occur  only  after 
chromosome  doubleness,  particularly  at  the  kinetochore,  has  developed 
to  a  certain  stage,  even  though  the  spindle  is  active  earlier.  It  has  also 
been  thought  that  a  special  sheath-like  differentiation  at  the  chromosome 
surface  undergoes  local  and  progressive  viscosity  changes  in  such  a  way 
as  to  result  in  endwise  movement.  In  spermatocytes  of  certain  fungus 
flies  (Sciara)  the  achromatic  figure  is  monopolar,  and  10  single  chromo- 
somes lie  scattered  mthin  it.  Of  the  10,  6  regularly  go  toward  the  single 
pole  and  4  away  from  it,  although  all  have  spindle  attachments  facing  the 
pole.  Furthermore,  the  4  which  pass  away  from  the  pole  and  are 
eventually  extruded  from  the  cell  are  always  the  same  4  out  of  the  set 
of  5  originally  contributed  by  the  male  parent.  This  indicates  clearly 
that  the  reactions  of  a  chromosome  in  the  spindle  are  determined  in  part 
by  specific  constitutional  features  of  the  chromosome  itself. 

All  the  foregoing  considerations  lead  to  the  conclusion  that  the 
behavior  of  chromosomes  at  anaphase  and  other  phases  of  mitosis  is 
brought  about  by  a  nicely  correlated  combination  of  forces,  even  though 
it  is  not  yet  possible  to  name  them  all  or  to  estimate  their  relative  impor- 
tance. To  a  certain  extent  we  know  well  ivhat  occurs:  the  equational 
separation  of  certain  key  materials  of  the  nucleus.  We  also  know  why 
this  occurs,  in  the  sense  that  we  can  state  its  biological  significance  with 
respect  to  ontogeny  and  heredity.  How  it  occurs  we  know  least  of  all. 
Cytologists,  now  that  they  may  take  advantage  of  new  developments  in 
physical  chemistry,  are  ever  more  confident  that  an  adequate  explanation 
of  chromosome  movement  can  some  day  be  reached. 

Time  Occupied  by  Cell  Division. — The  amount  of  time  required  for  a 
somatic  division  to  be  carried  through  and  the  rapidity  with  which 
divisions  succeed  one  another  are  found  to  vary  in  different  tissues  and 
organisms.  They  also  vary  with  temperature.  Mitosis  in  the  Trades- 
cantia  stamen  hair  occupies  about  30  minutes  at  45°C.,  75  minutes  at  25°, 
and  135  minutes  at  10°.     In  dividing  stigma  hair  cells  of  Arrhenatherum 


THE  DIVISION  OF  Till':  PROTOPLAST  73 

at  19°,  the  prophase  occupies  36  to  45  minutes,  the  metaphase  7  to  10, 
the  anaphase  15  to  20,  and  the  telophase  20  to  35;  total,  not  inchidin^ 
interphase,  78  to  110  minutes.  In  the  brown  alga,  Sphacelaria,  growing 
at  nearly  the  same  temperature,  the  process  requires  less  than  half  as 
much  time.  Mesenchyme  cells  of  the  chick  growing  in  tissue  cultin-es 
at  39°C.  pass  through  prophase  in  5  to  50  minutes,  usually  more  than  30; 
metaphase,  1  to  15,  usually  2  to  10;  anaphase,  1  to  5,  usually  2  to  3; 
telophase  and  cytokinesis,  32  to  133;  total,  70  to  180  minutes.  Choroidal 
cells  from  chick  embryos  and  cartilage  cells  from  adult  fowls  carry  through 
their  division  in  about  half  this  time  under  like  conditions.  In  fibroblasts 
from  a  l-da}-  mouse  in  a  2-da3'  tissue  culture,  about  10  minutes  elapse 
between  the  initiation  of  the  equatorial  furrow  during  anaphase  and  the 
completion  of  cytokinesis.  In  the  development  of  the  male  gametophyte 
and  gametes  from  microspores  of  the  water  fern  Marsilea,  there  are  nine 
successive  cell  divisions  and  then  a  transformation  of  certain  cells  into 
spermatozoids.  All  this  has  been  observed  to  occur  in  as  short  a  time 
as  10  to  12  hours;  hence  the  divisions  and  the  intervals  between  them 
must  be  of  short  duration. 

The  Shape  of  Cells  in  Tissues. — Obviously  the  shape  of  a  tissue  cell 
must  be  related  to  its  internal  differentiation  and  to  the  more  general 
conditions  pervading  the  tissue  or  organ  of  which  it  is  a  part.  It  is 
nevertheless  a  matter  of  considerable  interest  to  determine  what  shapes 
tissue  cells  assume  when  conditions  are  as  simple  and  uniform  as 
possible. 

Investigations  in  this  field  have  shown  that  in  a  flat  epithelium  or 
epidermis  the  cells  in  surface  view  tend  strongly  to  be  hexagonal  in  outline. 
If  a  flat  Plasmodium  with  its  nuclei  scattered  at  random  in  one  layer 
were  divided  into  uninucleate  cells  by  walls  with  minimal  surface  area,  a 
hexagonal  pattern  would  result.  Such  a  pattern  is  seen  in  the  cucumber 
epidermis.  When  one  of  the  hexagonal  cells  divides,  the  new  wall  forms 
stable  three-rayed  intersections  with  two  opposite  walls  of  the  hexagon, 
the  daughter  cells  being  pentagons,  and  two  of  the  adjacent  cells  becoming 
heptagons  (Fig.  50).  With  subsequent  divisions,  chiefly  of  the  larger 
cells  with  more  than  six  sides,  the  number  of  sides  per  cell  varies  still 
further  in  the  tissue,  but  the  general  average  remains  not  far  from  six. 

A  similar  play  of  forces  at  cytokinesis  in  a  uniform  three-dimensional 
Plasmodium  would  result  in  the  formation  of  a  mass  of  cells  each  having 
14:  sides  and  trihedral  intersections,  tetrahedral  angles  being  unstable 
and  rare.  If  the  space  w^ere  uniformly  divided  into  polyhedral  cells 
with  equal  volume,  minimal  surface  area,  edges  of  equal  length,  and  no 
intercellular  spaces,  each  cell  would  have  the  form  of  an  orthic  tetrakaide- 
cahedron,  which  has  8  hexagonal  and  (>  (luadrilateral  faces.  Cells  in 
uniform  tissue  mas.ses  such  as  pitii  approach  this  14-sided  form.     Further 


74 


FUNDAMENTALS  OF  CYTOLOGY 


divisions  disturb  the  regularity  of  the  pattern  more  than  they  do  in  an 
epithehum,  since  the  divisions  may  occur  in  so  many  different  planes,  but 
through  mutual  adjustments  of  the  plastic  walls  the  average  number  of 
sides  continues  to  be  close  to  14  so  long  as  the  cells  are  of  uniform  size. 
Cells  at  the  surface  of  the  mass  have  a  smaller  average  number  of  sides. 
Thd  development  of  intercellular  spaces  in  such  tissues  brings  further 
modifications  of  cell  shape. 

Light  on  the  problem  of  cell  shape  has  also  been  sought  through 
experiments  in  which  many  balls  of  lead  or  putty  were  compressed  in 
cylinders.  Although  the  resulting  mass  of  polyhedrons  did  not  originate 
as  a  tissue  does,  it  was  found  that  when  the  balls  were  of  uniform  size 
they  came  out  of  the  press  with  an  average  of  1-i  sides,  with  the  exception 
of  the  peripheral  ones  which  had  an  average  of  10.75.     After  mixtures 


Fig.  50. — n,  transverse  section  of  epidermal  cells  of  cucumber,  b,  diagram  of  cell 
division  in  a  simple  epithelium,  c,  model  of  an  orthic  tetrakaidecahedron.  Explanation 
in  text.      (After  F.  T.  Lewis.) 

of  large  and  small  balls  were  compressed,  the  average  number  of  sides 
was  more  than  14  for  the  large  ones  and  less  than  14  for  the  small  ones, 
the  average  number  for  all  taken  together  being  close  to  14.  A  method 
has  also  been  developed  for  making  wax  casts  of  cells  for  the  study  of 
their  shapes. 

This  is  but  a  brief  glimpse  of  another  field  of  cytological  research  that 
should  lead  to  a  better  understanding  of  the  role  of  cells  in  growing  and 
differentiating  tissues.  One  way  in  which  a  cell  affects  the  behavior  of 
its  neighbors  is  suggested  by  certain  geometrical  features  mentioned 
above:  the  division  of  a  given  cell  adds  one  side  to  each  of  two  others; 
cells  w^ith  more  than  the  average  number  of  sides  grow  larger  and  divide 
sooner  than  those  with  fewer  sides.  Hence  cell  division  is  not  merely  a 
multiplication  of  units;  it  is  also  a  factor  in  correlating  the  activities  of 
the  cells.  In  Chap.  II  we  stressed  the  point  that  the  behaVior  of  regions 
in  a  growing  mass  depends  in  part  upon  their  positions  in  the  whole. 
We  now  see  that  cell  partitions  within  a  tissue  affect  the  activities  of  the 
units  they  separate  not  only  by  virtue  of  their  constitution  and  consequent 
effect  upon  th(>  diffusion  of  materials,  but  also  through  the  geometrical 
form  they  impose  upon  the  cells  b.y  tending  to  develop  in  accordance  with 
the  laws  of  minimal  surface  area. 


CHAPTER  VI 
THE  CELL  WALLS  OF  PLANTS 

The  tissue  cells  of  plants,  like  those  of  animals,  are  separated  by 
intercellular  substance.  In  both  cases  each  cell  has  a  delicate  plasma 
membrane,  but  in  plants  each  has  in  addition  a  relativel}^  firm  wall 
between  the  membrane  and  the  intercellular  substance.  Such  cell  walls 
vary  greatly  in  degree  of  development  and  structural  complexity.  Their 
chief  constituent  is  cellulose,  and  with  this  other  materials  are  usually 
associated.  Surely  nobody  needs  to  be  reminded  of  the  importance  of 
cellulose  as  supporting  material  in  large  plants  or  of  the  varied  roles 
played  by  this  substance  in  our  modern  life. 


EesSii^ 


Fig.  51. — Diagrammatic  repre.sentation  of  successive  stages  (left  to  right)  in  formation 
of  plant  cell  wall  with  secondary  thickening,  a,  origin  of  cell  plate;  b,  cell  plate  transform- 
ing into  middle  lamella,  or  intercellular  substance  {ml) ;  c,  beginning  of  deposition  of 
primary  wall  layers  (1,  1);  d,  e,  f,  beginning  of  deposition  of  outer,  middle,  and  inner 
portions  (o,  m,  i)  of  secondary  wall  layers  (2,  2);  g,  completion  of  wall  thickening;  pm, 
plasma  membrane;  cyt,  cytoplasm.  {Based  on  researches  of  W.  A.  Becker,  I.  W.  Bailey, 
T.  Kerr,  and  others.) 

Development  of  the  Wall. — In  a  section  of  plant  tissue  under  a 
microscope  of  moderate  power  the  partition  between  any  two  cells 
appears  as  a  triple  structure  (Fig.  54,  a).  In  a  meristematic  tissue,  such 
as  that  in  the  root  tip  or  the  cambium,  the  three  layers  are  very  thin 
and  semifluid,  but  all  are  present:  each  cell  has  its  own  cellulose  wall 
lying  against  an  intervening  layer  of  noncellulosic  intercellular  substance. 
The  outer  wall  of  an  epidermal  cell  is,  of  course,  single.  We  shall  now 
recall  the  development  of  this  condition  as  described  in  the  foregoing 
chapter  (page  65)  and  then  proceed  with  an  account  of  the  further 
changes  that  transform  the  early  walls  into  the  elaborate  structures  seen 
in  such  mature  tissues  as  wood.  The  successive  stages  in  the  entire 
process  are  represented  diagrammatically  in  Fig.  51. 

75 


76 


FUNDAMENTALS  OF  CYTOLOGY 


Through  the  equator  of  the  cell  between  the  recently  formed  daughter 
nuclei  there  is  formed  a  continuous  fluid  film,  the  cell  -plate,  which  extends 
until  it  reaches  the  lateral  walls.  Physical  and  chemical  changes, 
including  the  deposition  of  pectin,  transform  this  into  a  somewhat  firmer 
layer  of  intercellular  substance,  the  middle  lamella.  The  protoplasts 
then  deposit  upon  each  side  of  the  middle  lamella  a  thin  primary  wall 
of  cellulose.  This  is  the  stage  observed  in  the  meristem.  The  primary 
wall  undergoes  some  thickening,  but  it  remains  very  plastic  during  the 
further  growth  and  divisions  of  the  cells.  In  some  tissues  no  further 
layers  are  added,  the  primary  layers,  with  certain  chemical  transforma- 
tions, becoming  the  walls  of  the  mature  tissue  cells. 

In  other  tissues,  notably  woody  ones,  additional  wall  layers  are  added, 
a  secondary  wall  of  cellulose  being  deposited  upon  each  primary  wall 
(Fig.   54,   h).     Each  secondary  wall  is   composed   characteristically  of 


'..-.■  s 


lO 


Fig.  52. — Four  stages  in  the  establishment  of  the  connection  between  the  middle  lamella 
of  a  newly  formed  wall  with  that  of  the  lateral  wall  of  the  divided  cell,  ml,  middle  lamella; 
1,  primary  wall  layer;  cyt,  cytoplasm.      {After  P.  Martens.) 

three  layers,  of  which  the  middle  one  is  commonl.y  the  thickest.  At  this 
stage  the  plasma  membranes  of  the  two  protoplasts  are  separated  by  a 
partition  in  which  there  can  be  distinguished  as  many  as  nine  layers: 
the  two  secondary  walls  each  composed  of  three  layers,  the  two  primary 
walls,  and  the  middle  lamella.  This  elaborate  structure  is  not  necessarilj^ 
uniform  over  the  entire  extent  of  the  wall,  however.  Here  and  there 
are  small  areas  in  which  no  secondary  layers  are  deposited,  leaving  pits 
in  which  only  a  delicate  membrane  separates  the  two  protoplasts.  This 
membrane  may  be  pierced  by  fine  pores,  and  in  some  cases  it  has  a  central 
thickening,  the  torus.  The  secondary  layer  may  overarch  the  margin 
of  the  membrane,  forming  the  bordered  pit  characteristic  of  certain 
vascular  cells  of  gymnosperms  (Fig.  53).  Another  form  of  localized 
deposition  is  seen  in  the  spiral  and  ring-like  thickenings  formed  by 
protoxylem  cells  before  their  elongation  has  been  completed. 

The  manner  in  which  the  several  layers  of  the  newlj^  formed  partition 
become  continuous  with  those  of  the  lateral  walls  of  the  original  meriste- 
matic  cell  is  illustrated  in  Fig.  52.  As  the  extending  cell  plate,  or  young 
middle  lamella,  meets  the  lateral  partition,  there  is  developed  at  its  edge 
a  swelling  with  a  minute  cavity.     This  gradually  extends  through  the 


THE  CELL  WALLS  OF  PLANTS  77 

lateral  primary  wall  until  it  unites  ^^^th  the  lateral  middle  lamella, 
the  small  cavity  in  its  margin  enlarging  as  an  intercellular  space  in  the 
midst  of  the  intercellular  substance.  Deposition  of  cellulose  meanwhile 
continues  on  all  sides  of  the  two  protoplasts,  including  the  new  middle 
lamella,  so  that  each  new  cell  has  its  own  continuous  primary  wall.  It  is 
only  after  cell  divisions  have  ceased  that  thick  secondar}^  wall  layers  are 
added. 

Although  cellulose  and  pectin  are  the  chief  constituents  of  cell  walls, 
other  materials  are  commonly  associated  with  them  in  mature  tissues. 
Some  of  these,  notably  lignin  and  suberin,  as  well  as  the  main  constituents 
themselves,  vary  greatlj^  in  relative  amount  in  the  various  layers  of  the 
wall  and  in  the  walls  of  different  tissues.  They  have  pronounced  effects 
upon  reactions  to  stains.     Lignified  cellulose,  which  ^^^      ^.^ 

contains  a  pentosan  {e.g.,  xylan)  and  an  organic  sub-    ^„___^^ — -^li^ 
stance  with  an  aromatic  nucleus,  stains  vigorously  •      •      I     • 

with  safranine  or  crystal  violet,  whereas  unlignified    ^'  ""*"  ^^1^;    i^^^ 
cellulose  walls  do  not.     One  should  not,  however,  /   ;"~\\       ^ 

rely  too  strongly  upon  staining  reactions  as  criteria    —      ("t^JPr; 1 

of  chemical  composition.     Suberin,  formed  in  special  vT  "l^       ?! 

abundance  in  corky  tissues,  is  an  aggregate  of  various    '—  —  -  --"5 

anhydrides  and  glycerides  of  certain  organic  acids.  ^^*^-   ,    53.    Dia- 

.     '  .    .  gram  of  bordered  pit 

Cutin  is  similar  in  composition  and  occurs  mainly  as  in  wall  of  conifer 
an  external  coating  on  epidermal  cells.  Other  or-  ^°?,'^"  ^'^•'^'"'  ^"  ^ " 
ganic  compounds,  such  as  tannins,  oils,  and  resins, 
are  also  deposited  in  old  cell  walls  and  are  largely  responsible  for  the  char- 
acters of  heartwood  in  trees.  Mineral  matter,  including  certain  salts  of 
silicon  and  calcium,  may  also  occur  in  wood,  and  the  location  of  ash  in 
incinerated  tissues  indicates  that  such  matter  is  restricted  largely  to  the 
primary  wall  layers.  Among  certain  algae  and  fungi,  chitin  and  keratin 
occur  as  wall  constituents. 

Minute  Structure  of  the  Cell  Wall. — The  results  of  chemical  studies 
and  physical  researches  with  X  rays  on  the  cellulose  wall  have  shown 
that  it  has  a  crystalline  structure,  i.e.,  it  is  composed  mainly  of  units 
arranged  in  a  regular  three-dimensional  pattern.  The  primary  unit  Is 
the  anhydrous  glucose  residue,  CeHioOs.  Such  residues  are  united  by 
primary  valencies  into  long  cellulose  chains,  and  these  are  linked  lat(M-ally 
by  secondary  forces  to  form  a  regular  space  lattice.  The  intermolecular 
cohesive  forces  result  in  the  formation  of  larger  groupings,  or  micelles. 
The  presence  and  the  nature  of  amorphous  materials  between  the  groups 
of  cellulose  chains  are  debated  points. 

As  a  result  of  the  regular  parallel  arrangement  of  the  submicroscopic 
chains  in  the  pnmary  and  secondary  cellulose  wall  layers,  these  layers  are 
anisotropic  in  contrast  to  the  isotropic  intercellular  substance.     This 


78 


FUNDAMENTALS  OF  CYTOLOGY 


Fig.  54. — Thickened  cell  walls  in  secondary  xylem  of  plants,  a,  wood  of  spruce  {Picea). 
b,  transverse  section  of  wood  of  Trochodeiidron;  the  primary  walls  are  deeply  stained,  the 
secondary  walls  and  the  intercellular  substance  are  not.  c,  transverse  section  of  wood  of 
Trochodendron  photographed  with  polarized  light  through  crossed  nicols;  outer  and  inner  por- 
tions of  secondary  wall  bright,  middle  lamella  and  thick  middle  portion  of  secondary  wall 
dark,      (a,  courtesy  of  U.  S.  Forest  Products  Laboratory;  b,  c,  after  T.  Kerr  and  I.  W.  Bailey.) 


THE  CELL   WALLS  OF  PLANTS  79 

can  be  seen,  for  example,  in  a  transverse  section  of  wood  viewed  between 
crossed  nicols  on  the  polarizing  microscope:  here  the  two  thin  layers  of 
the  secondary  wall  appear  bright,  while  the  thick  layer  between  them 
appears  dark  (Fig.  54,  c).  In  a  longitudinal  section  the  thin  layers  are 
dark  and  the  thick  one  bright.  This  shows  that  the  cellulose  chains, 
although  highly  variable  in  orientation,  tend  to  lie  more  nearly  parallel 
to  the  cell's  longitudinal  axis  in  the  thick  layer  and  more  nearly  at 
right  angles  to  it  in  the  thin  ones.  The  intercellular  substance  is  dark 
when  viewed  at  any  angle.  In  the  cotton  fiber  the  thin  primary  wall 
has  two  systems  of  chains  following  spiral  courses  in  opposite  directions 
in  a  matrix  of  pectic  and  waxy  material.  The  secondary  wall  is  much  the 
same,  except  that  the  chains  form  smaller  angles  with  the  longitudinal 
axis  and  show  more  reversals  of  direction. 

Under  some  circumstances,  as  when  the  cell  walls  are  swollen  or 
dried,  coarse  fibers  become  visible  in  the  wall  substance.  This  involves  a 
rupturing  of  the  system  of  cellulose  chains  and  may  not  indicate  accu- 
rately the  arrangement  that  the  chains  had  in  the  untreated  wall.  When 
properly  handled,  the  fresh  untreated  secondary  walls  of  some  cells  show 
microscopically  visible  striations  revealing  the  true  orientation  of  the 
chains.  The  orientation  may  also  be  shown  by  the  arrangements 
assumed  by  iodine  crystals  induced  to  form  within  the  wall  substance 
and  by  the  shape  of  cavities  resulting  from  enzymatic  activity  when 
fungus  hyphae  invade  thick  secondary  walls. 

The  relative  arrangement  of  cellulose  and  lignin  in  the  secondary  wall 
is  strikingly  shown  in  the  fiber  tracheids  of  certain  tropical  dicotyledons. 
When  the  tracheid  is  swollen,  delignified  with  cuprammonium  hydroxide, 
and  stained  with  Congo  red,  the  transverse  section  has  the  appearance 
shown  in  Fig.  55.  Comparison  with  longitudinal  sections  shows  the 
radiating  dark  lines  to  represent  a  sj^stcm  of  longitudinally  arranged, 
branching  plates  having  a  high  proportion  of  cellulose  and  a  low  propor- 
tion of  lignin,  the  lighter  lines  between  them  being  regions  in  which 
cellulose  is  less  and  lignin  more  abundant.  When  a  fiber  tracheid  is 
decellulosed  with  sulphuric  acid  and  stained  with  iron  alum-hematoxylin, 
a  similar  pattern  appears,  but  here  the  dark  regions  are  those  containing 
a  high  proportion  of  lignin  and  a  low  proportion  of  cellulose.  Both 
substances  are  continuous  throughout  the  wall,  but  their  relative  abun- 
dance varies  along  different  radii. 

Concentric  zones  appearing  in  the  secondary  wall  are  due  in  different 
cases  to  at  least  three  causes:  variations  in  the  cellulose  pattern,  variations 
in  the  intensity  of  lignification,  and  the  alternation  of  cellulosic  and 
noncellulosic  layers.  In  the  cotton  fiber  the  numerous  concentric  zones 
have  been  correlated  with  the  daily  metabolic  cycle,  a  compact  anisoti-opic 


80 


FUNDAMENTALS  OF  CYTOLOGY 


layer  being  formed  each  day  and  a  looser,  relatively  isotropic  layer  each 
night. 


^iittgs:!^ 


f>"€^it^\ 


'^:^^^'' 


W^; 


^/^ 


.^n 


^ 


Fig.  55. —  Minute  structure  of  secondary  cell  wall  ot  iiipariina.  Above,  delignified  fiber 
tracheid,  showing  radiating  pattern  in  the  remaining  cellulose.  Below,  fiber  tracheid  after 
removal  of  cellulose,  showing  radiating  pattern  of  ligniu.      {After  I.  W.  Bailey  and  T.  Kerr.) 

Plasmodesms. — The  cell  walls  in  plant  tissues  do  not  completel\' 
separate  the  protoplasts.     It  appears  to  l)e  generally  true  that  the  hitter 


THE  CELL   WALLS  OF  PLANTS 


81 


are  connected  by  numerous  delicate  protoplasmic  strands  known  as 
plasmodesms  passing  through  fine  channels  in  the  walls.  Where  they  are 
relatively  coarse  and  not  too  numerous,  they  may  show  plainly  in 
sections  (Fig.  56),  but  in  many  tissues,  particularly  in  meristems,  their 
extreme  delicacy  and  the  destructive  effects  of  technics  involving  dehydra- 
tion make  their  demonstration  very  difficult.  For  the  same  reasons  their 
mode  of  origin  has  not  yet  been  clearly  established. 

That  plasmodesms  are  actually  protoplasmic  strands  has  sometimes 
been  questioned,  an  alternative  view  being  that  they  are  merely  peculiar 


Fig.  56. —  Soctidii  i>i  fiKlDsjici  m  i>l  pci --iiriiiioii  { I)i<>si)i/roii) .  .showing;  j)la.>iii<)<it',Miis  cdii- 
neo.ting  the  cells  through  the  enormously  thickened  walls.  (Courti.iy  of  General  Biologiail 
Supply  HoHKc,  Inc.,  Chicago.) 


structural  features  of  tlie  wall.  Among  the  evidences  cited  in  favor  of 
their  protoplasmic  nature  aie  the  following:  they  occur  only  in  walls 
separating  two  protoplasts;  when  protoplasts  are  plasmolyzed,  the}'  often 
remain  connected  with  the  wall  by  numerous  fine  strands;  b^^  plasmoIysLs 
the  plasmodesms  may  be  withdrawn  from  their  channels;  their  staining 
reactions  are  like  those  of  protoplasm;  they  give  a  positive  test  for 
oxj^dase;  in  germinating  seeds  the  digestion  of  the  endosperm  walls 
proceeds  along  the  plasmodesms;  after  hardening  the  plasmodesms  in 
formalin  the  endosperm  walls  have  been  dissolved  with  sulphuric  acid, 
leaving  them  as  connections  between  the  undissolved  protoplasts;  the 


82  •  FUNDAMENTALS  OF  CYTOLOGY 

tobacco  mosaic  virus  passes  readily  through  walls  having  plasmodesms, 
but  not  into  the  guard  cells  of  stomates,  where  plasmodesms  are  appar- 
ently absent.  The  transfer  of  materials  from  cell  to  cell  by  way  of  the 
very  slender  plasmodesms  has  not  been  directly  observed,  although 
mass  movement  of  protoplasm  has  been  seen  occurring  through  larger 
pores  having  a  diameter  of  1.5  to  2/i  in  the  green  alga  C odium.  In  the 
red  algae,  peculiar  cell  connections  have  been  described,  but  actual 
protoplasmic  continuity  here  is  still  subject  to  doubt. 

It  is  probable  that  the  functional  significance  of  plasmodesms  is  to 
be  found  in  the  conduction  of  stimuli  promoting  correlation  and  in  the 
transfer  of  certain  materials  important  in  metabolism.  The  same 
interpretation  is  warranted  for  the  intercellular  bridges  in  animal  tissues. 
By  virtue  of  such  protoplasmic  continuity  a  tissue  or  a  complex  organism 
would  seem  to  be  better  able  to  function  consistently  as  an  individual 
than  it  w^ould  if  only  nonprotoplasmic  materials  separated  its  protoplasts. 
At  the  same  time  it  is  to  be  remembered  that  certain  correlating  factors, 
e.g.,  electrical  gradients  and  the  diffusion  of  dissolved  substances,  can 
exist  in  systems  that  are  partly  or  even  wholly  nonprotoplasmic. 

The  Formation  of  Cellulose  by  the  Protoplast.^ — This  topic  is  at 
present  a  highly  controversial  one,  but  it  is  included  here  because  of  its 
prominence  in  cytology  today  and  its  great  biological  interest.  For 
many  years  the  prevailing  view  has  been  that  cellulose  first  becomes 
visible  at  the  surface  of  the  protoplast,  where  it  is  deposited  as  successive 
layers  having  the  crystalline  structure  described  in  foregoing  pages. 

According  to  an  opposing  view,  which  has  come  into  prominence 
during  the  past  decade,  cellulose  first  appears  in  the  cytoplasm  in  the 
form  of  minute  ellipsoidal  bodies  having  a  size  of  about  0.5  by  1.5^. 
These  form  chain-like  aggregates  and  are  built  into  the  wall  along  with  a 
colloidal  material  that  cements  them  together.  Moreover,  the  wall  can 
be  broken  down  into  such  ellipsoidal  bodies  by  the  use  of  hydrochloric 
acid  and  centrifuged  out  of  the  mixture. 

In  recent  papers  on  the  ellipsoidal  particles  it  is  claimed  that  they  are 
produced  by  plastids.  In  the  green  alga  Halicysfis  ring-like  masses  of 
carbohydrate  are  formed  just  beneath  the  membranes  of  disc-shaped 
chloroplasts.  These  break  up  into  "mercerized"  cellulose  particles 
of  uniform  size  which  are  liberated  into  the  cytoplasm  when  the  plastid 
membranes  disintegrate.  They  are  then  built  into  the  developing 
wall  in  successive  layers.  In  the  cotton  fiber  there  are  small  disc- 
shaped colorless  plastids  in  which  cellulose  particles  appear  in  a  similar 
manner.  In  the  green  cells  of  the  leaves  and  stem  of  the  cotton  plant, 
both  starch-forming  chloroplasts  and  cellulose-forming  leucoplasts 
function  simultaneously. 


THE  CELL  WALLS  OF  PLANTS  83 

The  conflicting  evidences  and  interpretations  involved  in  the  con- 
troversy centering  about  the  ellipsoidal  bodies  cannot  be  reviewed  hei-e, 
but  the  subject  is  one  that  cytologists  will  continue  to  follow  with  th(> 
greatest  interest.  The  theory  of  the  origin  of  cellulose  in  j^lastids  is 
especially  intriguing  in  view  of  the  fact  that  starch,  which  resembles 
cellulose  so  closely  in  chemical  composition,  is  elaborated  in  such  cell 
organs.  A  completely  satisfying  answer  to  the  question  of  whether  these 
two  materials,  which  are  about  the  onl}^  solid  substances  deposited  in 
large  amounts  by  plant  protoplasts,  have  similar  or  dissimilar  cytological 
origins  would  indeed  be  a  major  and  welcome  achievement. 


CHAPTER  VII 
THE  CHROMOSOMES 

It  is  not  difficult  to  account  for  the  fact  tfiat  thie  chromosomes  have 
long  held  a  major  share  of  the  attention  of  cytologists.  They  are  indi- 
vidualized protoplasmic  units  present  in  definite  numbers  and  multiplying 
regularly  and  only  by  division.  Because  of  the  precision  with  which 
they  are  distributed  at  mitosis,  every  nucleus  of  the  developed  plant 
or  animal  body  has  a  descendant  of  every  chromosome  present  when 
development  w^as  initiated.  Furthermore,  when  reproduction  occurs 
the  chromosomes  are  passed  on  to  the  next  generation  through  the  spores 
or  gametes.  Their  physicochemical  composition  is  such  that  they  have 
specific  and  profound  effects  upon  the  course  of  development  and  hence 
upon  the  organism's  characters.  As  a  consequence  of  all  this,  they  play 
a  major  role  in  heredity. 

Some  of  the  above  points  were  brought  out  in  the  chapter  on  cell 
division.  It  was  also  showTi  there  that  the  chromosome  has  two  main 
structural  constituents:  the  chromonemata,  present  throughout  the 
entire  nuclear  cycle,  and  the  matrix,  which  is  conspicuous  only  during 
certain  phases  of  mitosis.  The  chromosome,  although  it  is  a  persistent 
individual  reproducing  by  division  in  every  mitotic  cj'cle,  passes  in  each 
cycle  through  a  series  of  structural  changes  that  alter  its  appearance 
profoundly'.  At  metaphase  and  anaphase  it  is  clearly  evident  as  a  dis- 
tinct individual,  whereas  in  the  metabolic  stage,  when  its  chief  functions 
are  being  performed,  it  is  rarely  possilile  to  determine  its  limits. 

In  this  chapter  we  shall  consider  in  greater  detail  the  form  and  the 
structure  of  chromosomes,  chiefly  in  somatic  tissues,  and  discuss  the 
constitution  of  the  chromosome  complement,  or  outfit  of  chromosomes 
making  up  a  given  nucleus. 

Somatic  Chromosomes. — The  general  morphology  of  somatic  chromo- 
somes is  best  displayed  at  the  metaphase  and  anaphase  of  mitosis.  If 
special  technical  methods  are  used,  much  can  also  be  learned  about  their 
structure  at  these  stages,  but  so  far  the  most  reliable  information  of  this 
kind  has  come  from  chromosomes  passing  through  meiosis.  Chromo- 
somes may  dilTer  greatly  in  size  in  different  organisms,  in  unlike  tissues, 
and  in  some  degree  in  plants  grown  in  different  nutrient  solutions. 
Fixation  often  affects  their  size.  Nearly  all  chromosomes  at  anaphase 
lie  between  1  and  20fi  in  length.     It  is  easy  to  understand  why  investiga- 

84 


THE  CHROMOSOMES  '  85 

tors  have  preferred  larger  ones,  like  those  of  amphibians  and  liliaceous 
plants,  for  studies  of  chromosomal  constitution.  Special  mention  will 
be  made  of  the  giant  salivary-gland  chromosomes  of  certain  insect 
tissues  later  in  the  chapter. 

The  form  and  the  structure  of  a  typical  somatic  anaphase  chromosome 
are  represented  in  Fig.  57.  It  is  an  elongate  body  consisting  of  matrix 
and  two  spiral  chromonemata  recentl}^  formed  by  division.  The  two 
may  be  so  closely  associated  that  they  seem  to  be  one,  or  they  may 
appear  as  clearly  separate  threads  more  or  less  twisted  about  one  another. 
They  represent  the  chromatids  which  will  separate  in  the  anaphase  of 


Fig.  57. — A  typical  somatic  chromo- 
some at  anaphase  of  mitosis.  Semi- 
diagrammatic,  c,  kinetochoie;  ch,  chro- 
monema;  h,  heterochromatic  region;  m, 
matrix;  s,  satellite;  no,  nucleolus  organizer. 


■\ 


'^^■^ 


\ 


\ 


\f« 


y 


/ 


Fig.  58. — Chromosome  G  in  meiotic 
prophase  in  maize,  showing  its  nucleolus 
organizer  {no)  in  contact  with  the  large 
nucleolus;  short  region  to  right  of  it  is  the 
satellite,     c,  kinetochore. 


the  next  mitotic  cycle.  Along  the  chromonemata  are  small  lumps,  the 
chromomeres.  These  are  rarel}^  evident  in  preparations  of  anaphase 
chromosomes  even  when  the  matrix  has  been  rendered  transparent,  but 
during  the  prophase,  when  the  threads  have  less  chromatic  material, 
they  frequently  show  clearl3\  They  are  best  studied  in  meiotic  pro- 
phases (Figs.  58,  64,  6;  78). 

An  important  feature  of  the  chromosome  is  the  specialized  region 
at  which  its  reactions  with  the  spindle  mechanism  seem  to  be  largely 
centered.  This  region  is  called  the  kinetochore.  (Other  terms  in  the 
literature  are  centromere,  primary  constriction,  and  kinomere.)  The  kine- 
tochore commonly  appears  as  a  relatively  achromatic  region.  In  certain 
large  chromosomes  it  can  be  seen  to  be  traversed  by  two  slender  strands 
which  evidently  represent  the  chromonemata.  In  the  double  metaphase 
chromosome  there  are  accordingly  four  (Fig.  59).  In  some  instances  a 
minute  body,  the  kinosome,  has  been  made  out  at  the  middle  of  each 


86  '  FUNDAMENTALS  OF  CYTOLOGY 

strand.  The  portions  of  the  chromosome  on  either  side  of  the  kineto- 
chore  are  known  as  arms.  These  are  equal  or  unequal  in  length  depending 
upon  the  kinetochore's  position,  which  is  constant  for  a  given  chromo- 
some. Telokinetic  chromosomes,  i.e.,  those  with  terminal  kinetochores, 
have  been  reported  in  animals,  but  they  seem  to  be  very  rare  in  plants. 
A  number  of  supposedly  telokinetic  chromosomes  have  been  shown  to 
have  a  minute  second  arm.  The  present  tendency  is  to  regard  the 
telokinetic  condition,  at  least  in  plants,  as  an  abnormality  that  does  not 
long  persist.  In  metaphase  and  especially  in  anaphase  the  chromosome 
tends  to  be  bent  at  the  kinetochore. 

Chromosomes  may  have  more  or  less  prominent  "secondary  con- 
strictions" in  one  or  both  of  their  arms.  Special  methods  may  reveal 
more  of  these  than  appear  after  ordinary  treatments.  Commonly  one 
chromosome  of  each  of  the  genomes,  or  basic  outfits  composing  a  nucleus, 
has  in  one  arm  an  especially  prominent  secondary  constriction  with 
which  there  is  associated  a  particular  function,  the  organization  of  the 
nucleolus.  The  small  segment  of  the  chromosome  distal  to  this  con- 
striction is  called  a  satellite.  In  the  anaphase  the  exact  extent  of  the 
nucleolus  organizer,  or  specialized  region  directly  concerned  in  the  develop- 
ment of  the  nucleolus,  is  not  evident,  for  the  chromosome  is  very  compact 
and  no  nucleolus  is  present.  Its  features  appear  much  more  clearly  in 
certain  plants  during  the  meiotic  prophase,  when  the  chromosome  is 
extended  and  devoid  of  matrix  (Figs.  58;  64,  a).  During  the  telophase, 
as  the  matrix  loses  its  stainability  and  disappears,  the  nucleolus  makes 
its  appearance  in  connection  with  the  chromonemata  at  or  near  the 
constriction.  Hence  the  number  and  the  position  of  the  nucleoli  in 
the  resulting  metabolic  nucleus  are  dependent  upon  the  number  and 
location  of  the  nucleolus-forming  chromosomes  in  the  telophase.  It  is 
known  that  the  material  for  the  nucleoli  is  derived  from  all  the  chromo- 
somes present,  but  in  some  manner  it  is  collected  or  organized  as  a 
nucleolus  only  at  the  nucleolus  organizer.  In  the  ensuing  prophase  the 
nucleolus  commonly  disappears,  partially  or  completely,  as  the  matrix 
accumulates  and  becomes  highly  stainable. 

Another  important  feature  of  chromosomal  constitution  is  heteropyk- 
nosis. This  term,  which  means  "difference  in  density,"  refers  to  the 
condition  present  when  all  or  a  definite  part  of  a  chromosome  remains 
denser  and  more  highly  chromatic  than  the  other  chromosomes  or  parts 
through  the  nuclear  cycle.  In  the  anaphase  the  stained  chromosome 
may  exhibit  this  feature  weakly  or  not  at  all,  but  during  the  telophase 
the  heterockromatic  part  retains  its  compactness  and  stainability  while 
the  euchromatic  parts  undergo  the  usual  telophasic  transformation  (Figs. 
60;  64,  d).  Most  commonly  it  is  the  regions  near  the  kinetochore  that 
are  heteropyknotic.     In  some  plants  this  makes  it  possible  to  estimate 


TIII'J  CHROMOSOMES 


87 


the  number  ot"  chromosoine.s  in  a  metabolic  nucleus  by  counting  such 
heterochromatic  masses  (euchromocenters)  (Fig.  16).  Other  such  regions 
may  occur  elsewhere  in  the  chromosome,  the  nucleolus  organizer  fi-e- 
quentl}^  having  this  character.  Structurally,  such  portions  seem  to  be 
regions  in  which  the  chromonemata  are  more  closely  coiled,  and  by 
treatment  vnih.  NH4CI  it  has  been  found  possible  to  relax  the  coils. 

The  significance  of  heteropyknosis  is  not  yet  fully  evident.  Recent 
researches  oji  the  giant  chromosomes  in  the  salivary  glands  of  the  fruit 
fly  {Drosoyhila)  indicate  some  connection  between  heterochromatic 
regions  and  the  nucleic  acid  cycle.  It  is  thought  to  have  a  role  in  the 
synthesis  of  the  thymonucleic  acid  in  the  nucleus  and  also  to  affect  the 
ribonucleic  acid  content  of  the  cytoplasm  of  the  egg.  Since  the  nucleolus 
also  contains  ribonucleic  acid  compoimds,  it,  too,  appears  to  be  involved 


'W I  ^ 


Fig.  59. — Portion  of  somatic  chromo- 
some of  Trillium  at  metaphase,  showing 
structure  of  kinetochore.  Description  in 
text.      {AflrrL.  W.  Sharp.) 


Fig.  60. — Two  cells  recently  formed  by 
division  of  one  in  root  tip  of  onion.  Chro- 
mocenters  at  opposite  poles  of  the  pair  of 
nuclei. 


in  this  chemical  cycle.  This  is  stronglv  emphasized  by  the  origin  of  the 
nucleolus  in  direct  connection  with  the  nucleolus  organizer,  which  is 
typicall}'  heterochromatic,  and  by  the  reciprocal  relationship  existing 
between  the  chromosomal  and  nucleolar  cycles:  the  nucleolus  appears 
as  the  telophase  chromosomes  gradually  lose  their  stainability,  and  it 
disappears  as  the  prophase  chromosomes  regain  it.  This  cy(;lic  change 
has  alwaj'S  puzzled  cytologists,  and  now  it  seems  that  a  solution  of  the 
puzzle  is  being  fovmd. 

In  the  chapter  on  cell  division,  reference  was  made  to  an  uncertainty 
regarding  the  number  of  chromonemata  actually  present  in  a  chromo- 
some. Some  observers  do  not  admit  the  presence  of  more  than  one  at 
anaphase  and  telophase,  many  hold  that  there  are  two,  and  some  believe 
that  there  are  four,  each  possibly  having  its  own  individual  matrix. 
Since  visual  observation  involves  the  interpretation  of  stmctures  so  near 
the  limit  of  visibility,  more  refined  techniques  have  been  brought  to 
bear  upon  the  problem.     The  most  promising  of  these  has  been  irradia- 


88  FUNDAMENTALS  OF  CYTOLOGY 

tion  with  X  rays,  which  are  capable  of  causing  breaks  in  the  chromo- 
nemata.  The  procedure  is  as  follows.  Cells  are  irradiated  at  whatever 
stage  it  is  desired  to  ascertain  the  chromonema  number,  e.g.,  during  the 
telophase  or  the  metabolic  stage.  They  are  afterwards  allowed  to  grow 
until  the  nuclei  have  had  time  to  reach  the  anaphase  of  the  succeeding 
mitosis.  The  anaphase  figures  are  then  examined  for  broken  chromo- 
somes or  chromatids,  and  from  the  types  of  abnormality  observed 
inferences  are  drawn  regarding  the  number  of  chromonemata  present 
at  the  time  the  breakage  was  induced. 

The  possible  types  of  breakage  and  their  effects  upon  the  appearance 
of  the  anaphase  chromosomes  are  shown  diagrammatically  in  Fig.  61. 
If  the  chromosome  has  only  one  chromonema  and  it  is  broken  bj^  the 
X  ray,  both  of  the  separating  chromatids  at  anaphase  should  lack  a 
portion,  since  they  were  formed  by  splitting  of  a  thread  already  broken. 
This  is  a  so-called  chromosome  break.  The  part  lost  may  lie  near  by. 
If  there  are  two  chromonemata  at  the  time  of  breakage,  a  variety  of 
results  may  appear  at  anaphase:  (1)  a  normal  chromatid  may  be  seen 
separating  from  a  deficient  one,  with  a  single  fragment  lying  near  by; 
this  indicates  the  break  of  but  one  of  the  two  chromonemata — a  chro- 
matid break.  (2)  Two  deficient  chromatids  may  be  seen  separating, 
with  a  double  fragment  near  by ;  this  looks  like  the  result  of  a  chromosome 
break,  but  it  could  be  due  to  the  breakage  of  two  associated  chromatids 
by  the  same  X-ray  ''hit,"  It  is  known  that  two  threads  may  thus  be 
broken  even  when  they  are  more  than  O.Ijli  apart.  (3)  A  chromatic 
"bridge"  may  appear  at  anaphase  as  a  result  of  a  reunion  of  the  broken 
ends  of  the  two  associated  chromatids,  giving  a  chromosome  with  two 
kinetochores  which  may  pass  toward  opposite  poles.  If  there  are  four 
chromonemata  at  the  time  of  breakage  and  only  one  of  them  is  broken, 
a  chromatid  with  two  equal  longitudinal  halves  may  be  seen  separating 
from  one  wdth  unequal  halves.     This  is  a  half-chromatid  break. 

The  results  obtained  with  this  method  thus  far  by  different  workers  are 
equivocal.  Some  find  evidence  for  the  presence  of  two  chromonemata  at 
somatic  telophase  and  a  division  of  these  into  four  either  in  the  late 
metabolic  stage  or  very  early  in  the  prophase.  Others  conclude  that 
there  is  but  one  chromonema  in  the  telophase,  this  becoming  doubled 
late  in  the  metabolic  stage.  To  what  extent  such  discrepancies  are  due 
to  differences  in  the  type  or  condition  of  the  materials  used,  or  to  varia- 
tions in  the  procedures,  remains  to  be  determined.  An  interesting  piece 
of  evidence  is  the  appearance  of  two  satellites  and  sometimes  two  nucleoli 
side  by  side  on  a  telophase  chromosome. 

It  has  been  suggested  that  the  process  of  doubling  may  involve  a 
succession  of  reactions  extending  over  a  considerable  period  of  time  and 
that  various  phases  of  the  process  may  be  affected  by  different  agencies 


THE  CHROMOSOMES 


89 


Metabolic  Stage  Prophase 


Metophase 


Anaphase 


CHROMOSOME 
BREAK 


CHROMATID 
BREAKS 


HALF- 

CHROMATID 

BREAK 


Fig.  61. — Diagram  illustrating  the  metliod  of  investigating  the  number  of  chromonemata 
in  a  chromosome  by  means  of  X  rays.     Explanation  in  text. 


90 


FUNDAMENTALS  OF  CYTOLOGY 


and  conditions.  Furthermore,  it  is  not  known  to  what  degree  the 
threads  may  be  longitudinally  compound  below  the  range  in  which  X  rays 
can  affect  their  parts  individually.  It  may  therefore  be  helpful  to  dis- 
tinguish provisionall,y  three  levels  of  doubling:  (1)  elementary  doubling, 
in  which  the  ultimate  longitudinal  constituents  (protein  chains?)  of 
the  chromosome  become  duplicated  or  multiplied,  probably  through 
the  formation  of  new  ones  close  to  the  old  ones  b}^  a  process  analogous  to 
crystallization  or  polymerization ;  (2)  effective  doubling,  in  which  the  thread 
somehow  reaches  a  stage  at  which  a  given  agency  such  as  X  rays  may 
affect  one  longitudinal  fraction  and  not  another;  (3)  visible  doubling,  in 
which  a  thread  appearing  single  under  the  microscope  becomes  double 
by  a  process  that  looks  like  a  real  splitting. 

Studies  on  chromosome  structure  are  complicated  by  the  fact  that 
the  chromonemata  are  spirally  coiled  in  some  degree  at  all  stages  of 


^  hi 


Anaphase  Telophase  Early  Prophase  Lafe  Prophase  Metaphase 

Fig.  62. — Diagram  of  the  chronionema  coiling  cycle  through  mitosis.  While  the  gyres 
(a)  of  one  series  are  relaxing  and  disappearing,  a  new  series  (b)  is  developed;  thus  two  coiling 
cycles  overlap  in  the  mitotic  cycle.  Further  explanation  hi  text.  (Based  on  mitosis  in 
Trillium  m.icrospore  as  described  by  A.  H.  Sparrmv.) 


the  somatic  nuclear  cj^cle.  During  this  cycle  the  changes  undergone 
appear  to  be  somewhat  as  follows  (Figs.  02,  63).  In  anaphase  the  two 
spirally  coiled  chromonemata  recently  formed  by  division  in  the  prophase 
(page  61)  lie  mostly  close  together  and  twisted  about  each  other.  In 
the  enlarging  telophase  nucleus  they  tend  to  separate  somewhat  and 
relax  their  coils,  although  the  number  of  spiral  turns,  or  gyres,  remains 
about  the  same  as  it  was  in  anaphase.  In  the  following  early  prophase 
these  gyres  begin  to  disappear,  but  before  the  uncoiling  is  completed 
each  of  the  constituent  chromonemata  (chromatids)  begins  independently 
to  form  numerous  new^  gyres.  In  the  advancing  prophase  these  new 
gyres  become  fewer  and  larger  while  the  chromatids  gradually'  untwist. 
By  the  end  of  the  prophase  the  chromatids  have  lost  their  twists  and  old 
gyres  (relic  coils)  and  have  developed  individual  matrices.  At  this 
time  the  chromonemata,  with  their  new  gyres  now  closer  together,  reveal 
the  doubleness  which  becomes  effective  in  the  next  mitotic  cycle.  The 
metaphase  chromosome  thus  consists  of  two  chromatids  which  have 
become  nearly  or  completely  untwisted  and  in  each  of  wliich  there  are  two 


THE  CHROMOSOMES 


91 


chromonemata  in  the  form  of  a  double-stranded  spiral  like  that  of  tlic 
preceding  anaphase. 

Meiotic    Chromosomes. — The    next    chapter   is    to    be    devoted    to 
chromosome  beha\'ior  during  meiosis,  the  process  by  which  a  reduction 


FiCx.  63. — Four  stages  in  mitosis  in  microspores  of  Trillium  grandijloium.  1,  propliase: 
twists  and  relic  coils  (large  gyres)  from  previous  cy(-le  still  present;  small  gyres  developing 
in  each  chromatid.  2,  later  prophase:  new  gyres  in  chromatids  now  larger  and  fewer.  3, 
metaphase.  4,  anaphase.  See  text  and  Fig.  62.  P>om  temporary  acetocarmine  smears. 
{After  A.  H.  Sparrow.) 

in  the  number  of  chromosomes  is  accomplished  at  a  certain  point  in  the 
life  cycle.  In  the  present  section  we  shall  merely  review  a  few  facts 
that  will  serve  to  complete  our  description  of  fundamental  chromosome 
structure. 


92 


FUNDAMENTALS  OF  CYTOLOGY 


Meiotic  chromosomes  are  especially  favorable  for  the  study  of 
chromonemata,  first  of  all  because  in  higher  plants  they  occur  in  cells 
(microsporocytes)  that  lie  more  or  less  free  from  each  other  in  the  anther. 
They  can  therefore  be  easily  pressed  out  on  a  slide  in  large  numbers 
for  study  in  the  living  condition  or  for  special  treatments.  In  the  pro- 
phase of  the  first  of  the  two  nuclear  divisions  in  the  microsporocyte 
they  are  usually  longer  and  straighter  than  at  any  other  period  in  the 
life  cycle;  moreover,  thej^  contain  very  little  stainable  material  except 
in  the  chromomeres.  Hence  in  some  plants,  e.g.,  maize,  the  more  minute 
structural  features  of  different  chromosomes    can  be  clearly  seen  and 


\ 


Fig.  64. — Chromosomes  (synapsed  pairs)  of  maize  at  mid-prophase  in  microsporocytes. 
a,  chromosome  6  attached  to  nucleolus  by  its  nucleolus  organizer  (compare  Fig.  58).  b, 
portion  of  chromosome  8,  showing  chromomeres.  c,  chromosome  7  with  heterochromatic 
region  next  to  kinetochore  at  right,  knob  at  left,  d,  B-type  chromosome  seen  in  certain 
strains;  euchromatic  region  above,  and  heterochromatic  region  below,  e,  portion  of 
chromosome  9  in  a  strain  heterozygous  for  knob  size.  /,  chromosome  9  with  terminal 
knob.      (After  B.  McClintock.) 

closely  compared  (Figs.  58,  79).  One  result  of  such  studies  on  plant 
microsporocytes  and  animal  spermatocytes  has  been  to  show  that  the 
various  chromomeres  tend  to  appear  in  regular  and  constant  patterns 
in  particular  chromosomes.  In  maize  and  its  relatives  large  chromatic 
knobs  also  occupy  definite  positions.  This  characteristic  longitudinal 
differentiation  of  the  chromosome  suggests  a  corresponding  functional 
differentiation,  and  this  conception  is  borne  out  b}^  the  results  of  cyto- 
genetical  studies. 

In  the  meiotic  prophase  other  structural  features,  such  as  kinetochores, 
heterochromatic  regions,  and  nucleolus  organizers,  also  stand  out  clearly 
(Fig.  64).  The  nucleolus  organizer  in  maize  appears  as  a  swollen  hetero- 
chromatic region  immediately  proximal  to  the  secondary  constriction  in 
the  shorter  arm  of  chromosome  6.  In  the  meiotic  prophase  the  nucleolus 
formed  at  the  preceding  telophase  is  still  present  in  contact  with  it. 
That  the  heterochromatic  region  rather  than  the  constriction  itself  acts 
as  the  organizer  is  shown  by  the  fact  that  in  abnormal  cells  which  have 


THE  CHROMOSOMES 


93 


lost  the  satellite,  the  constriction,  and  part  of  the  organizer  the  remaining 
portion  of  the  organizer  forms  a  nucleolus. 


1  »» 


?/  .  a  **fl^ 


Fig.  65. — Spiral  chromonemata  in  chromosomes,  a,  chromonemata  in  microsporocjrte 
of  Trade scaiitia  after  removal  of  chromosome  matrix  with  hot  water.  {After  T.  Sakamura.) 
b,  chromosomes  in  microsporocyte  of  Trillium,  showing  the  major  coils  and  in  certain 
regions  (at  top,  faintly)  the  minor  coils.  {Photograph  by  A.  W.  S.  Hunter.  After  C.  L. 
Huskins.)  c,  nucleus  of  a  protozoan  {Spirotrichonympha) ;  each  of  the  four  chromosomes  is 
attached  to  the  nuclear  membrane  by  a  fiber.     Compare  Fig.  66.      {After  L.  R.  Cleveland.) 


Fig.  66. — Three  drawings  of  a  telophase  nucleus  of  a  protozoan  {H olomastigot aides) . 
a  shows  the  major  and  minor  coils  of  the  chromonemata;  b  shows  only  the  major  coils; 
c  shows  the  larger  sup^rcoils  which  result  from  the  elongation  of  the  chromosomes.  Com- 
pare Fig.  6.5,  c.      {Courtesy  of  L.  R.  Cleveland.) 

In  some  plants,  e.g.,  Trillium  and  Tradescantia,  the  meiotic  chromo- 
somes at  metaphase  and  anaphase  are  extremely  large.  Their  coiled 
chromonemata  appear  \^ith  admirable  clarity  in  good  preparations 
(Fig.  65),  and  they  can  easily  be  watched  while  being  suljjectod  to  various 


94  FUNDAMENTALS  OF  CYTOLOGY 

experimental  treatments.  Thus  by  treatment  with  warm  water  the 
matrix  can  be  dissolved-  away,  leaving  the  chromonemata  intact  on 
the  slide  (Fig.  65,  a).  In  some  plants  it  has  been  found  that  the  chro- 
monemata in  meiotic  chromosomes  are  doubly  coiled,  i.e.,  the}'  not  only 
form  the  large  "major"  spiral  so  easily  seen  but  have  in  addition  a 
minute   "minor"  spiral  running  throughout  their  length   (Fig.   65,   h). 


Sciara  ocellaris 


Fig.  67. — The  salivary-gland  chromosomes  of  a  fungus  gnat  {Sciara).  Each  of  the  four 
consists  of  two  homologous  members  of  the  diploid  somatic  complement  in  intimate  lateral 
union;  note  the  doubleness  at  end  1  of  chromosome  A.  From  an  iron-acetocarmine  smear 
preparation.      Magnification,  575  X.      (Photograph  by  O.  0.  Heard.      After  C.  W.  Metz.) 

The  same  condition  has  been  found  in  certain  somatic  chromosomes 
(Figs.  65,  c;  66). 

Salivary-gland  Chromosomes. — That  the  chromosome  possesses  a 
definite  longitudinal  differentiation  in  structure  and  functional  effect 
is  most  convincingly  shown  by  the  amazing  chromosomes  in  the  salivary 
glands  and  certain  other  larval  tissues  of  the  two- winged  flies  (Diptera). 
These  chromosomes  were  first  observed  in  1881,  but  it  is  only  during 
the  past  10  years  that  they  have  become  well  enough  known  to  be  of 
service  to  the  cytologist  and  cytogeneticist.  It  was  a  stroke  of  good 
fortune  to   find   them   in  the   Diptera.     Many   years   of  cytogenetical 


THE  CHROMOSOMES 


95 


research  had  made  the  fruit  fly,  DrosopMla  mdanogaster,  the  most  impoi- 
tant  animal  in  that  field,  yet  further  progress  in  the  correlation  of  the 
genetical  phenomena  with  chromosome  behavior  seemed  to  be  blocked 
along  some  lines  by  the  small  size  of  the  fly's  chromosomes.  All  this  is 
now  changed.  The  giant  salivary-gland  chromosomes  have  characters 
that  render  them  almost  ideal  for  the  purpose,  and  as  a  result  the  science 
of  c>i^^ogenetics  has  received  a  groat  stimulus.  They  arc  also  furnishing 
xaluable  new  information  regarding  the  fundamental  structure  and 
composition  of  chromosomes.     It  is  fortunate  that  the  cells  containing 


Fig.  68. — Arrangement  of  salivary 
chromosomes  in  the  nucleus  of  Drosophila 
melanogaster.  The  arms  of  the  chromo- 
somes extend  from  the  chromocenter 
formed  by  their  heterochromatic  portions. 
(After  T.  S.  Painter.) 


«»;-•-  «• 


M^l 


Fig.  69. — Nucleus  of  living  cell  in 
salivary  gland  of  a  fly  (Chironomus) ,  show- 
ing giant  chromosomes.      (After  H.  Bauer.) 


them  are  so  located  that  the  skilled  investigator  can  prepare  them  for 
study  by  simple  and  rapid  methods. 

The  first  striking  character  of  salivary-gland  chromosomes  is  their 
great  size  (Fig.  67).  They  are  usually  between  70  and  110  times  as  long 
as  the  chromosomes  in  the  oogonial  cells.  When  moderately  stretched 
for  the  study  of  certain  structural  details  they  may  be  150  times  the  length 
of  the  oogonial  chromosomes,  the  longest  chromosomes  of  the  complement 
then  reaching  a  length  of  about  half  a  millimeter.  In  some  flies  all  the 
chromosomes  lie  well  separated  in  the  nucleus.  In  others,  including 
Drosophila  melanogaster,  the  heterochromatic  portions  about  their 
kinetochores  are  all  grouped  into  a  single  mass  known  as  the  chromoccitlcr 
(Fig.  08). 


96  FUNDAMENTALS  OF  CYTOLOGY 

No  less  striking  is  the  visible  structure  of  the  salivaiy  chromosomes, 
and  it  is  largely  this  feature  which  is  responsible  for  their  great  value  in 
cytogenetics.  Even  in  the  living  and  unstained  nucleus  it  can  be 
seen  that  they  have  conspicuous  transverse  bands  (Fig.  69) .  The  longest 
chromosome  in  Drosophila  melanogaster  has  more  than  2000  of  thesc^ 
bands.  In  fixed  matei'ial  prepared  with  the  acetocarmine  and  Feulgen 
techniques  the  bands  stain  vigorously,  leaving  the  interband  regions 
weakly  stained  or  colorless.  The  differentiation  with  the  latter  technique 
is  particularly  sharp,  showing  that  the  thymonucleic  acid  is  restricted 
almost  entirel}^  to  the  bands.  Ultraviolet-absorption  studies  lead  to  the 
same  conclusion. 

Each  band  appears  to  be  composed  of  one  or  more  discs  of  chromatic 
granules  extending  across  the  cylindrical  body  of  the  chromosome. 
These  granules  have  been  called  chromomeres,  although  their  \'ariation 
in  size  and  number,  even  in  a  given  band,  indicates  that  here  this  term 
does  not  designate  units  all  of  the  same  rank.  One  general  interpretation 
placed  upon  the  whole  chromosome  is  that  it  consists  of  a  large  number — 
dozens  or  even  many  hundreds — of  chromonemata  that  have  multiplied 
as  the  nucleus  grew  without  any  mitoses  to  separate  them,  their  chromo- 
meres remaining  closely  associated  or  united  laterally  as  the  discs. 
Studies  of  the  earlier  stages  in  the  development  of  salivary-gland  nuclei 
lend  some  support  to  this  view.  If,  however,  the  chromonemata  in  the 
fully  developed  salivary  chromosome,  which  may  have  a  thousand  or 
more  times  the  volume  it  had  before  enlargement,  are  comparable  to  the 
original  chromonema,  they  must  be  present  in  very  great  numl^ers ;  more- 
over, if  an  ordinary  chromonema  were  extended  to  the  length  of  the 
salivary  chromosome  without  the  addition  of  new  material,  it  would  be  of 
submicroscopic  thickness.  The  longitudinal  fibrils  seen  connecting  the 
discs  in  stretched  chromosomes  probably  do  not,  therefore,  represent  indi- 
vidual chromonemata.  Some  workers  regard  them  as  large  bundles  of 
chromonemata,  wiiile  others  interpret  them  as  distortions  of  an  alveolar 
structure  pervading  the  chromosome  (Fig.  70) .  The  finer  structure  of  the 
salivary  chromosome  is  at  present  a  ver}^  controversial  subject,  although 
it  is  agreed  that  the  transverse  bands  are  natural  features  having  the 
cytogenetic  usefulness  indicated  below-. 

Of  the  greatest  importance  is  the  fact  that  the  bands  form  a  pattern 
that  is  constant  for  a  given  chromosome.  On  the  basis  of  differences  in 
size,  spacing,  and  other  characteristics  of  the  bands  it  is  possible  for  the 
investigator  to  distinguish  particular  regions  of  the  various  chromosomes. 
Data  of  this  kind  have  been  recorded  in  pictorial  "chromosome  maps." 
The  usefulness  of  band  patterns  to  the  cytogeneticist  should  be  obvious. 
In  later  chapters  it  will  be  shown  how  variations  in  genetical  characters 
and  even  differences  betwx^en  races  and  species  may  be  correlated  with 


THE  CHROMOSOMES 


97 


differences  in  band  pattern,  so  that  it  becomes  possible  to  assign  contribut- 
ing causes  of  certain  characters  to  particular  chromosomal  regions.  Thus 
the  salivary-gland  chromosome  map  becomes  to  the  specialist  a  sort  of 
biological  spectrum  indicating  the  organism's  genetical  constitution, 
much  as  an  absorption  or  a  bright-line  spectrum  reveals  the  chemical 
composition  of  an  inorganic  body. 

Chromosome  Complements. — Any  group  of  chromosomes  composing 
a  nucleus,  whatever  their  number  or  kind,  is  a  chromosome  complement. 
The  simplest  typical  complement  is  one  made  up  of  several  members 
differing  variously  in  form  and  function  but  acting  together  as  a  complete 
and  harmonious  system;  such  a  complement  is  a  genome,  or  set.     Since 


a< 


— .—— o- 


■oooo— •■— -co 


^^oO^^CX> 


Fig.  70. — Drawings  and  diagrams  illustrating  (a)  the  theory  that  the  salivary-gland 
chromosome  consists  of  many  reduplicated  chromonemata  with  their  chromomeres  and 
(6)  the  theory  that  it  has  an  alveolar  structure  with  chromatic  matter  variously  distributed 
within  it.      (a,  after  T.  S.  Painter  and  A.  Griffen;  b,  after  C.  W.  Metz.) 

only  one  chromosome  of  each  kind  is  present,  the  nucleus  (or  tissue,  or 
organism)  with  such  a  complement  is  said  to  be  monoploid. 

A  nucleus  may  contain  one  to  many  genomes.  In  typical  cases, 
sexually  reproducing  organisms  exhibit  an  alternation  of  two  chromosome 
numbers  in  the  course  of  the  life  cycle.  Two  gametes,  each  monoploid, 
unite  to  form  a  zygote  W'hich  has  Qvavy  kind  of  chromosome  in  duplicate 
and  is  therefore  diploid.  In  higher  animals  and  plants  this  diploid  con- 
dition is  maintained  by  equational  mitoses  throughout  the  development 
of  the  body.  When  the  animal  produces  gametes,  the  diploid  number 
is  reduced  by  the  process  of  meiosis  to  the  monoploid  number,  each 
gamete  having  a  single  complete  genome  which  may  include  members 
from  both  of  the  original  genomes.  In  the  plant  this  reduction  in  number 
occurs  when  spores  are  produced.  The  monoploid  number  is  then 
maintained   through    the    development    of   the    gametophyte   and    the 


98 


FUNDAMENTALS  OF  CYTOLOGY 


gametes  it  produces.  In  plants  having  such  an  alternation  of  gameto- 
])hyti('  and  sporophytic  generations  in  the  hfe  cycle,  therefore,  this 
alternation  is  typically,  though  not  invariai)ly,  correlated  with  an  alterna- 
tion of  monoploidy  and  diploidy  in  the  nuclei. 

In  subsequent  chapters  we  shall  describe  in  detail  the  cytological 
features  of  such  typical  cycles  and  review  other  cycles  of  quite  different 
types.  We  shall  also  deal  with  cases  in  which  the  nuclei  show  an  alterna- 
tion not  of  one  and  tw^o  genomes,  but  rather  of  two  and  four,  or  three  and 
six,  or  four  and  eight.  Such  a  condition,  which  is  known  as  polyploidy, 
is  largely  responsible  for  the  high  chromosome  numbers  observed  in  many 
organisms,  especially  among  plants.     Because  of  the  frequent  occurrence 


^  Crepis  pulcherrima 


4€ 


Crepis  parviflora 


^D, 


^i  Crepis  virens 


/?,  A  1^^  iEz       Crep/s  rhoeadifolia 


fl       B       C     D      E 

I   ll 


C       D       E 


tll/ll/ 


Fig.  71. — The  chi-omosomes  of  four  species  of  Crepis.     Genome  at  right;  diploid  comple- 
ment from  root  cell  at  left.      {Crepis  virens  =  C.  capillaris.)      {After  M.  Navashin.) 

of  polyploidy  here  and  there  among  organisms,  it  is  advisable  to  use 
the  general  terms  gametic  number  and  zygotic  number  for  the  reduced  and 
unreduced  chromosome  numbers  in  life  cycles. 

Nuclei  with  different  numbers  of  genomes  tend  to  have  different 
numbers  of  nucleoli.  This  is  because  a  given  genome  commonly  includes 
but  one  chromosome  with  a  nucleolus  organizer.  As  a  result,  the  nuclei 
in  ordinary  tissues  often  show  as  many  nucleoli  as  there  are  genomes, 
although  the  correlation  is  disturbed  by  the  tendency  of  the  fluid  nucleoli 
to  fuse  if  they  come  in  contact.  The  character  has,  however,  in  many 
instances  been  a  useful  one  in  estimating  the  number  of  genomes 
present. 

Most  known  genomes  consist  of  relatively  few  members:  among 
flowei-ing  plants  12,  8,  and  7  are  the  most  frequent  numbers.  Crepis,  a 
genus  of  composites,  has  been  especially  valuable  in  studies  involving 
chromosome  complements  because  of  the  small  number  and  distinct 


THE  CHROMOSOMES  99 

form  of  the  chromosomes  composing  its  genomes  (Fig.  71).  Crepis 
capillaris  has  a  genome  of  only  3  members,  all  of  which  differ  in  their 
length  and  in  kinetochore  location,  while  one  of  them  has  a  satellite. 
In  Datura,  another  genus  prominent  in  c.ytogenetics,  there  are  12  members 
(Fig.  72).  The  genome  has  2  members  in  some  fungi,  4  or  5  in  some 
species  of  Crepis,  7  in  the  garden  pea,  8  in  the  onion,  9  in  the  cabbage, 
10  in  maize,  12  in  many  conifers,  19  in  some  willows,  2  or  more  in  aphids, 
6  in  the  house  fly,  12  in  various  salamanders,  19  in  the  cat,  30  in  the 
cow,  sheep,  and  horse,  and  24  in  man  (Fig.  73)  and  the  Rhesus  monkey. 
Somatic  nuclei,  of  course,  contain  double  these  numbers. 

In  a  diploid  chromosome  complement  the  two  members  constituting 
each  pair  of  similar  chromosomes  are  said  to  be  homologous.     They  have 


e 


M  15  n 


Fig.     72. — The     genome     of     Datura  Fig.    73. — Diploid    complement    of    48 

stramonium    frona    a    monoploid    root    tip.  chromosomes      from      human      spermato- 

The  ends  of  the  vaiious  chromosomes  are  gonium.      {After  O.  Minouchi  and  T   Ohta.) 
designated    by   arbitrary   numbers.      {After 
S.  Satina,  D.  Bergner,  and  A.  F.  Blakeslee.) 

an  ultimate  common  origin  and  affect  the  same  group  of  reactions  in 
the  life  of  the  organism.  As  a  general  rule  the  chromosomes  of  the 
complement  in  a  somatic  cell  may  occupy  any  relative  position  in  the 
nucleus  or  the  mitotic  figure  without  respect  to  their  homologies.  In 
exceptional  cases,  however,  notably  in  Diptera,  there  is  a  strong  tendency 
for  the  homologues  to  lie  rather  near  each  other.  This  is  well  shown  in  the 
ganglion  cells  and  spermatogonia  of  Drosophila  (Fig.  74).  The  somatic 
complement  of  D.  melanogaster  has  8  members :  two  large  V-shaped  pairs, 
one  very  minute  pair,  and  one  pair  of  sex  chromosomes  (XX  in  the  female, 
XF  in  the  male).  In  the  salivary  glands  and  certain  other  larval  tissues 
the  pairing  becomes  very  intimate,  each  giant  chromosome  being  in 
reality  two  homologous  members  in  close  union.  Such  a  condition, 
which  resembles  the  synapsis  normally  occurring  in  the  meiotic  jirophase, 
is  not  known  to  occur  elsewhere  in  somatic  nuclei.  This  phenomenon  is 
exceedingly  useful  to  the  cytogeneticist,  for  it  often  makes  it  possible 
for  him  to  compare  very  minutely  the  chromosomes  of  two  strains  or 
species  after  a  cross  has  been  made  between  them  (page   184).     It  is 


100 


FUNDAMENTALS  OF  CYTOLOGY 


surely  regrettable  that  other  groups  of  organisms  do  not  have  giant 
chromosomes. 

Conclusions. — The  chromosome  should  be  thought  of  as  a  persistent 
individual  that  reproduces  only  by  division  and  in  this  sense  maintains 
its  individuality  throughout  successive  nuclear  and  life  cycles.  In  every 
nuclear  cycle  it  passes  through  a  series  of  alterations  which  may  obscure 
its  continuity  although  they  do  not  disprove  it.  There  is  no  evidence  that 
individualized  masses  of  matrix  persist,  but  all  the  evidence  obtained 
directly  and  indirectly  {e.g.,  by  X-ray  alterations  induced  during  the 
metabolic  stage)  indicates  that  the  chromonema  with  its  characteristic 
longitudinal  differentiation  in  structure  and  function  does  persist.     The 


Iff 


Fig.  74. — Chromosomes  of  Drosophila  melanogaster.  a,  prophase  in  nucleus  of  giant 
cell  of  ganglion  of  female.  Proximal  ends  of  X-chromosomes  separated  from  distal  por- 
tions by  nucleolar  material;  kinetochores  marked  1.  b,  chromosomes  at  metaphase  of 
mitosis  in  ganglion  cell  of  male,  c,  X-  and  F-chromosomes  associated  with  nucleolus 
during  prophase.      (After  B.  P.  Kaufmann.) 

chromonemata  in  the  metabolic  stage  are  relatively  free  from  enveloping 
matrix  and  lie  more  exposed  to  the  other  nuclear  materials,  which  indi- 
cates that  the  chromosomes  exert  their  effects  upon  cell  activities  mainly 
at  this  time.  Their  compact  arrangement  within  a  matrix  appears  to  be 
significant  in  connection  with  mitotic  distribution  rather  than  with 
metabolism.  Later  on  we  shall  point  out  how  alterations  occur  in  the 
organization  of  the  chromonema  from  time  to  time,  its  parts  being 
rearranged  or  exchanged  with  those  of  other  chromonemata,  but  these 
changes  are  not  such  as  to  invalidate  the  basic  concept  of  chromosomal 
individuality  and  continuity. 

The  various  nuclear  materials  or  ultimate  units  necessar}-  to  the 
normal  life  of  the  organism  are  nearly  everywhere  carried  in  several 
chromosomes  rather  than  in  only  one  or  a  very  large  number.  This 
small  group,  or  genome,  is  to  be  regarded  as  an  organized  system  of 
interdependent  members,  and  not  as  a  simple  collection  of  materials. 
Studies  on  altered  chromosomes  show  that  the  primary  requisite  is  the 
presence  of  the  right  assortment  of  vmits  or  materials  and  that  their 


THE  CHROMOSOMES  101 

relative  positious  in  some  instances  affect  tlicir  action;  the  number  of 
chromosomes  in  which  they  are  carried  seems  to  be  a  matter  of  minor 
importance.  This  is  probably  one  reason  for  the  lack  of  any  general 
correlation  between  the  numbers  of  chromosomes  composing  genomes 
and  the  relative  complexity  of  the  organisms  in  which  these  genomes  are 
found. 


CHAPTER  VIII 
MEIOSIS 

In  all  organisms  reproducing  sexually  the  doubling  of  the  gametic 
chromosome  number  (diplosis)  by  the  union  of  the  nuclei  of  two  gametes 
is  compensated  by  a  halving  of  the  resulting  zygotic  number  (haplosis) 
at  some  other  point  in  the  life  cycle.  This  quantitative  alternation  is  in 
itself  a  matter  of  considerable  interest,  but  the  full  significance  of  the 
changes  involved  can  be  appreciated  only  when  one  is  aware  of  the  peculiar 
and  orderly  manner  in  which  the  reduction  in  number  is  accomplished 
and  of  the  effect  that  the  alteration  may  have  upon  the  capacities  of  the 
nuclei  that  result.  "Chromosome  reduction"  means  not  merely  the 
change  from  the  zygotic  to  the  gametic  number,  but  also,  more  specificall}^, 
the  segregation  (disjunction)  of  the  two  chromosomes  composing  each 
homologous  pair  in  the  zygotic  complement. 

These  changes  are  brought  about  by  two  successive  nuclear  divisions 
in  the  course  of  which  the  chromosomes  are  actually  divided  only  once, 
and  the  whole  process  constitutes  meiosis.  A  nucleus  undergoing  meiosis 
consequently  gives  rise  to  a  c^uartet  of  nuclei,  each  of  which  has  the 
gametic  chromosome  number.  Any  cell  in  which  meiosis  is  initiated 
may  be  termed  a  meiocyte.  In  most  animals  the  meiocj^tes  are  the  pri- 
mary spermatocytes,  each  of  which  produces  a  quartet  of  spermatozoa, 
and  the  primary  oocytes,  each  of  which  produces  an  egg  and  three  (or  two) 
polar  bodies.  In  most  plants  the  meiocytes  are  sporocytes,  each  of  which 
produces  a  quartet  of  spores. 

In  this  chapter  we  shall  give  first  a  preparatory  general  account  of  the 
behavior  of  the  chromosomes  -wdth  special  reference  to  their  distribution  to 
the  resulting  four  nuclei.  This  will  be  followed  by  a  detailed  account  of 
the  changes  occurring  at  each  of  the  rather  well-marked  stages  character- 
istic of  meiosis.  For  convenience  the  first  and  second  meiotic  divisions 
will  often  be  referred  to  simply  as  /  and  II.  Only  the  ordinary  diploid- 
monoploid  cycle  will  be  considered  here,  discussion  of  chromosome  behav- 
ior in  polyploid  plants  being  deferred  to  a  later  chapter. 

Distribution  of  Chromosomes  in  the  Meiotic  Divisions. — Meiosis 
begins  in  a  nucleus  with  a  diploid  chromosome  complement.  The  two 
genomes  were  brought  together  at  the  previous  gametic  union;  hence  the 
two  chromosomes  composing  each  homologous  pair  in  the  offspring  of  a 
cross  are  derived  one  from  each  parent  (see,  however,  page  216).     The 

102 


ME  IDS  IS  103 

two  hoinologucs  iiifluciu'C'  the  saiiu^  grouj)  of  reactions  and  characters 
in  tlie  oi-ganisni,  but  their  conii)osition  varies  in  such  a  way  thattheii- 
influence  ma.y  be  either  the  same  or  in  certain  respects  different. 

The  distribution  of  the  chromosomes  in  the  two  meiotic  divisions  L'^ 
shown  diagrammatically  in  Fig.  75.  The  remarkable  alterations  in  form 
undergone  by  the  chromosomes  during  the  successive  stages  are  not 
represented  here;  these  will  be  described  in  the  following  section.  A 
diploid  number  of  6  is  arbitrarily  chosen.  The  members  of  the  two  gen- 
omes are  distinguished  by  shading  and  bv  large  and  small  letters.  Dots 
in  the  uppermost  nuclei  indicate  location  of  kinetochores. 

Referring  to  the  first  column  in  the  diagram,  we  see  that  the  six 
chromosomes  are  arranged  in  no  particular  order  in  the  nucleus.  In  the 
prophase  of  the  first  meiotic  division  the  tw-o  chromosomes  of  each 
homologous  pair  approach  each  other  and  become  very  intimately 
associated,  but  they  do  not  actually  fuse;  this  is  sy^iapsis.  The  chromo- 
somes are  now  in  the  bivalent  condition.  Xot  long  afterw^ard  each  of  the 
members  of  the  s.ynapsed  pair  becomes  visibl.y  double  apparently  by 
splitting;  the  bivalents  now  have  the  form  of  tetrads,  the  four  members  of 
each  tetrad  being  chromatids.  At  the  end  of  prophase  /  the  nucleus  still 
has  all  the  chromosomes,  but  they  appear  as  the  monoploid  number  of 
tetrads. 

In  metaphasc  /  the  tetrads  are  arranged  in  the  equator  of  the  spindle, 
and  in  the  anaphase  each  of  them  separates  into  tw^o  dyads,  or  pairs  of 
chromatids,  which  pass  to  the  two  poles.  In  the  diagram  the  arrangement 
at  metaphase  is  such  that  sister  chromatids  (those  formed  by  the  recent 
splitting)  pass  to  the  same  pole;  hence  it  is  the  members  previously 
brought  together  by  synapsis  which  separate  here,  and  this  form  of 
separation  is  called  disjunction. 

Each  of  the  resulting  nuclei  then  undergoes  a  second  meiotic  division. 
Here  the  two  chromatids  composing  each  dyad  separate  equationally 
(along  the  recent  split)  and  paiss  poleward.  Meiosis  thus  results  in  a 
quartet  of  nuclei  in  each  of  w^hich  there  is  one  complete  genome  with  three 
members.  The  four  chromatids  of  each  tetrad  now  lie  in  four  different 
nuclei. 

Several  features  of  the  process  thus  far  described  should  be  carefully 
noted,  for  it  will  be  necessary  to  qualify  present  statements  after  other 
modes  of  distribution  have  been  considered.  (1)  As  illustrated  in  column 
1,  the  first  division  is  disjunctional  and  the  second  equational  for  all  the 
chromosomes.  (2)  In  the  resulting  quartet  of  nuclei  there  are  genomes  of 
two  kinds  with  respect  to  the  derivation  of  their  members  from  the  two 
original  genomes.  (3)  The  arrangement  shown  at  metaphase  /  is  only 
one  of  several  possible  ways  in  which  the  chromosomes  could  be  arranged 
and  still  have  all  the  separation  disjunctional  at  anaphase.     With  three 


104 


FUNDAMENTALS  OF  CYTOLOGY 


^ti 


Fig.  75. — Distribution  of  chromosomes  in  meiosis.      Explanation  in  text. 


MEIOSIS  105 

chromosome  pairs  the  number  of  such  possible  arrangements  is  four;  hence 
a  nucleus  at  the  close  of  meiosis  might  have  any  one  of  eight  possible  types 
of  genome:  A  B  C ,  a  b  c,  A  B  c,  a  b  C ,  A  b  c,  a  B  C ,  A  b  C ,  a  B  c.  A  gi\(Mi 
(jiuartet  would  have  two  of  these  types.  There  are  both  cytological  and 
genetical  evidences  that  this  randomness  of  orientation  at  metaphase  /  does 
prevail.  With  more  chromosome  pairs  the  nimiber  of  possible  types  of 
genome  would  of  course  be  greater,  the  formula  for  this  number  being 
2",  where  n  equals  the  number  of  pairs.  (4)  These  various  genomes  differ 
qualitatively^  and  therefore  in  their  effects  upon  reactions  and  characters, 
only  to  the  extent  that  the  chromosomes  in  the  original  diploid  comple- 
ment differed  from  their  respective  homologues.  If  there  had  been  no 
such  differences  originally,  the  genomes  in  the  quartets  would  all  be 
qualitatively  alike  in  spite  of  differences  in  the  derivation  of  their  mem- 
bers; hence  meiosis  does  not  always  result  in  nuclei  differing  in  actual 
constitution.  Ordinarily,  however,  there  are  some  original  differences, 
so  that  the  genomes  eventually  resulting  from  meiotic  chromosome  dis- 
tribution do  show  qualitative  differences.  * 

Turning  now  to  the  second  column  of  Fig.  75,  we  see  what  would 
result  if  the  tetrads,  instead  of  all  separating  disjunctionally  in  division  /, 
were  to  be  so  oriented  at  metaphase  that  separation  would  be  equational 
for  at  least  one  of  them  (the  Bb  pair  in  the  diagram).  Sister  chromatids 
of  such  a  tetrad  would  separate  equationall}'  to  opposite  poles,  and 
disjunction  would  follow  at  division  II.  The  other  tetrads  would 
disjoin  at  /,  wnth  equational  separation  at  //.  The  result  would  be  a 
quartet  of  nuclei  with  four  tj'pes  of  genome  rather  than  two.  Equational 
division  of  whole  tetrads  at  /  may  indeed  occur,  but  it  is  now  thought 
that  it  must  at  least  be  verj^  exceptional,  the  reported  cytological  evidence 
for  it  having  received  a  new  interpretation. 

The  third  column  of  Fig.  75  illustrates  the  interpretation  now  generally 
placed  upon  cytological  and  genetical  evidence  indicating  the  occuri-ence 
of  equational  separation  in  division  /.  In  one  of  the  tetrads  is  shown  a 
chiasma,  i.e.,  a  place  at  which  two  of  the  four  chromatids  actually 
exchange  corresponding  portions.  Such  crossing  over  is  a  normal  feature 
of  meiosis  in  most  organisms  and  commonly  occurs  in  all  the  tetrads; 
moreover,  a  single  tetrad  may  have  more  than  one  such  exchange.  It 
is  only  for  the  sake  of  simplicity  that  the  diagram  shows  onl}'  one  in 
the  whole  complement.  Crossing  over  complicates  the  process  of 
meiosis,  but  the  complication  must  be  faced  because  it  affords  an 
explanation  of  certain  genetical  phenomena  to  be  discussed  in  later 
chapters. 

If,  now,  all  the  tetrads  are  oriented  in  the  spindle  at  metaphase  /  so 
that  kinetochores  of  sister  chromatids  face  the  same  pole,  the  proximal 
portion   (near  the   kinetochores)   of  the  tetrad   with  the   chiasma   will 


106  FUNDAMENTALS  OF  CYTOLOGY 

separate  disjunctionally  in  I  and  equationally  in  //  (prereduction), 
while  the  distal  portion  beyond  the  chiasma  separates  equationally  in  I 
and  disjunctionally  in  II  (postreduction).  The  proximal  portions  of  the 
chromatids  eventually  lie  in  the  four  (quartet  nuclei  precisely  as  they  do 
in  column  1.  The  distal  portions  also  lie  in  the  four  nuclei.  The 
complication  introduced  by  crossing  over  appears  when  the  relative 
positions  occupied  by  the  two  portions  are  considered. 

In  the  few  cases  supported  by  adequate  cytological  evidence  (visibly 
unlike  homologues;  data  on  chiasmata)  and  genetical  evidence  (distribu- 
tion of  genetical  factors),  it  appears  that  the  four  chromatids  behave  as 
described  above:  the  regions  near  the  kinetochores  disjoin  in  /,  while 
equational  division  in  I  occurs  only  in  certain  regions  determined  by 
crossovers.  If  these  latter  regions  show  visible  differences  in  the  two 
homologues,  their  postreduction  is  evident,  but  it  does  not  follow  that 
the  whole  tetrad  separates  in  this  way.  Hence  reduction  in  the  strict 
sense  (disjunction)  does  not  take  place  in  all  portions  of  the  complement 
or  even  in  all  portions*  of  the  same  tetrad  at  the  same  division.  It 
is  only  after  both  meiotic  divisions  have  been  carried  through  that 
chromosome  reduction  is  complete.  It  is  only  then  that  all  disjunction 
is  finished,  and  only  then  does  each  nucleus  have  the  reduced  number  of 
single  chromosomes. 

The  points  brought  out  in  this  section  may  now  be  summarized.  In 
meiosis  each  chromosome  enters  into  synapsis  with  its  homologue  and  also 
splits  longitudinally,  giving  thus  a  tetrad  composed,  of  four  chromatids. 
The  four  chromatids  of  every  tetrad  are  distributed  iri  two  divisions  to  the  four 
nuclei  formed  at  the  close  of  meiosis.  Each  chromosome  {or  portion  of  a 
chromosome)  is  disjoined  from  its  homologue  {reduction)  in  one  of  the 
divisions  and  divided  equationally  in  the  other.  It  is  probable  that  dis- 
junction in  the  first  division  is  the  rule  for  the  kinetochores  and  near-by 
regions,  the  second  divisio7i  therefore  being  equational  for  these  regions.  This 
order  can  be  reversed  in  other  regions  when  crossovers  occur. 

Each  of  the  four  nuclei  of  the  resulting  quartet  contains  a  single  genome 
composed  of  members  from  one  or  both  of  the  genomes  of  the  original  diploid 
complement.  The  four  nuclei  are  qualitatively  alike  or  unlike  depending 
upon  the  amount  of  difference  between  homologous  chromosomes  in  the 
original  complement.  Every  kind  of  chromosomal  unit  is  present  singly 
instead  of  in  duplicate  in  each  nucleus. 

Detailed  Account  of  the  Phases  of  Meiosis. — Most  of  the  significant 
features  peculiar  to  meiosis  are  found  in  the  prophase  of  the  first  division ; 
when  these  are  understood  the  subsequent  stages  present  few  difficulties. 
The  phases  of  the  entire  process  will  now  be  described  in  order  (see  Fig. 
76).  It  is  to  be  remembered  that  the  details  of  meiotic  chromosome 
behavior  vary  a  good  deal  in  different  organisms  and  that  the  purpose  of 


MEIOSIS 


07 


TELOPHASE    I 


PROPHASE    XL 


METAPHASE    U  ANAPHASE    H  TELOPHASE    H 

Fig.  76. —  Diagram  of  stages  of  meiosis.     Explanation  in  text. 


108 


FUNDAMENTALS  OF  CYTOLOGY 


this  account  will  be  best  sei-ved  by  confining  attention  largely  to  those 
features  more  or  less  common  to  all  of  them. 

Leptotene  Stage. — The  chromonemata,  after  having  presented  a  rather 
confused  picture  since  the  premeiotic  telophase,  become  more  distinct 
from  one  another  in  the  early  meiotic  prophase  and  appear  as  very  long 
and  slender  threads.  They  are  present  in  the  diploid  number,  and  each 
represents  a  chromosome.  Because  of  their  great  attenuation  and  the 
scarcity  of  enveloping  matter,  their  chromomeres  show  plainly.  Whether 
their  singleness  is  actual  or  only  apparent  because  of  their  slenderness  is  a 
debated  point.  In  some  meiocytes,  notably  in  animals,  the  threads  may 
all  be  oriented  with  one  end  toward  the  same  side  of  the  nucleus,  forming 
a  so-called  bouquet  (Fig.  77). 

Zygotene  Stage. — The  leptotene  threads  now  become  very  closely 
associated  laterally  in  pairs,  each  of  them  cohering,  though  not  actuall>' 


Fig.  77. — Stages  in  meiosis  in  spermatocyte  of  salamander.      1,  leptotene  threads  developing: 
2,  pachytene  stage;  3,  diplotene  stage;  4,  metaphase  I. 

fusing,  with  its  homologue  (Fig.  78).  This  selective  pairing,  or  synapsis, 
begins  at  one  or  more  points,  often  at  the  ends  or  the  kinetochore,  and 
gradually  extends  "zipper-hke"  until  it  is  complete.  The  extension  and 
the  slenderness  of  the  chromonemata,  which  reach  their  maximum  length 
at  leptotene-zygotene,  are  believed  to  bear  a  causal  relation  to  the  synaptic 
union.  In  some  nuclei,  notably  in  those  with  threads  arranged  in  a 
"bouquet,"  one  portion  may  be  occupied  by  paired  threads  while  the  rest 
shows  only  threads  still  unpaired ;  this  is  the  amphitene  condition.  As  the 
threads  pair,  they  immediately  begin  to  shorten  and  thicken. 

It  appears  likely  that  synapsis  is  facilitated  by  the  arrangement 
assumed  by  the  chromosomes  at  the  close  of  the  last  premeiotic  anaphase. 
All  the  members  of  the  complement  then  lie  more  or  less  parallel,  with 
their  kinetochores  directed  toward  the  pole,  so  that  in  the  resulting 
telophase  nucleus  their  arrangement  is  not  a  haphazard  one.  Further- 
more, in  some  organisms  a  loosel}^  paired  arrangement  of  homologues  is 
evident  during  the  premeiotic  mitoses  or  even  earlier  (page  99). 

Pachytene  Stage. — After  synaptic  pairing  has  been  completed,  the 
nucleus  is  said  to  be  in  the  pachytene  stage,  for  the  threads  are  then 


MEIOSIS 


109 


noticeably  thicker  than  in  the  leptotene  (thin-thread)  stage.  Tiie  thick 
double  pachytene  threads  are  present  in  the  monoploid  number,  and 
each  of  them  is  bivalent,  since  it  consists  of  two  homologous  chromosomes 
in  synaptic  association  (Figs.  77-79).  They  may  be  arranged  at  random 
or  in  the  bouquet  position. 

Late  in  the  pachytene  stage  each  of  the  two  chromosomes  of  each 
synapsed  pair  becomes  visibly  double,  presumabl>^  because  of  the  enlarge- 


'^-^ 


WJ 


'%-'. 


Flu.  78.- — Leptotene,  zygotene,  pachytene,  and  very  early  diplotene  stages  in  niicrosporo- 
cytes  of  Trillium.     Arrows  indicate  chiasniata.      {After  C.  L.  Huskins  and  S.  G.  Smith.) 

ment  of  the  structures  concerned.  If  the  doubleness  reported  in  the 
premeiotic  telophase  has  persisted,  although  invisible,  this  represents  its 
reappearance.  The  pachytene  threads  are  now  quadruple:  each  is  a 
tetrad  of  chromatids. 

Diplotene  Stage. — In  each  of  the  tetrads  the  four  chromatids  begin  to 
separate,  one  pair  of  sister  chromatids  from  the  other  two,  as  though  the 
synaptic  force  holding  them  together  were  being  replaced  by  a  repulsive 
force.     At  one  or  more  points  they  are  prevented  from  separating  by 


no 


FUNDAMENTALS  OF  CYTOLOGY 


chiasmata,  where  two  of  the  chromatids  have  exchanged  portions  by 
crossing  over  (Fig.  81).  A  tetrad  with  one  chiasma  thus  has  the  form  of 
an  X,  while  one  with  two  at  or  near  its  ends  appears  as  an  0.  When  there 
are  several,  the  regions  between  them  form  a  series  of  such  openings,  one 
of  which  usually  includes  a  region  on  ])oth  sides  of  the  kinetochore.  The 
number  and  the  location  of  chiasmata  may  differ  characteristically  in 
different  organisms.  In  general  there  are  more  in  long  chromosomes 
than  in  short  ones. 

During  the  diplotene  stage  the  tetrads  continue  the  shortening  begun 
in  the  pachytene  stage.     This  involves  a  coiling  of  the  chromonemata 


Fig.    79. — Pachytene    stage    in    micro-  Fig.  80. — Diplotene  stage  in  speraiato- 

sporocyte  of  maize.  Chromosome  6  at-  cyte  of  scorpion  (Tityus  hahiensis).  The 
tached  to  nucleolus  above;  chromosome  1  members  of  one  bivalent  are  very  loosely 
looped  over  6;  chromosome  3  at  upper  left.  associated.  The  large  black  spots  are 
{After  B.  McClintock.)  extraneous    material    in    the    preparation. 

{Courtesy  of  F.  G.  Brieger  and  E.  A.  Graner.) 

within  the  matrix,  which  begins  to  be  more  evident  at  this  time.  With 
such  changes  the  chromosomes  pass  gradually  into  the  diakinesis  stage. 

Diakinesis  Stage. — This  stage  is  characterized  by  the  presence  of 
compact  tetrads  lying  well  spaced  out  in  the  nucleus,  often  near  its 
membrane.  This  is  therefore  a  favorable  stage  for  counting  the  chromo- 
somes. They  have  continued  to  shorten  as  their  chromonemata  have 
continued  to  coil,  sometimes  into  both  major  and  minor  spirals.  The 
matrix  has  become  abundant,  giving  them  smoother  contours.  They 
have  the  form  of  X's,  V's,  O's  and  other  more  complex  shapes  depending 
upon  the  number  and  location  of  chiasmata.  Since  the  matrix  tends  to 
form  a  common  mass  about  any  two  closely  associated  chromonemata,  the 
tetrads  often  look  merely  double  instead  of  ciuadruple.  Commonly  th(; 
one  having  the  nucleolus  organizers  is  in  contact  with  the  nucleolus. 

In  many  organisms  the  tetrads  show  fewer  chiasmata  than  they  did  in 
the  diplotene  stage.     This  is  due  to  a  process  called  tenninalization ,  in 


MEIOSIS  1 1 1 

which  the  four  chromatids,  after  opening  out  two  by  two  to  give  the 
diplotene  .stage,  continue  to  open  into  the  chiasma  on  its  proximal  side 
while  the  opening  on  its  distal  side  closes,  the  result  being  a  gradual 
movement  of  the  chiasma  along  the  tetrad,  even  to  its  end. 

The  meiocytes,  or  cells  in  which  all  the  foregoing  prophasic  changes 
occur,  are  in  most  cases  relatively  large  cells  with  lafge  nuclei.  Their  size 
increases  through  a  portion  if  not  all  of  the  prophase.  In  plant  sporocytes 
the  increase  is  moderate,  while  in  animal  spermatocj^tes  it  is  often  greater 
and  involves  a  temporary  ''diffusion"  of  the  chromosomes  at  about  the 
diplotene  stage.  The  animal  oocyte  undergoes  an  enormous  inci-ease  in 
size  at  this  stage,  developing  most  of  the  features  that  are  to  characterize 
the  egg  which  it  eventually  becomes.  The  oocyte  nucleus  becomes  wery 
large  during  this  "growth  period,"  its  chromosomes  sending  out  thready 
processes  in  all  directions  and  losing  their  stainability-  This  suggests  a 
synthetic  function  comparable  to  that  performed  during  the  metabolic 


^'^ 


Fig.  81. — Tetrads  in  advauced  diplotene  .stage  from  .spermatocytes  of  grassliopper.      They 
show,  respectively,  one,  two,  three,  and  four  chiasmata.      {After  F.  A.  Janssens.) 

stage,  when  the  chromonemata  are  again  in  extensive  contact  with  the 
other  substances  in  the  nucleus.  Eventually  the  chromosomes  again 
become  compact  and  stainable  and  assume  the  form  characteristic  of  the 
diakinesis  .stage. 

Metaphase  I. — At  the  close  of  the  diakinesis  stage  the  achromatic 
figure  is  developed,  and  the  tetrads  become  arranged  in  its  equator  with 
their  kinetochores  facing  its  two  poles.  In  a  lateral  view  of  the  metaphase 
figure  they  appear  about  as  they  did  at  late  diakinesis  except  for  the  more 
clearly  evident  location  of  their  kinetochores  and  sometimes  their  greater 
compactness.  When  viewed  from  the  direction  of  the  spindle  pole  they 
can  easily  be  counted  unless  they  are  very  long  and  crowded.  Large 
chromosomes  show  their  coiled  chromonemata  particularly  well  at  this 
stage  and  at  anaphase,  many  of  the  best  studies  on  minute  structure 
having  been  made  on  such  chromosomes.  For  example,  some  genera  of 
plants  show  a  "double-coiled"  condition  in  their  chromonemata  only  at 
this  time. 

Of  special  interest  is  the  fact  that  longitudinal  doubleness  (the  so-called 
tertiary  split)  may  become  visible  in  the  chromonema  of  each  chromatid 
at  metaphase  and  anaphase  /,  the  tetrad  therefore  having  eight  lialf- 
chromatids.  In  other  cases  it  is  first  seen  during  division  //.  Occasion- 
ally it  has  been  demonstrated  as  early  as  diakinesis  or  even  the  diplotene 


112  FUNDAMENTALS  OF  CYTOLOGY 

stage.  The  results  of  X-ray  studies  suggest  that  it  is  present  below  the 
limit  of  visibility  much  earlier.  In  any  event  it  represents  the  plane  of 
anaphasic  separation  in  the  first  postmeiotic  mitosis.  In  a  plant  this 
would  be  the  first  mitosis  in  the  spore,  in  an  animal  the  first  mitosis  in  the 
fertilized  egg. 

Anaphase  I. — The  tetrads  now  separate  into  dyads  which  begin  to 
move  apart  toward  the  opposite  poles  of  the  spindle.  Often  there 
appears  to  be  considerable  resistance  in  the  region  of  a  chiasma  to  the 
disjunctive  forces,  so  that  the  tetrad  may  elongate  and  assume  an  odd 
shape  (Fig.  82).  Eventually  the  two  dyads  become  free.  Meanwhile  the 
two  chromatids  composing  each  of  them  commonly  widen  out  from  each 


J 

Fig.  82. — Late  metaphase  /  in  microsporocyte  of  peony  (Paeonia),  showing  the  five 
tetrads  about  to  separate  into  dyads.  In  the  third  the  kinetochores  are  at  the  points  above 
and  below;  a  chiasma  is  present  in  each  arm.  In  the  fourth  and  fifth  the  kinetochores  are 
at  the  sharp  angles;  subterminal  chiasmata  are  present  at  the  equator.      {After  K.  Sax.) 

other  except  at  the  kinetochore  (Fig.  84).  Thus  a  dyad  with  a  nearly 
terminal  kinetochore  appears  as  a  single  V,  while  one  with  a  median 
kinetochore  is  a  double  V.  As  already  pointed  out,  genetical  evidence 
indicates  that  the  two  chromatids  of  a  dyad,  at  least  in  the  region  of  the 
kinetochore,  are  as  a  rule  sisters.  This  interpretation  has  further  cyto- 
logical  support  in  the  chromosomes  of  an  amphibian,  w^hich  show^  the 
kinetochore  of  the  dyad  not  yet  divided  at  this  time,  although  it  has  two 
kinosomes  and  two  tractile  fibers.  Its  division  is  completed  in  //.  In 
maize  also  tw^o  tractile  fibers  can  be  seen  extending  from  each  dyad  in  I. 
At  the  end  of  the  anaphase  the  two  groups  of  dyads  form  compact  groups 
at  the  poles. 

Telophase  I  and  Interkinesis. — The  polar  groups  of  chromosomes  at  the 
close  of  anaphase  /  nearly  always  undergo  a  certain  amount  of  telophasic 
transformation  similar  to  that  seen  in  somatic  nuclei.  In  most  cases, 
however,  the  alteration  is  not  carried  far  enough  to  form  fine-textured 
metabolic  nuclei,  the  individual  chromosomes  often  being  discernible  at 
least  in  part  up  to  the  beginning  of  prophase  //.  The  abbreviation  of 
interkinesis,  or  stage  between  divisions  /  and  II,  reaches  an  extreme  in 


MEIOSIS  113 

certain  animal  oocytes,  where  the  chroniosomc^s  at  the  close  of  anaphase  / 
immediately  become  arranged  in  new  spindles  for  division  //.  In  ordi- 
nary interkinetic  nuclei  where  the  chromosomes  can  still  be  seen,  the  two 
chromatids  of  each  dyad,  although  more  slender  than  during  the  anaphase, 
continue  to  remain  closely  associated  at  the  kinetochore  and  widened  out 
elsewhere ;  hence  they  tend  to  appear  as  X's  with  arms  varying  in  length 
according  to  kinetochore  position. 

Cytokinesis  does  not  invariably  follow  division  /.  In  the  micro- 
sporocytes  of  many  vascular  plants  and  in  the  meiocytes  of  certain  lower 
plants  the  two  nuck^i  lie  in  a  common  mass  of  cytoplasm  and  undergo 
division  //,  after  which  the  meiocyte  is  divided  simultaneously  into  four 
cells  (Fig.  101,  4)-     In  other  plant  meiocytes,  cytokinesis  does  occur  aftei- 


1 

1  2  3  4 

Fig.  83. — Chromosomes  in  first  meiotic  division  in  microsporocytes  of  Trade scantia. 
1,  bivalent  at  metaphase  about  to  disjoin;  in  each  half  the  apparently  single  thick  spiral  is 
actually  composed  of  two  chromonemata  which  represent  the  chromatids.  2,  a,  b,  c,  meta- 
phase bivalents;  the  two  spiral  chromonemata  in  each  of  the  dyads,  which  are  about  to 
disjoin  upward  and  downward,  are  separating  from  each  other  laterally  (a  and  b  are  two 
prints  from  the  same  negative).  3,  one  dyad  at  anaphase;  the  two  chromatid  spirals  now 
lie  side  by  side.-  4,  four  dyads  at  anaphase;  in  each  of  them  the  two  chromatids  remain 
closely  associated  only  near  the  kinetochore.      (After  K.  Sax  and  L.  M.  Humphrey.) 

I  (Fig.  84).  In  animals  the  primary  spermatocyte  is  divided  into  two 
secondary  spermatocytes  in  which  division  //  then  takes  place.  The 
primary  oocyte  divides  very  unequally  to  form  a  minute  polocyte  (polar 
body)  and  the  secondary  oocyte;  division  II  follows  in  the  latter  but  not 
alwaj^s  in  the  former  (Fig.  91).  Further  c,ytological  features  of  these 
reproductive  stages  are  to  be  added  in  the  next  three  chapters. 

Prophase  II. — This  prophase  is  much  simpler  than  prophase  I.  When 
the  transformation  of  the  chromosomes  in  telophase  /  has  not  been  carried 
very  far,  prophase  //  consists  in  little  more  than  their  resumption  of  a 
more  compact  form  in  each  of  the  daughter  nuclei  of  division  7.  The  two 
chromatids  of  each  dyad  tend  to  retain  in  some  degree  the  divergent 
position  they  first  assumed  in  anaphase  /,  so  that  their  association,  except 
at  the  kinetochore,  is  usually  looser  than  that  observed  in  somatic 
prophases. 

Metaphase  II  and  Anaphase  II. — The  dyads  in  the  two  nuclei  now  take 
up  positions  \vith  their  kinetochores  at  tin;  equators  of  newly  formed 


114 


FUNDAMENTALS  OF  CYTOLOOY 


.spindles.  In  many  cases  they  are  longer  than  they  were  in  metaphase  /, 
and  this,  together  with  their  simpler  structure,  causes  division  //  to 
resemble  a  somatic  mitosis  much  more  closely  than  division  I  does.     In 


Fig.  84. — Stages  in  meiosis  in  angiospeiuii  microsporocytes.  1,  transverse  section  of 
lily  anther  with  sporocytes.  2,  metaphase  I  in  May  apple.  3-5,  anaphase  /,  metaphase 
II,  and  anaphase  II  in  lily.      Cliromosome  doubleness  is  obscured  by  fixation  in  4. 

the  anaphase  the  chromatids  of  each  dyad  move  apart  to  the  spindle  poles, 
thus  completing  the  meiotic  distribution  of  the  chromosomes. 

Telophase  II. — The  four  groups  of  chromosomes  now  reorganize  as  a 
quartet  of  nuclei.     Each  of  them  contains  one  complete  genome,  this 


Fig.  85. — Meiosis  and  syngamy  in  a  threadworm  {Ascaris).  1,  metapliase  of  first 
meiotic  division  in  oocyte;  two  tetrads  present.  The  sperm  has  already  entered  the  egg  (at 
left);  the  smaller  dark  body  is  its  nucleus.  2,  3,  second  meiotic  division;  first  polar  body 
disintegrated  above;  sperm  chromosomes  near  center  of  egg.  4,  sperm  and  egg  nuclei 
about  to  unite;  first  polar  body  above  and  second  one  below  because  of  rotation  of  egg. 
During  these  stages  a  perivitelline  space  develops  between  the  thickened  wall  and  the  egg. 

genome  being  composed  of  one  chromatid  (now  a  chromosome)  from  each 
of  the  tetrads  present  in  metaphase  I.  Each  chromosome  has  the 
chromonemal   doubleness  which   will  become  effective  in  the  ensuing 


MEIOSIS 


115 


postmeiotic  division.  Cytokinesis  follows  in  typical  cases,  giving  a 
quartet  of  uninucleate  cells  (spores;  spermatids  soon  to  become  sper- 
matozoa; egg  and  polocytes). 

Problems  of  Meiosis. — The  foregoing  account  of  meiosis  will  serve  as  a 
l)rercquisite  to  our  later  discussion  of  the  mechanism  of  M(?ndelian  hered- 
ity, but  it  does  not  full.y  indicate  the  prominent  position  that  the  ]:)rocess 
holds  in  present-da}^  cytological  research.  Meiosis,  like  practically  (»very 
other  process  in  the  organism,  continues  to  present  numerous  problems, 
the  solution  of  which  would  shed  needed  light  upon  phenomena  occurring 
at  various  stages  of  the  life  cycle.  We  should 
therefore  consider  for  a  moment  a  few  such 
questions  now  engaging  the  special  attention  of 
cytologists. 

What  initiates  meiosis?  What  are  the  physi- 
ological conditions  associated  with  the  change 
from  the  ordinary  somatic  type  of  nuclear  divi- 
sion to  the  meiotic  type,  and  what  relation  do 
these  conditions  bear  to  the  prior  changes  that 
bring  on  the  reproductive  phase  in  the  organism? 
Experimental  treatments  frequently  result  in  the 
partial  or  complete  replacement  of  the  meiotic 
b}^  the  somatic  type  of  division  in  meiocytes,  and 
various  suggestions  have  been  made,  for  example, 
regarding  the  apparent  effects  of  a  retardation 
or  an  acceleration  of  the  prophasic  process  upon 
the  character  of  the  division.  So  far,  however, 
the  main  question  has  received  no  satisfactory^ 
answer. 

What  causes  synapsis?  The  synaptic  reac- 
tion occurs  between  apparently  single  chro- 
mosomes having  the  peculiar  constitutional 
relationship  designated  as  homology,   and  only 

under  physiological  conditions  that  are  essentially  normal.  It  is 
manifested  primarily  between  corresponding  minute  portions  or 
units  in  the  two  threads,  synapsis  being  normal  in  all  respects 
only  when  the  various  units  or  portions  in  the  two  have  the  same 
serial  order.  Moreover,  if  three  or  more  homologous  chromosomes 
are  present  in  the  meiocyte,  only  two  as  a  rule  synapse  closely  at  any  given 
region,  as  though  the  synaptic  force  were  somehow  neutralized  by  the 
union  of  two  homologous  portions  (Fig.  86).  In  the  "somatic  synapsis" 
of  salivary-gland  chromosomes  three  or  more  do  unite  closely.  These 
phenomena  remind  one  of  electrical  attractions,  the  agglutination  of 
bacteria,  the  formation  of  one-strain  groui^s  of  myxobacteria  at  the  time 


Fig.  80. — Trivalent 
chromosome  in  meiotic  pro- 
phase in  maize.  Each 
member  shows  its  two 
chromatids  in  regions  not 
in  synapsis.  The  kineto- 
chores  of  all  three  members 
lie  in  contact.  {From  a 
preparation  by  B.  Mc- 
Clintock.) 


116  FUNDAMENTALS  OF  CYTOLOGY 

of  fruiting  in  mixed  cultures,  and  especially  the  regular  pair-by-pair  luiion 
of  nuclei  of  two  strains  in  the  multinucleate  reproductive  organs  of  certain 
fungi.  Here  again  we  have  valuable  suggestions  but  no  complete 
explanation  of  the  important  feature  we  wish  to  understand. 

What  causes  diplotene  opening?  The  fact  that  the  synaptic  mates  in 
the  tetrad  tend  to  separate  soon  after  each  of  them  becomes  visibly  double 
suggests  a  causal  connection  between  these  two  events.  The  hypothesis 
that  single  threads  attract  while  double  ones  repel  one  another  has  been 
prominent  in  cytology  for  some  time.  At  present  it  appears  doubtful 
that  singleness  in  itself  is  a  decisive  factor  in  bringing  homologues  into 
proximity,  since  doubled  homologues  also  move  together  in  some  instances. 
Moreover,  it  is  visible  doubling  only,  and  probably  not  actual  doubling, 
that  immediately  precedes  the  opening  of  the  chromatids  at  diplotene. 
With  regard  to  repulsions,  it  seems  more  likely  that  they  are  an  important 
factor,  not  only  in  diplotene  opening,  but  also  in  the  spacing  of  the 
chromosomes  at  diakinesis  and  metaphase  and  in  the  widening  out  of 
associated  chromatids  at  anaphase  I. 

Does  coiling  have  a  role  in  meiosis?  In  meiosis,  as  in  somatic  nuclear 
cycles,  coiling  makes  it  possible  for  long  chromonemata  to  be  carried  in 
chromosomes  of  compact  form  during  their  segregation  into  daughter 
groups.  A  special  meiotic  role  of  factors  influencing  coiling  is  indicated 
by  the  complete  uncoiling  and  great  attenuation  of  the  chromonemata  in 
the  leptotene  stage.  The  hypothesis  has  been  advanced  that  it  is  just 
this  condition,  brought  on  by  a  stronger  or  more  prolonged  action  of 
certain  physiological  factors,  that  makes  complete  synapsis  possible,  the 
chromonemata  at  this  one  stage  in  the  life  cycle  being  in  a  condition 
permitting  forces  acting  over  short  distances  between  individual  pairs  of 
homologous  units  to  bring  two  long  series  of  such  units  into  close  asso- 
ciation. The  physical  causes  of  coiling  are  being  sought  in  order  to 
improve  our  understanding  of  the  chromosomal  changes  from  diplotene 
onward,  notably  shortening  and  chiasma  terminalization,  and  to  gain 
insight  into  the  molecular  architecture  and  growth  of  the  chromonemata. 

What  is  the  mechanism  of  crossing  over?  That  two  of  the  four 
chromatids  actually  do  exchange  corresponding  portions,  in  all  probability 
at  a  chiasma,  has  been  proved  with  heteromorphic  homologues,  i.e., 
homologous  chromosomes  differing  here  and  there  in  certain  visible  fea- 
tures which  make  it  possible  to  identify  particular  regions  of  the  chromo- 
somes before  and  after  the  exchange.  How  the  exchange  is  actually 
accomplished  is  still  an  unsolved  problem.  It  is  known  that  breakage  of 
chromonemata  may  be  induced  by  such  agencies  as  X  rays  and  that 
under  certain  circumstances  freshly  broken  ends  tend  to  unite  in  new 
patterns.  It  is  thei-efore  a  logical  assumption  that  something  of  the  kind 
may  occur  naturally  in  crossing  over,  the  precision  with  which  correspond- 


MEIOSIS 


11 


ing  portions  are  exchanged  being  a  consequence  of  their  orderly  arrange- 
ment in  synapsis.  On  the  other  hand,  theories  have  been  propounded  to 
account  for  crossing  over  without  actual  breakage.  The  idea  is  that  new- 
threads  are  developed  parallel  to  the  old  ones  immediately  after  the 
chromomeres  of  each  synaptic  mate  have  doubled  by  division  in  the 
pachytene  stage,  and  that  when  the  synaptic  mates  have  a  sharp  twist  at 
any  given  point  two  of  the  resulting  chromatids  may  contain  chromomeres 


;X: 


II  (I 
II  II 
II        ■  p 

p  I 

p  p 

p  I  p 


12  3  4  5 

Fig.  87. — Diagram  illustrating  theory  of  crossing  over  involving  formation  of  new 
strands  between  newly  formed  chromomeres.  1,  original  chromosomes.  2,  chromosomes 
with  half-twist.  3,  chromomeres  doubled.  4,  new  strands  developing;  between  the  third 
and  fourth  pairs  of  chromomeres  they  result  in  crossover  chromatids.  5,  the  tetrad  of 
chromatids  after  crossing  over.      {Based  on  theory  of  J.  Belling.) 

from  different  mates  on  either  side  of  this  point  because  the  new  chro- 
momeres form  the  new  unions  with  their  nearest  neighbors  (Fig.  87). 
Under  this  theory,  crossing  over  is  a  concomitant  of  chromosome  division. 
Finally,  there  is  the  problem  of  the  relation  of  meiosis  to  the  phenomena 
of  genetics.  So  far  as  major  features  such  as  synapsis,  disjunction, 
random  assortment,  and  crossing  over  are  concerned,  this  problem  has 
been  solved,  as  will  be  shown  in  the  later  chapters  of  the  book.  Numer- 
ous other  questions,  however,  remain  open,  and  it  is  hoped  that  these 
will  be  answered  by  fui-ther  refinements  in  our  knowledge  of  the  constitu- 
tion and  behavior  of  meiotic  chromosomes. 


CHAPTER  IX 
CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS 

One  of  the  major  cytological  crises  in  the  reproduction  of  organisms, 
viz.,  raeiosis,  has  just  been  described  in  some  detail.  Another  major 
crisis,  the  fusion  of  nuclei  in  the  union  of  gametes,  has  also  received  brief 
mention.  It  now  becomes  necessary  to  relate  these  processes  more  closely 
to  life  cycles  by  describing  the  various  other  events  that  precede,  accom- 
pany, and  succeed  them  in  the  reproductive  phases.  This  will  be  done 
in  three  chapters.  Again  we  should  be  reminded  that  such  events 
occur  in  numerous  variations  in  different  organisms  and  that  our  descrip- 
tions are  designed  to  include  examples  that  are  fairly  representative  of 
what  occurs  in  each  class  of  organism  considered,  even  though  no  single 
species  can  be  expected  to  show  all  the  stages  precisely  as  described.  We 
consider  first  the  animals. 

The  Germ  Cells. — The  term  germ  cells  is  applied  in  the  case  of  animals 
to  those  specialized  cells  whose  ultimate  descendants  are  to  be  female  or 
male  gametes  (eggs  or  spermatozoa)  and  often  certain  accessory  cells,  but 
not  somatic  cells.  When  the  specialization  first  becomes  recognizable, 
there  may  be  but  one  'primordial  germ  cell,  or  several,  or  a  considerable 
number  of  them.  It  is  a  striking  fact  that  the  differentiation  of  these  cells 
from  the  somatic  cells  occurs  very  early  in  the  ontogeny  of  the  organism. 
They  can  be  distinguished  during  larval  stages,  and  in  some  animals  it  has 
been  determined  that  they  are  set  apart  from  the  somatic  cells  in  one  of 
the  earliest  cleavage  divisions  of  the  fertilized  egg.  In  extreme  cases, 
notably  certain  insects,  the  cytoplasm  of  the  future  germ  cells  can  even  be 
distinguished  in  one  end  of  the  yet  undivided  egg  (Fig.  88).  In  the  case 
illustrated  one  of  the  eight  nuclei  formed  by  the  third  embryonal  mitosis 
enters  this  specialized  cytoplasm,  which  is  then  cut  off  as  the  primordial 
germ  cell  from  the  larger  somatic  portion  of  the  young  embryo. 

The  primordial  germ  cells,  whatever  their  number  and  time  of  origin 
from  embryonic  tissues,  commonly  pass  through  a  period  of  multiplica- 
tion. After  their  divisions  cease,  they  migrate  to  the  site  of  the  future 
ovaries  or  testes.  These  organs  then  develop  and  incorporate  within 
them  the  sperm  cells,  which  undergo  a  new  series  of  divisions.  In  the 
ovary  these  multiplying  cells  are  called  oogonia;  in  the  testis  they  are 
called  spermatogonia.  Hermaphroditic  animals  may  have  separate 
ovaries  and  testes,  or  both  oogonia  and  spermatogonia  may  be  present  in 

118 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS 


1J9 


one  organ.  Accompan\4ng  the  germ  cells  are  certain  accessory  cells,  such 
as  the  nurse  cells  in  the  insect  ovar,y  and  the  Sertoli  cells  in  the  mammalian 
testis.  In  some  cases  such  special  cells  represent  transformed  germ  cells, 
while  in  others  they  are  derived  from  other  tissues.  Up  to  this  stage  the 
nuclear  divisions  in  all  the  cells — somatic,  germ,  and  accessory — are  of 
the  equational  type,  every  nucleus  having  a  diploid  chromosome  com- 
plement like  that  of  the  zygote  nucleus  from  which  they  have  all  descended. 


Fig.  88. — Oogenesis  and  early  embryogeny  in  a  fly  (Aliastor).  1,  section  through  ovary. 
At  the  top  is  a  nurse  chamber  with  several  nuclei;  with  it  is  associated  a  young  oocyte. 
Earlier  stages  in  the  development  of  this  condition  are  seen  below.  2,  oocyte  nearly  full 
grown;  note  "pole  plasm "  at  its  lower  end.  3,  third  embryonal  mitosis  following  syngamy, 
showing  three  of  the  four  division  figures  (the  small  figure  is  a  dividing  polar  body  nucleus) . 
The  pole  plasm  is  being  provided  with  a  nucleus.  4,  pole  plasm  with  its  nucleus  cut  off  as 
the  primordial  germ  cell;  nuclei  above  continuing  to  divide  without  cytokinesis.  5, 
primordial  germ -cell  has  divided  into  eight  oogonia  (four  shown);  main  portion  of  embryo 
undergoing  superficial  cleavage  into  cells.  {From  R.  Hegner:  The  Germ-cell  Cycle  in 
Animals,  The  Macmillan  Com-pany.) 

Spermatogenesis. — After  the  spermatogonia  in  the  testis  have  ceased 
multiplying,  there  is  initiated  a  series  of  changes  peculiar  to  this  stage  of 
the  life  cycle.  The  spermatogonia  commonly  enlarge  somewhat  and 
are  then  termed  primary  spermatocytes.  Each  of  them  then  undergoes 
two  successive  divisions  which  are  mciotic  in  character :  each  spermatocyte 
divides  into  two  secondary  spermatocytes,  and  these  immediately  divide 
into  four  spermatids.  The  chromosomes  in  these  divisions  behave  accord- 
ing to  the  scheme  described  in  the  previous  chapter,  the  nuclei  in  the 
quartet  of  spermatids  each  having  one  genome,  or  monoploid  complement, 
in  place  of  the  diploid  complement  present  in  the  spermatogonia  and  the 
somatic  tissues. 

In  the  two  spermatocyte  divisions  the  various  cytoplasmic  inclusions, 
in  particular  the  Golgi  bodies  and  chondriosomes,  are  usually  distributed 
rather  equally  to  the  four  spermatids.  This  results  from  a  tendency'  of 
such  inclusions  to  be  grouped  near  the  equator  of  the  cell  or  about 


120 


FUNDAMENTALS  OF  CYTOLOGY 


the  poles  of  the  mitotic  figure,  so  that  cytokinesis  separates  them  into 
more  or  less  equal  groups  (Fig.  28).  Only  in  a  few  known  cases  does  this 
involve  an  actual  division  of  individual  chondriosomal  bodies.  In  some 
spermatocytes  the  Golgi  bodies  form  a  conspicuous  mass,  the  idiosome, 
about  the  centrioles,  and  this  breaks  up  into  smaller  portions  during  the 
divisions.  The  centrioles  that  function  in  the  second  meiotic  division 
have  usually  become  doubled  by  the  time  the  division  is  completed.  As  a 
result  of  these  events  each  spermatid  consists  of  cytoplasm,  a  nucleus  with 
the  gametic  chromosome  number,  chondriosomes,  one  or  more  Golgi 
bodies,  a  pair  of  centrioles,  and  frequently  other  inclusions. 


Fig.  89. — Stages  in  the  transformation  of  the  animal  spermatid  into  a  spermatozoon. 
1,  spermatid  of  an  insect  (Brochyviena).  {Redrawn  from  R.  H.  Bowen.)  2-5,  spermatids 
of  the  guinea  pig;  ga,  Golgi  apparatus;  gr,  Golgi  remnant;  a,  acrosome;  n,  nucleus;  m, 
mitochondria  (in  Brochymena  they  form  a  nebenkern);  c',  c^,  centrioles;  png,  postnucloar 
granules;  pnc,  postnuclear  cap;  mp,  middle  piece.  {From  J.  B.  Gatenby  and  H.  W.  Beams, 
based  on  studies  by  Gatenby,  Vejdovsky,  and  Meves.) 

The  transformation  of  the  spermatid  into  a  spermatozoon,  or  sperm,  is 
known  as  sperniiogenesis.  This  process  involves  a  very  remarkable  series 
of  changes  which  have  been  found  to  occur,  with  minor  variations,  in 
animals  of  many  types.  Each  of  the  spermatid  components  behaves  in  a 
characteristic  manner  (Fig.  89).  From  one  of  the  two  centrioles  a  slender 
filament  grows  out  and  pierces  the  cell  membrane;  this  is  the  axial  fila- 
ment of  the  future  tail  of  the  spermatozoon.  As  the  filament  continues  to 
grow,  the  centrioles  become  modified  and  connected  Anth  the  nucleus  in 
various  ways. 

The  chondriosomes  in  mammalian  spermatids  become  grouped  in  a 
region  near  the  posterior  pole  of  the  nucleus,  where  they  form  a  compact 
sheath  about  the  proximal  portion  of  the  axial  filament.  In  insect 
spermatids  the  chondriosomes  usually  unite  into  a  single  large  mass,  the 
nebenkern,  which  soon  divides  and  elongates  as  two  long  strands  wound 
about  the  axial  filament  in  the  lengthening  spermatid. 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS 


121 


^"' 


/-- 


The  Golgi  material  commonly  forms  a  single  large  body,  the  acroblast, 
situated  near  the  posterior  pole  of  the  nucleus. 
Most  often  the  acroblast  appears  like  a  heavily 
staining  cup  holding  a  less  stainable  material. 
Soon  it  begins  to  move  along  the  nucleus  to  the 
anterior  pole  of  the  cell  and  then  continues  its 
migration  back  along  the  other  side,  following  a 
path  previously  taken  by  the  centrioles.  At 
some  stage  of  this  migration  a  small  droplet  of 
matter  appears  to  exude  from  the  acroblast. 
The  droplet  enlarges  and  organizes  as  the 
acrosome,  a  specialized  structure  at  the  tip  of  the 
spermatozoon,  and  a  portion  of  it  often  differ- 
entiates as  a  pointed  'perforatorium.  What  re- 
mains of  the  acroblast,  now  known  as  the  Golgi 
remnant,  moves  backward  along  the  developing 
tail  and  is  eventually  lost  from  the  cell  in  a  mass 
of  protoplasm  sloughed  off  at  the  close  of  spermio- 
genesis.  In  species  having  no  acroblast  the 
acrosomal  material  appears  to  come  from  numer- 
ous small  Golgi  bodies. 

Conspicuous  modifications  of  the  spermatid 
nucleus  ordinarily  appear  after  most  of  the 
changes  described  above  have  been  carried  out. 
Its  chromatic  matter  gradually  becomes  con- 
centrated, often  against  the  nuclear  membrane 
in  the  anterior  region,  while  its  achromatic  com- 
ponent decreases  in  amount.  Eventuall}^  the 
nucleus  becomes  a  dense  and  apparently  homo- 
geneous body  closely  united  anteriorly  with  the 
acrosome  and  posteriorly  with  one  or  both  of  the 
centrioles,  from  which  the  axial  filament  extends. 
The  cytoplasm  lengthens  out  along  with  the 
axial  filament  and  chondriosomal  filament 
sheaths. 

The  mature  spermatozoon,  or  male  gamete, 
consists  typically  of  two  main  parts;  the  head, 
comprising  the  nucleus,  the  acrosome,  a  surface 
membrane,  probably  cytoplasmic  in  origin,  and 
occasionally  other  elements;  and  the  tail,  which 
is  made  up  of  the  axial  filament,  the  filament 
sheaths,  and  a  small  amount  of  residual  cyto- 
plasm.    In    many    cases,    notably    mammalian 


Fig.  90. — Diagram  of 
typical  mammalian  sper- 
matozoon, a,  acrosome;  n, 
nucleus;  c,  c',  centrioles  and 
their  derivatives;  w,  cell 
membrane;  d,  chondrioso- 
mal matter;  /,  axial  fila- 
ment of  tail;  TO,  middle 
piece;  p,  principal  piece  of 
tail;  c,  end  piece  of  tail. 
{After  R.  H.  Bowen.) 


122 


FUNDAMENTALS  OF  CYTOLOGY 


sperms,  the  basal  portion  of  the  tail  is  more  or  less  distinctly  differenti- 
ated as  a  middle  piece  containing  the  centrioles  and  chondriosomal  ele- 
ments in  a  sheath  of  undifferentiated  cytoplasm  (Fig.  90) .  Posterior  to 
this  is  a  principal  piece,  with  a  thin  sheath  but  no  undifferentiated 
cytoplasm,  and  an  end  piece,  which  represents  the  naked  end  of  the  axial 
filament. 

The  spermatozoa  of  many  animals  reveal  in  their  external  form  little 
or  no  evidence  of  the  above  differentiations,  but  taper  so  gradually  toward 
one  or  both  ends  that  no  subdivision  into  parts  is  possible  on  such  a  basis. 
Furthermore,  some  animals,  e.g.,  certain  crustaceans  and  spiders,  have 
spermatozoa  with  no  tails.  Except  for  the  lack  of  development  of  a  motor 
apparatus,  the  changes  within  the  spermatid  in  such  instances  are  funda- 
mentally similar  to  those  in  spermiogenesis  of  the  ordinary  type. 


Fig.  91. — Maturing  egg  of  a  worm   (Cerebratulus) .      1,  anaphase  of  first  meiotic 
with  first  polar  body  budding  off.     2,  polar  bodies  completed;  egg  nucleus  near 


division 
center. 


Oogenesis. — In  the  ovarj^  the  oogonia  enlarge  somewhat  and  become 
primary  oocytes.  It  is  in  these  cells  that  meiosis  is  initiated.  Enlarge- 
ment continues,  especially  during  the  remarkable  "growth  period"  which 
comes  at  about  the  time  the  chromosomes  are  in  the  late  pachytene  and 
the  diplotene  stages  of  the  first  meiotic  prophase  (page  111).  During  the 
growth  period  the  oocyte  becomes  supplied  with  nutritive  materials 
(yolk)  and  develops  other  features  characterizing  the  egg.  In  some 
animals,  groups  of  nurse  cells  or  a  surrounding  layer  of  cells,  the  follicular 
epithelium,  have  a  part  in  these  activities. 

At  the  close  of  the  growth  period  the  chromosomes  assume  the  compact 
form  characteristic  of  diakinesis  and  lie  scattered  in  the  enormous  oocyte 
nucleus  (the  germinal  vesicle) .  The  relatively  small  first-division  spindle 
assumes  a  position  perpendicular  to  the  cell  membrane,  and  at  anaphase 
and  telophase  the  first  polocyte,  or  polar  body,  is  budded  off  with  one  of 
the  daughter  nuclei  (Figs.  91,  85).     The  nucleus  remaining  in  the  second- 


CYTOLOGY  OF  REPRODICTION  IN  ANIMAL:S  123 

ary  oocyte  undergoes  the  second  meiotic  division,  usually  at  once,  forming 
a  second  polocyte  with  one  of  the  daughter  nuclei.  The  other  daughtei- 
nucleus  of  this  division  remains  in  the  now  mature  ovum,  or  egg.  The  first 
polocyte  may  or  may  not  divide  to  complete  the  quartet  of  cells  expected 
after  meiosis.  At  the  close  of  these  meiotic  divisions,  or  maturation 
divisions,  as  they  are  frequently  called,  the  nucleus  of  the  ovum  has  the 
gametic  number  of  chromosomes. 

The  mature  ovum,  or  female  gamete,  is  bounded  by  a  delicate  vitelline 
membrane  and  sometimes  by  additional  jelly-like  layers.  In  some  ani- 
mals a  thick  layer  of  nutritive  albumen  is  deposited  about  the  ovimi,  and 
around  this  may  later  be  added  further  structures,  such  as  the  shell 
membrane  and  calcareous  shell  of  the  bird's  egg.  The  egg  of  a  bird  at  the 
time  it  is  laid  is  therefore  more  than  an  egg.  The  ovum  proper  has  been 
enormously  distended  by  great  amounts  of  yolk  material,  its  surface  mem- 
brane lying  at  the  outer  boundary  of  this  yolk.  Most  of  its  protoplasm 
has  taken  the  form  of  a  flattened  yolk-free  mass,  the  g^erminal  disc,  lying 
just  beneath  the  membrane  at  one  side  (this  side  lies  uppermost  in  an  egg 
at  rest  in  an  incubator).  It  is  surrounded  by  "white"  and  a  shell  which 
form  no  part  of  the  egg  proper.  If  the  egg  is  a  fertile  one,  fertilization  was 
accomplished  before  these  modifications  and  additions  appeared,  and  the 
development  of  the  resulting  embryo  has  already  advanced  to  the  blasto- 
derm stage  (page  128).  This  development  is  resumed  when  the  egg  is 
incubated. 

Syngamy. — The  term  syngamy  denotes  the  sexual  union  of  two 
gametes,  regardless  of  their  relative  structure  and  behavior  and  the  nature 
of  the  consequences.  When  one  gamete  is  large  and  apparently  passive 
while  the  other  is  small  and  active,  the  process  is  referred  to  as  the  fertili- 
zation of  one  gamete,  the  egg,  by  the  other,  the  sperm,  since  the  arrested 
development  of  the  egg  is  thereupon  resumed.  The  induction  of  develop- 
ment by  experimental  agencies  is  accordingly  called  artificial  fertilization, 
or  artificial  parth(;nogenesis.  Although  syngamy  and  fertilization  are 
often  used  as  interchangeable  terms,  it  should  always  be  remembered  that 
normal  gametic  union  in  any  case  is  a  mutual  reaction  and  that  it  is  the 
fusion  product  that  proceeds  wdth  development. 

Syngamy  in  most  animals  includes  typically  the  entrance  of  the  sperm 
into  the  egg,  a  structural  transformation  of  the  sperm  often  accompanied 
by  related  structural  changes  in  the  egg,  a  union  of  the  gametic  nuclei, 
and  certain  physiological  alterations  in  the  egg,  some  of  which  are 
initiated  even  before  the  sperm's  entrance  has  been  completed.  In  some 
animals  the  rhythmic  movements  of  the  tail  which  bring  the  sperm  to  the 
egg  continue  after  the  two  gametes  have  come  into  contact  and  suggest 
a  boring  action  instrumental  in  gametic  union.  In  other  cases,  however, 
such  movements  cease  as  soon  as  the  sperm  reaches  the  egg  membrane,  or 


124 


FUNDAMENTALS  OF  CYTOLOGY 


even  while  it  is  still  separated  from  the  membrane  bj^  a  layer  of  jelly 
surrounding  the  egg;  nevertheless  the  sperm  enters,  as  though  it  were 
being  acted  upon  by  some  force  resident  in  the  egg. 

In  a  marine  annelid  worm  (Nereis)  it  has  been  shown  how  the  egg 
behaves  during  the  entrance  of  the  sperm.  If  the  sperm  reaches  the  egg  in 
the  brief  period  during  which  the  egg  is  fertilizable,  the  union  of  the  two 
proceeds  as  follows  (Fig.  92).  All  but  one  of  the  many  sperms  which 
may  have  attached  themselves  to  the  egg  are  usually  carried  away  from 
its  surface  by  a  jelly  which  flows  out  from  an  alveolar  zone  just  beneath 


Fig.  92.^-Entrance  of  spermatozoon  into  egg  of  N'ereis.  Only  the  sperm  head  is  drawn 
in  by  the  fertilization  cone;  the  middle  piece  and  tail  remain  outside.  (From  F.  R.  Lillie: 
Problems  of  Fertilization,  University  of  Chicago  Press.) 

the  membrane.  From  the  inner  region  of  the  egg  a  transparent /pr^///2a- 
tion  cone  then  extends  across  this  zone  and  touches  the  membrane  at  the 
point  where  the  single  remaining  sperm  is  about  to  penetrate  it.  The 
perforatorium  pierces  the  membrane  and  becomes  attached  to  the  cone 
which  is  then  withdrawn  toward  the  inner  region  of  the  egg,  carrying  the 
head  of  the  sperm  with.  it.  The  sperm's  middle  piece  and  tail  are  left 
outside  the  egg.  In  some  sea  urchins  both  head  and  middle  piece  enter, 
while  in  most  animals  the  whole  sperm  passes  in. 

In  different  animals  there  is  considerable  variation  in  the  stage  of  the 
egg's  maturation  at  the  time  when  the  sperm  enters.  In  sea  urchins  and 
starj&sh  both  meiotic  divisions  have  been  completed,  the  sperm  thus 
entering  a  fully  matured  egg.  The  frog's  egg  is  entered  during  the  meta- 
phase  of  the  second  meiotic  division.  Entrance  occurs  during  the  first 
metaphase  in  certain  annelids,  insects,  and  mollusks,  and  even  slightly 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS 


125 


earlier  in  the  threadworm,  Ascaris  (Fig.  85).  In  these  latter  cases,  there- 
fore, the  cell  entered  is  an  oocyte  in  terms  of  nuclear  condition  but  a 
female  gamete  in  terms  of  power  to  undergo  impregnation.  It  will  be 
pointed  out  shortly  that  the  union  of  the  gametes  may  in  turn  be  followed 
so  quickly  by  the  first  embryonal  division  that  the  latter  process  is  begun 
before  the  former  is  completed.  This  overlapping  of  the  events  com- 
prising the  general  processes  of  meiosis,  syngamy,  and  embryogeny  com- 
plicates the  cytological  study  of  reproduction  in  many  animals. 

One  of  the  first  visible  effects  of  syngamy  in  many  eggs  is  an  elevation 
of  the  vitelline  membrane  which  begins  at  the  point  of  sperm  entrance 
and  extends  rapidl}^  over  the  egg,  forming  the  so-called  fertilization 
membrane.     This  change  and  certain  further  alterations  in  the  egg  have 


Fig.  93. — Diagram  of  syngamy  and  cleavage  in  an  animal  that  completes  meiosis  before  the 
entrance  of  the  spermatozoon. 

been  found  to  occur  in  Nereis  even  when  the  sperm  is  removed  from  the 
egg  immediately  after  it  begins  to  penetrate.  In  some  animals  the 
perivitelline  space  beneath  the  raised  membrane  is  A\dde  enough  to  allow 
the  rotation  of  the  main  body  of  the  egg  within  it.  After  the  fertilization 
membrane  has  been  formed,  no  more  sperms  enter.  This  is  not  due 
simply  to  the  presence  of  a  mechanical  barrier,  but  also  to  a  new  physio- 
logical state  in  the  egg  cytoplasm,  for  sperms  will  not  enter  membraneless 
fragments  of  eggs  in  this  stage  of  development. 

The  behavior  of  the  sperm  and  egg  nuclei  (the  pronuclei)  during 
syngamy  is  illustrated  in  Fig.  93.  Soon  after  entering  the  egg,  the  sperm 
nucleus  commonly  begins  to  enlarge  and  reveal  a  structure  more  like 
that  of  an  ordinary  nucleus.  Meanwhile  the  pronuclei  approach  each 
other  and  meet.  By  this  time  the  sperm  nucleus  has  often,  though  not 
always,  assumed  a  size  and  structui-e  aljout  like  that  of  the  egg  nucleus. 
The  two  now  proceed  to  fuse.  As  they  do  so  both  may  have  a  metabolic 
type  of  structure,  the  resulting  diploid  nucleus  later  entering  prophase^ 


126  FUNDAMENTALS  OF  CYTOLOGY 

and  carrying  out  the  first  embryonal  mitosis.  In  other  cases  both  pro- 
nuclei have  passed  independently  through  most  or  all  of  the  prophasic 
stages  before  meeting;  hence,  when  their  membranes  disappear  their 
two  genomes  lie  together  in  the  achromatic  figure  of  the  first  embryonal 
mitosis,  and  they  first  become  enclosed  in  a  common  nuclear  membrane 
at  the  telophase  of  this  mitosis.  This  more  or  less  independent  behavior 
of  the  genomes  w^th  no  intimate  nuclear  union  before  the  first  embryonal 
mitosis  is  known  as  gonomery.  In  some  organisms  the  two  genomes 
continue  to  act  as  two  visibly  distinct  groups  of  chromosomes  through 
several  embryonal  mitoses. 

This  close  association  of  syngamy  with  the  first  embryonal  mitosis  is 
emphasized  further  by  the  behavior  of  the  centrioles.  The  origin  of 
the  centrioles  seen  at  the  poles  of  the  achromatic  figure  in  the  first 
embryonal  mitosis  is  not  altogether  clear,  but  the  characteristic  appear- 
ance of  an  aster  with  a  centriole  in  the  cytoplasm  near  the  base  of  the 
sperm  head  is  significant.  Either  the  centriole  is  that  known  to  have 
been  incorporated  in  the  sperm  during  spermiogenesis,  or  the  sperm 
in  some  way  induces  the  formation  of  an  aster  and  centriole  by  the  egg 
cytoplasm.  It  has  been  shown  that  by  treating  echinoderm  eggs  with 
certain  chlorides  the  formation  of  numerous  asters  can  be  induced  in  the 
cytoplasm  and  that  two  of  these,  especially  if  they  originate  near  the 
nucleus,  can  function  in  mitosis.  Whatever  the  origin  of  the  aster  in 
normal  syngamy,  it  divides  to  two  which  occupy  the  poles  of  the  first 
embryonal  mitotic  figure.  The  entrance  of  the  sperm  is  not  merely  a 
necessary  preliminary  to  syngamic  nuclear  union;  it  also  affects  the 
processes  leading  to  the  division  of  the  cell. 

Syngamy  has  two  effects  of  cardinal  importance.  The  first  of  these  is 
activation,  by  which  certain  physiological  processes  are  set  in  motion  or 
greatly  accelerated.  In  most  cases  this  leads  to  the  immediate  develop- 
ment of  the  cell  into  a  new  individual,  but  in  some  animals  and  plants 
the  cell  develops  certain  protective  coats  and  enters  a  dormant  state 
from  which  it  emerges  later  under  the  appropriate  environmental  condi- 
tions. In  cither  event  there  is  a  profound  physiological  change  at  the 
time  of  syngamy.  This  is  not  dependent  upon  the  union  of  the  gametic 
nuclei,  for  not  only  are  some  of  the  results  of  the  change  manifested  long 
before  the  union  occurs,  but  complete  activation  maj^  be  induced  by 
various  physical  and  chemical  treatments  in  the  absence  of  sperms. 
Furthermore,  some  animal  eggs  are  naturally  parthenogenetic,  undergoing 
complete  development  without  syngamy  regularly  in  successive  life 
cycles.  The  egg  has  accordingly  been  termed  an  inde'pendently  activahle 
system  which  contains  everything  necessary  to  development,  even 
though  a  stimulus  of  one  kind  or  another  is  ordinarily  required  to  initiate 
its  fiu'ther  activity. 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS  127 

The  second  important  effect  of  syngamy  is  diplosis,  the  doubling  of 
the  number  of  chromosomes  by  the  union  of  the  two  gametic  nuclei. 
In  syngamy  two  genomes  with  the  monoyloid  chromosome  iiumher  are 
combined  into  a  diploid  chromosome  complement,  each  kind  of  chromosome 
then  being  present  in  duplicate.  Every  chromosome  of  this  complement 
divides  equationally  at  every  somatic  mitosis  in  the  development  of  the  result- 
ing new  individual,  so  that  every  nucleus  in  this  individual  contains  a 
descendant  of  every  chromosome  originally  present  in  the  zygote.  The 
peculiar  significance  of  these  facts  with  respect  to  hereditj^  is  evident 
when  it  is  borne  in  mind  that  the  two  genomes  usually  come  from  two 
parent  individuals,  that  they  may  exert  somewhat  different  influences 
upon  the  characters  developed,  and  that  they  are  to  be  reshuffled  by 
meiosis  to  form  new  genomes  before  the  next  sexual  generation  is 
produced. 

Cleavage. — The  rapid  succession  of  meiosis,  syngamy,  and  cleavage 
in  many  animals  has  required  frequent  mention  of  cleavage  in  the  fore- 
going descriptions.  This  process,  also  called  segmentation ,  is  one  of  much 
cytological  interest  and  a  few  features  of  its  early  stages  will  be  sketched 
briefly.  The  subsequent  course  of  embryogeny  lies  beyond  the  scope  of 
this  book.  It  is  the  early  cleavage  divisions  that  furnish  such  excellent 
material  for  studies  on  mitosis  and  especially  of  cytokinesis  in  animals 
(page  69). 

The  animal  egg  commonly  shows  a  polarity  of  such  a  nature  that  one 
region,  the  "animal  pole,"  is  physiologically  more  active  than  the 
diametrically  opposite  region,  the  "vegetal  pole."  Also,  eggs  of  different 
animals  differ  greatly  in  the  amount  and  location  of  their  yolk  material. 
These  features,  to  mention  only  two,  exert  a  strong  influence  upon  the 
determination  of  the  various  cleavage  patterns  encountered  in  different 
classes  of  animals. 

The  geometrically  most  regular  cleavage  pattern  is  found  in  eggs 
having  their  yolk  uniformly  distributed  throughout  the  cell  (homolecithal 
eggs).  Among  echinoderms,  for  example  (Fig.  94,  A),  the  first  cleavage 
division  is  meridional  (through  the  two  poles),  the  second  meridional 
at  right  angles  to  the  first,  the  third  equatorial,  and  the  several  following 
divisions  in  such  planes  as  to  result  in  a  spherical  mass  of  cells  (blastomeres) 
of  uniform  size.  As  development  proceeds  this  sphere  becomes  a  hollow 
hlastula,  and  this  in  turn  is  converted  into  a  gastrula  by  an  invagination 
which  begins  at  the  vegetal  pole. 

The  egg  of  the  frog  is  somewhat  telolecithal,  i.e.,  its  yolk  tends  to  be 
denser  in  the  region  of  the  vegetal  pole  than  near  the  animal  pole.  The 
first  and  second  cleavage  divisions  occur  as  in  the  homolecithal  egg,  but 
the  third  division  is  unequal,  giving  four  small  cells  (micromeres)  at  the 
animal  pole  and  four  larger  ones  (macromeres)  at  the  vegetal  pole  (Fig. 


128 


FUNDAMENTALS  OF  CYTOLOGY 


94,  B).  From  this  stage  onward  the  macromeres  divide  at  a  slower  rate 
than  the  micromeres.  Invagination  to  form  the  gastrula  here  begins  at 
the  side  of  the  blastula  where  the  regions  of  large  and  small  cells  meet. 

The  eggs  of  birds,  reptiles,  squids,  and  bony  fish  are  very  strongly 
telolecithal,  the  yolk  being  very  densely  packed  throughout  most  of  the 
cell  but  absent  from  the  small  germinal  disc  at  the  animal  pole.  The 
cleavage  divisions  are  restricted  to  this  relatively  thin  layer  of  protoplasm 
and  do  not  extend  through  the  bulk  of  the  cell  (Fig.  94,  C).  After  a  few 
divisions  have  occurred,  the  3^oung  embryo  has  the  form  of  a  plate  of  cells, 
the  blastoderm,  lying  against  a  large  yolk  mass,  the  distinctness  of  the 


/0' 

■•■©^ 

':°W'^^:%. 

Fig.  94. — Three  types  of  cleavage  in  animal  egg.s.     Sections  of  eggs  in  early  cleavage  stages 
above;  surface  views  of  later  stages  below.     Explanation  in  text. 


boundary  between  the  two  regions  showing  some  variation.  This  is 
called  meroblastic  cleavage,  in  contrast  to  the  holoblastic  tj^je  which 
extends  through  the  whole  egg. 

A  further  type  of  cleavage  is  observed  among  insects  (Fig.  88).  The 
first  few  mitoses  in  the  fertilized  egg  are  not  accompanied  by  cytokinesis, 
the  embrj'o  being  coenocytic  during  its  earliest  stages.  Soon  after  the 
primordial  germ  cell  has  been  set  apart,  the  multiplying  nuclei  in  the 
somatic  portion  of  the  embryo  move  to  the  periphery  along  with  small 
masses  of  cytoplasm,  leaving  the  central  region  holding  most  of  the  yolk 
(the  centrolecithal  condition).  Cytokinesis  occurs  between  the  periph- 
eral nuclei,  but  the  cells  so  formed  remain  open  on  the  side  toward  the 
yolk.  Further  divisions  of  these  cells  produce  the  ventral  plate  from 
which  most  of  the  embryo  arises. 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS  129 

In  the  foregoing  examples  it  is  evident  that  there  is  some  correlation 
between  the  type  of  cleavage  and  such  visible  features  as  the  location  of 
3^olk.  The  correlation  is  so  far  from  complete,  however,  that  this  featun; 
cannot  be  regarded  as  more  than  a  contributing  cause  of  cleavage  pat- 
terns. Exhaustive  studies  have  shown  that  the  positions  assumed  by  the 
cleavage  spindles  and  hence  of  the  resulting  partitions  are  determined 
mainly  by  some  fundamental  protoplasmic  organization  which  it  is  not 
yet  possible  to  describe. 

A  most  important  aspect  of  cleavage  is  the  relation  it  bears  to  the 
internal  differentiation  of  the  embryo.  It  is  evident  that  differentiation 
has  proceeded  much  further  in  some  animals  than  in  others  at  a  given 
stage  of  cleavage.     It  has  long  been  known  that  in  a  coelenterate  (Cly(ia) 


Fig.  95. — Embryo  of  rabbit  in  eight-cell  stage;  five  of  the  cells  (blastomeres)  visible  iii  the 
.section.      {Courtesy  of  General  Biological  Supjily  House,  I  tic.,  Chicago.) 

one  of  the  first  16  blastomeres  may  produce  a  complete  embryo,  whereas 
in  a  ctenophore  (Beroe)  it  has  been  observed  that  the  larva  is  incomplete 
if  a  portion  of  the  egg's  protoplasm  has  been  removed  even  before  the 
first  cleavage  division.  In  cases  like  the  latter  the  egg,  before  its  cleavage 
or  even  before  its  fertilization,  may  have  a  definite  promorphology,  i.e., 
it  has  developed  an  internal  organization  which  in  some  manner  fore- 
shadows the  morphology  of  the  young  embryo.  Hence  to  the  three 
processes  which  sometimes  follow  each  other  so  closely-  as  to  overlap — 
meiosis,  syngamy,  and  cleavage — we  may  now  add  a  fourth,  embryonic 
differentiation. 

In  some  eggs  an  internal  differentiation  can  be  detected  in  the  pattern 
assumed  by  certain  visible  substances.  This  pattern  may  be  cut  up  in 
various  ways  by  the  successive  cleavage  furrows,  the  ability  of  any 
isolated  blastomere  to  produce  a  whole  embryo  or  only  a  part  of  one 
being  in  some  measure  dependent  upon  the  elements  of  the  pattern  it 


130  FUNDAMENTALS  OF  CYTOLOGY 

includes.  The  inference  that  cleavage  accompanies  internal  differentia- 
tion but  does  not  produce  it  is  borne  out  by  the  striking  fact  that  the 
larva  of  an  annelid  worm  has  been  seen  to  develop  its  characteristic  form 
and  structure  to  a  certain  stage  even  when  the  cleavage  divisions  were 
suppressed  altogether  by  treatment  with  KCl.  Further  discussion  of 
these  phenomena  would  lead  us  into  the  fascinating  but  extremely  difficult 
field  of  developmental  mechanics. 

Parthenogenesis. — The  development  of  an  egg  into  an  embryo 
without  syngamy  is  called  'parthenogenesis.  An  individual  so  derived  is  a 
parthenote.  This  mode  of  reproduction  occurs  frequently  in  lower 
animals,  notably  insects,  lower  crustaceans,  and  rotifers.  In  some 
species,  parthenogenesis  is  the  only  mode  of  reproduction,  male  indi- 
viduals being  unknown,  while  in  others  a  series  of  parthenogenetic 
generations  is  succeeded  under  the  proper  environmental  conditions  by 
individuals  that  reproduce  sexually.  Eggs  of  several  other  groups, 
including  echinoderms,  mollusks,  amphibians,  and  even  mammals 
(rabbit),  in  which  parthenogenesis  does  not  normally  occur,  have  been 
made  to  undergo  parthenogenetic  development,  at  least  to  a  certain 
stage,  by  treating  them  variously  with  hypertonic  sea  water,  fatty  acids, 
alkaloids,  foreign  blood  serum,  and  a  number  of  other  agencies.  Only 
exceptionally  do  these  artificially  induced  parthenotes  reach  the  stage  of 
metamorphosis  or  of  sexual  maturity. 

Some  animal  parthenotes  have  the  gametic  chromosome  number, 
normal  meiosis  having  occurred  in  the  development  of  the  egg.  In  bees 
and  ants,  for  example,  such  eggs  are  capable  of  either  sexual  or  partheno- 
genetic development:  when  syngamy  occurs  the  result  is  a  diploid  female, 
whereas  parthenogenesis  leads  to  the  development  of  a  monoploid  male. 
In  the  majority  of  cases  the  parthenotes  are  diploid,  as  in  aphids  and 
rotifers.  The  eggs  developing  in  this  manner  arise  from  oocytes  in 
which  there  are  no  meiotic  divisions,  or  there  may  be  a  single  division 
which  is  equational  in  character.  These  diploid  eggs  are  usually  incap- 
able of  fertilization. 

There  is  every  reason  to  believe  that  the  parthenogenetic  mode  of 
development  in  these  organisms  has  been  derived  from  the  normal  sexual 
cycle,  involving  a  suppression  of  meiosis  in  the  diploid  type  and  an  adjust- 
ment of  development  to  a  different  nuclear  constitution  in  the  monoploid 
type.  The  suppression  of  meiosis  is  suggested  by  the  various  observed 
conditions  intermediate  between  meiosis  and  ameiosis  and  between 
facultative  and  obligatory  parthenogenesis,  and  also  by  the  fact  that  in 
certain  plants  parthenogenesis  occurs  only  after  a  failure  of  meiosis. 
That  the  ability  to  undergo  somatic  development  with  a  single  genome 
may  have  been  slowly  acquired  is  indicated  by  the  fact  that  monoploid 
parthenotes  occasionally  encountered  among  plant  and  animal  species 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS  131 

do  not  develop  so  well  as  the  diploid  zj-gotes.  Moreover,  when  the 
parthenogenetic  development  of  monoploid  animal  eggs  is  induced  by 
artificial  means,  the  few  individuals  that  are  successful  in  developing 
through  metamorphosis  have  almost  invariably  become  diploid.  In  the; 
mitoses  occurring  in  parthenogenetic  frog  embryos,  for  instance,  both  the 
monoploid  and  the  diploid  numbers  are  found,  indicating  a  gradual 
doubling  jjrocess.  Evidently  the  embryos  not  undergoing  this  change 
fail  to  survive.  Among  salamanders  obtained  from  fertilized  eggs 
subjected  to  low  temperatures  there  are  some  that  are  monoploid  at 
least  up  to  the  stage  at  which  ovaries  with  oocytes  are  developed. 

Development  in  the  monoploid  condition  is  therefore  a  possibility  in 
animals  as  it  is  in  plants,  provided  the  conditions  favoring  it  are  present. 
As  a  rule  monoploid  animals  derived  by  such  means  are  almost  if  not 
completely  sterile.  Among  natural  parthenotes,  however,  fertility  may 
obtain.  In  the  male  honey  bee,  for  example,  functional  sperms  are 
produced  after  a  single  spermatocyte  division  which  is  equational  in 
character.  All  these  facts  indicate  that  in  the  evolution  of  various  groups 
of  organisms  there  have  been  adjustments  of  the  reproductive  process 
to  a  considerable  range  of  variations  in  the  nuclear  cycle.  This  is  further 
evident  in  plants,  most  of  which  have  reproductive  cycles  differing 
widely  from  those  in  animals. 

Protozoa. — The  Protozoa  are  a  very  large  group  of  very  small  organ- 
isms. They  are  so  diversified  in  structure  and  type  of  life  cycle  that  long 
chapters  would  be  required  to  describe  their  cytology  fully;  hence  this 
discussion  must  be  restricted  to  a  few  representative  features  of  special 
interest. 

Protozoa  are  characteristically  unicellular,  i.e.,  the  body  has  the 
general  structure  of  a  typical  protoplast,  with  cji^oplasm,  membranes, 
and  one  nucleus.  Some  protozoologists  prefer  to  regard  them  as  non- 
cellular,  meaning  that  they  are  not  subdivided  into  compartments  but 
simply  have  the  structure  necessary  to  a  small  but  complete  mass  of 
protoplasm.  A  tendency  to  become  coenocj^tic  or  multicellular  does 
appear  here  and  there  in  these  animals,  but  evolution  within  the  group 
has  proceeded  mainly  along  other  lines.  It  is  customary  to  say  that 
Protozoa  are  small  because  they  are  unicellular,  but  as  we  compare 
their  frequently  elaborate  structure  and  behavior  ^^^th  what  is  seen  in 
Metazoa  something  may  be  gained  from  the  concept  that  they  arc 
unicellular  because  they  are  small.  Effective  differentiation  in  very  small 
masses  can  occur  without  a  multiplicity  of  nuclei  and  cells.  Cell  division 
serves  them  in  reproduction,  but  not  in  the  building  of  the  individual 
body. 

Nuclear  conditions  vary  widely  in  the  Protozoa.  Most  protozoans 
have  a  single  nucleus,  but  probably  in  all  groups  the  binucleate  and 


132 


FUNDAMENTALS  OF  CYTOLOGY 


multinucleate  conditions  can  be  found.  In  the  infusorian  cell  there  are 
commonly  nuclei  of  two  kinds:  one  or  more  micronuclei,  which  divide 
mitotically  and  are  concerned  chiefly  with  reproduction,  and  a  large 
macronucleus,  which  divides  by  constriction  and  is  concerned  in  the 
physiological  activities  of  the  cell,  including  the  mating  reaction  (Fig.  96). 
A  comjjonent  of  many  nuclei  in  Protozoa  and  flagellates  is  the  endosome, 
a  large,  compact  central  mass  which  in  different  cases  appears  to  contain 
nucleolar  matter,  chromatin,  or  both  of  these  substances.     At  the  time 


Fig.  96. — Fission  in  Paramecium  caudatum.  1,  individual  with  single  macroniicleus  and 
.single  uiicronucleus  above  it.  2,  the  macronucleus  is  elongating  and  the  inioronucleus  has 
divided,  constriction  beginning  at  middle  of  cell.  3,  the  macronucleus  has  divided,  and 
fission  is  nearly  completed.      (Courtesy  of  General  Biological  Supply  House,  Inc.,  Chicago.) 

of  nuclear  division  it  elongates  and  constricts  into  two  (Fig.  97).     Its 
function  is  not  understood. 

The  aspects  presented  by  protozoan  nuclei  at  the  time  of  division 
are  often  very  difficult  to  interpret.  The  nucleus  may  be  very  compact 
and  seem  to  undergo  a  simple  mass  division,  or  it  may  show  a  cloud  of 
small  chromatic  granules  instead  of  chromosomes  with  obvious  indi- 
viduality. In  many  cases,  however,  chromosomes  in  definite  numbers 
and  essentially  like  those  in  Metazoa  have  been  demonstrated,   ^\dth 


Fig.  97. — Mitosis  in  a  flagellate  (Heteronema) ,  showing  endosome 
chromosomes   is   longitudinal;   late   separation   at   one   end   makes 
(After  J.  B.  Loefer.) 


The  division  of  the 
it    appear   transverse. 


kinetochorcs,  spiral  chromonemata,  longitudinal  division,  the  association 
of  nucleoli  with  certain  members  of  the  complement,  meiosis,  and  even 
polyploidy.  To  what  extent  the  less  definite  types  of  structure  and 
behavior  should  be  regarded  as  primitive  or  as  special  modifications  of 
the  condition  generally  prevalent  in  organisms  nobody  can  say.  The 
achromatic  figure  also  presents  itself  in  many  forms,  with  and  without 
asters  and  centrioles.  Centrioles  in  some  groups  attain  an  astonishing 
size  and  form  (Fig.  99). 


CYTOLOGY  OF  REPRODUCTION  IN  ANIMALS 


133 


The  neuromotor  apparatus  is  an  unusually  interesting  specialization  in 
manj^  protozoan  and  flagellate  cells.  In  some  it  is  absent;  in  others  it 
consists  of  a  flagellum,  a  basal  granule  {blepharoplasi) ,  and  a  strand 
(rhizoplast)  connecting  the  latter  with  a  centrosome  at  the  nucleus 
(Fig.  98).  Sometimes  a  single  body  acts  as  both  centrosome  and  bleph- 
aroplast.  In  more  elaborate  forms  there  is  a  central  mass,  the  motoriuni, 
from  which  strands  run  out  to  the  several  swimming  oi-gans.  In  Ciliata 
there  are  also  numerous  ^'ery  fine  strands  (the  "siher-line  system")  run- 
ning beneath  the  rows  of  cilia  and  near  the  stinging  organs,  or  trichocysts. 

Digestion  in  the  more  highly  differentiated  Protozoa  may  occur  in  a 
special  tract  running  from  mouth  to  anus.  Special  contractile  and 
supporting  structures  also  occur  (Fig.  9).  Far  from  being  simple,  such 
protozoans  are  the  most  complicated  single  cells  known. 


Fig.  98.— 
A  flagellate 
[M enoidium)  . 
Explanation  in 
text.  [After  R. 
F.  Hall.) 


Fig.  99. — Mitosis  during  fission  in  a  protozoan  (Barbulanympha). 
Longitudinal  section  of  anterior  end  of  cell.  The  two  large  elongate 
centrioles  are  connected  anteriorly  with  the  two  flagellated  areas  and 
posteriorly  with  the  achromatic  figure.  Some  of  the  astral  rays  con- 
nect at  the  nuclear  membrane  with  intranuclear  chromosomal  fibers. 
(After  L.  R.  Cleveland.) 


The  asexual  reproduction  of  a  protozoan  by  fission  may  appear  to  be  a 
simple  process  externally,  but  in  the  more  highly  organized  species  it 
involves  a  very  complicated  series  of  changes.  Not  only  does  the 
nucleus  divide  mitotically,  but  the  components  of  the  motor  apparatus 
and  the  other  specialized  regions  are  also  doubled  variousl}^  bj-  division 
and  ne\^  formation.  A  prominent  part  in  the  process  is  usually  played 
bj'-  the  centrosomes  or  blepharoplasts  (Fig.  99). 

Sexual  reproduction  in  these  small  animals  involves  nuclear  divisions 
and  fusions  which  in  manj^  cases  have  not  been  fully  interpreted.  In 
some  genera,  however,  the  details  of  the  process  have  been  made  out 
sufficiently  well  to  show  that  in  certain  fundamentals  it  corresponds  to 
the  meiosis-syngamy  cycle  seen  in  Metazoa.  These  changes  may  occur 
entirely  within  one  individual  cell  (autogamy) .  Syngamy  involving  two 
individuals  may  include  the  complete  fusion  of  morphologically  similar 
or  dissimilar  gametes,  or  only  a  mutual  exchange  of  nuclei  between 
individuals  in  temporary  conjugation  (Fig.  100).  In  Paramecium  cauda- 
tum  the  latter  type  of  process  occurs  as  follows: 


134 


FUNDAMENTALS  OF  CYTOLOGY 


The  two  animals  become  united  by  their  ventral  sides  and  the  macronucleus 
of  each  begins  to  degenerate,  while  the  micronucleus  divides  twice  to  form  four 
spindle-shaped  bodies.  Three  of  these  degenerate,  forming  the  "corpuscles  de 
rebut,"  which  play  no  further  part.     The  fourth  divides  into  two,  one  of  which. 


Fk;.    100. — Diagram    illustrating   nuclear    behavior    during    conjugation   in   Paramecium. 
Explanation  in  text.      {From  T.  H.  Morgan,  after  G.  X.  Calkins.) 

the  "female  pronucleus,"  remains  in  the  body,  while  the  other,  or  "male  pro- 
nucleus," passes  into  the  other  animal  and  fuses  with  the  female  pronucleus. 
Each  animal  now  contains  a  cleavage-nucleus  equally  derived  from  both  the 
conjugating  animals,  and  the  latter  soon  separate.  The  cleavage-nucleus  in 
each  divides  three  times  successively,  and  of  the  eight  resulting  bodies  four 


CYTOLOGY  OF  REPliODLCTlON  IN  ANIMALS  135 

become  macronuclei  and  four  micronuclei.  By  two  succeeding  fissions  the  four 
macronuclei  are  then  distributed,  one  to  each  of  the  four  resulting  iiKhvichmls. 
In  some  other  species  the  micronuclei  are  equallj^  distributed  in  like  manner, 
but  in  P.  caudatum  the  process  is  moye  complicated,  since  three  of  them  degener- 
ate, and  the  fourth  divides  twice  to  produce  four  new  micronuclei.  In  either 
case  at  the  close  of  the  process  each  of  the  conjugating  individuals  has  given  rise 
to  four  descendants,  each  containing  a  macronucleus  and  a  micronucleus  derived 
from  the  cleavage-nucleus.  From  this  time  forward  fission  follows  fission  in  the 
usual  manner,  both  nuclei  dividing  at  each  fission,  until,  after  many  generations, 
conjugation  recurs.     (Wilson.) 

In  these  Infusoria  there  is  also  another  process,  endomixis,  in  which 
the  nuclear  reorganization  closely  parallels  that  in  conjugation:  it  resem- 
bles autogamj^  in  occurring  entirely  within  one  individual,  but  it  differs 
from  both  autogamj^  and  conjugation  in  involving  no  nuclear  fusion.  If 
the  first  two  divisions  of  the  original  micronucleus  here  accomplish  a 
reduction  in  chromosome  number,  as  they  evidently  do  in  conjugation, 
it  would  seem  that  the  diploid  number  should  be  restored  sooner  or  later. 
This  point  has  not  been  cleared  up  by  cytologists.  Both  conjugation 
and  endomixis  (in  species  having  it)  have  beneficial  effects  upon  the 
vigor  of  the  race.  In  cultures  the  organisms  continue  to  multiply  by 
fission  as  long  as  either  process  occurs  at  intervals,  but  when  conditions 
inducing  senile  change  arise,  their  vitality  decreases  and  the  cultures 
die  out  if  neither  process  takes  place.  Finally,  it  is  of  interest  to  know 
that  researches  on  Paramecium  and  other  genera  indicate  the  presence 
of  a  genie  basis  of  inheritance  among  Infusoria  paralleling  in  some  degree 
that  found  in  Metazoa.  The  problem  of  analyzing  the  experimental 
data  is,  however,  greatly  complicated  by  the  occurrence  of  several  types 
of  nuclear  reorganization,  the  presence  of  macronuclei,  and  other  factors. 
Intensive  studies  now  in  progress  promise  to  show  to  what  extent  genet- 
ical  principles  founded  on  data  derived  from  higher  animals  and  plants 
are  applicable  to  Protozoa. 

With  the  above  points  in  mind  it  becomes  unsatisfactory  to  regard 
Protozoa  merely  as  simple  or  primitive  organisms.  They  are  small,  but 
not  therefore  simple.  Their  organization  may  not  be  so  complex  as  that 
of  frogs,  but  for  animals  of  their  size  it  is  probably  fully  as  effective. 
Which  of  their  peculiar  characters  are  actually  primitive  can  scarcely 
be  stated  with  confidence  at  present.  Perhaps  it  is  best  to  think  of 
them  as  did  their  discoverer,  Anton  van  Leeuwenhoek,  simply  as  "little 
animals." 


CHAPTER  X 
CYTOLOGY  OF  REPRODUCTION  IN  ANGIOSPERMS 

The  life  cycles  of  vascular  plants,  brj^ophytes,  and  many  thallophytes 
are  complicated  by  the  presence  of  an  alternation  of  generations.  3y  this 
it  is  meant  that  the  cycle  comprises  two  phases  of  vegetative  development : 
one  of  these,  the  sporophyte,  develops  from  the  zygote  and  produces 
spores;  the  other,  known  as  the  gametophyte,  develops  from  the  spore  and 
produces  gametes.  Since  the  chromosome  number  is  reduced  at  sporo- 
genesis  and  doubled  at  syngamj^,  the  sporoph\i:e  normally  has  twice  as 
many  chromosomes  as  the  gametophyte. 

The  two  "generations "  differ  greatly  in  relative  degree  of  development 
in  the  various  plant  groups.  In  most  bryophytes  the  gametophyte  is 
the  more  conspicuous:  a  moss  plant,  for  example,  is  a  gametophyte,  the 
sporophyte  being  small  and  short-lived.  In  ferns  this  relationship  is 
reversed,  the  gametophyte  being  so  small  that  it  commonly  escapes  notice. 
In  flowering  plants  the  gametophyte  is  still  more  obscure  and  must  be 
studied  \wiih  the  microscope. 

The  cycles  are  complicated  further  by  variations  in  the  type  of  sex 
differentiation.  In  mosses  and  ferns  the  sperms  and  eggs  may  be  pro- 
duced by  the  same  gametophyte  (monoecism,  homothallism) ,  or  they  may 
occur  on  different  ones  (dioecism,  heterothallism) .  In  seed  plants,  male 
and  female  gametophytes  are  always  distinct  and  arise,  respectively, 
from  microspores  and  megaspores.  These  spores  may  be  produced  by 
the  same  sporophj^te,  in  which  case  the  plant  is  homophytic  (monoecious 
or  hermaphroditic).  In  other  species  they  are  produced  by  different 
sporophytes;  such  species  are  heterophytic  (dioecious).  The  cytological 
basis  of  these  sexual  conditions  will  be  discussed  in  Chap.  XII. 

The  present  chapter  will  be  devoted  entirel}^  to  the  angiosperms,  for 
these  are  the  plants  that  have  long  been  most  prominent  in  cytological, 
cytogenetical,  and  cytotaxonomic  researches. 

The  Flower. — Under  the  appropriate  physiological  conditions  the 
angiosperm  sporophyte  produces  flowers.  The  typical  flower  is  a  group 
of  parts — pistil,  stamens,  petals,  and  sepals — of  which  the  first  two  kinds 
are  directly  concerned  in  sexual  reproduction.  In  the  anthers  of  the 
stamens,  microspores,  later  becoming  pollen,  are  formed.  In  the  ovarian 
portion  of  the  pistil,  ovules  with  megaspores  are  produced,  and  it  is  there 
that  the  subsequent  stages  of  female  gametophyte  development,  syngamy, 
embryogeny,  and  seed  formation  are  carried  out. 

136 


CYTOLOGY  OF  REPRODUCTION  IN  ANGIOSPERMS  137 

The  many  families  of  angiosperms  show  almost  innumerable  variations 
in  floral  type.  Such  characteristics  as  the  size,  arrangement,  and  struc- 
ture of  parts  often  determine  the  suitability  of  a  species  for  cytological 
oi-  cytogenetical  investigation.  Of  special  importance  is  the  type  of  sex 
differentiation  present.  Some  angiosperms,  such  as  willows,  the  red 
campion,  and  the  date  palm,  are  heterophytic  (dioecious),  the  flowers 
on  one  plant  having  only  pistils  or  only  stamens.  Others  are  homophytic : 
of  these,  some  are  monoecious,  having  pistillate  flowers  and  staminate 
flowers  on  the  same  plant,  as  in  maize,  while  others  are  hermaphroditic, 
having  pistils  and  stamens  in  the  same  flower,  as  in  buttercups,  tulips, 
and  apple  trees.  Both  unisexual  and  bisexual  flowers  ma}^  be  borne  by 
the  same  plant  (some  maples),  and  various  other  arrangements  are  known. 
In  some  flowers  bearing  both  pistils  and  stamens  the  two  maj^  not  be 
functional  at  the  same  time,  so  that  cross-pollination  is  favored  or  made 
necessary  even  though  the  plant  is  structurally  bisexual.  Furthermore, 
some  bisexual  plants  are  self-sterile,  producing  seeds  only  after  cross- 
pollination.  The  mode  of  pollination,  i.e.,  whether  b}^  \^•ind  or  by  insects, 
is  often  a  matter  of  importance  in  designing  experimental  procedures. 

Microsporogenesis  and  the  Male  Gametophyte  (Figs.  101,  103). — 
The  anther  commonlj"  differentiates  internally  into  three  regions:  an 
outer  wall  consisting  of  several  layers  of  cells,  a  nutritive  tapetum  of 
one  layer,  and  a  central  mass  of  sporogenous  cells.  The  sporogenous 
cells  eventually  enlarge  as  microsporocytes,  round  up  from  one  another, 
and  lie  in  a  fluid  filling  the  enlarged  anther.  Each  microsporocyte  then 
divides  into  four  microspores.  The  two  nuclear  divisions  here  are 
meiotic,  each  nucleus  of  the  resulting  quartet  containing  the  reduced 
chromosome  number.  In  some  plants,  cytokinesis  occurs  after  division  / 
and  again  after  division  II,  but  in  most  species  it  does  not  take  place 
until  after  //,  the  spherical  cell  then  dividing  simultaneously  into  four 
tetrahedral  spores  (quadripartition) .  The  shape  of  the  spores  in  the 
quartet  often  reveals  which  mode  of  cytokinesis  has  occurred.  The  walls 
of  the  microspores  become  greatly  thickened,  the  characteristic  patterns 
formed  often  being  useful  in  the  identification  of  species.  In  many 
plants  this  wall  thickening  involves  the  activity  of  the  tapetum.  This 
tissue  sometimes  breaks  down  into  a  tapetal  Plasmodium  which  flows 
in  among  the  young  spores  and  deposits  materials  upon  them.  The  wall 
consists  typically  of  two  distinct  layers:  the  thickened  exine  and  AAathin 
this  an  inline. 

The  male  gametophyte  of  angiosperms  is  structurally  very  simple. 
Its  development  begins  mth  the  division  of  the  microspore  into  a  small 
generative  cell  and  a  large  tube  cell.  The  generative  cell  may  lie  against 
the  spore  wall  at  one  side,  or  it  may  be  completely  enclosed  by  the 
cytoplasm  of  the  tube  cell.     The  generative  cell  divides  into  two  male 


138 


FUNDAMENTALS  OF  CYTOLOGY 


gametes.  This  division  may  occur  before  the  anther  opens,  as  in  maize, 
or  at  some  later  stage ;  hence  the  pollen  grains  shed  from  the  anther  and 
transferred  to  a  stigma  may  contain  two  or  three  cells.     The  further 


Fig.  101. — Microsporogenesis  and  development  of  male  gametes  in  purslane  (Portulaca) . 
1,  section  of  anther  with  microsporocytes;  2,  metaphase  /  in  microsporocyte;  3,  anaphase 
//,  with  spindles  at  right  angles  to  each  other;  4,  cytokinesis  beginning;  5,  detail  of  develop- 
ing partition  wall;  6,  microspore;  7,  division  of  microspore  nucleus;  8,  generative  cell 
formed;  9,  young  pollen  grain,  with  tube  nucleus  and  elongate  generative  cell;  10,  generative 
cell  dividing  in  pollen  grain;  11,  pollen  grain  with  two  male  gametes  and  tube  nucleus;  12, 
male  gametes  from  older  pollen  grain;  13,  germinating  pollen  grain;  tube  nucleus  entering 
pollen  tube.      {After  D.  C.  Cooper.) 

behavior  of  this  small  male  gametophyte  will  be  described  below  in  the 
section  on  syngamy. 

Megasporogenesis  and  the  Female  Gametophyte. — The  pistil 
consists  typically  of  an  ovary,  a  more  or  less  elongated  style,  and  a 
sticky  or  hairy  stigma  upon  which  the  pollen  will  be  received.     In  the 


CYTOLOGY  OF  REFRODUCTION  IN  ANGIOSFERMS 


139 


ovary  are  one  or  more  ovules.  Each  ovule  consists  of  a  central  portion, 
the  nucellus,  surrounded  by  one  or  two  integuments  with  an  opening,  the 
micropyle.     In  the  nucellus  a  subepidermal  cell,  either  at  once  or  after 


Fig.  102. — Typical  ovule.s,  niega.sporogenesis,  and  female  gametophyte  development  in 
angiospeims.  1-10  from  lettuce;  11-15  from  bloodroot.  1-6,  development  of  megaspore 
quartet  from  mega.sporocyte  in  ovule.  7-15,  development  of  female  gametophyte  from  one 
surviving  megaspore  of  quartet,  a,  antipodal  cells;  e,  egg;  i,  integument;  m,  micropyle;  7i, 
nucellus;  p,  polar  nuclei;  s,  synergids.      {From  preparations  by  J.  Einset.) 


division,  enlarges  and  differentiates  as  a  megasporocyte  (Fig.  102).  This 
cell  divides  into  a  quartet  of  megaspores,  each  wdth  the  reduced  chromo- 
some number.  Often  the  outer  cell  of  the  two  present  after  division  / 
does  not  divide  again,  so  that  the  quartet  is  incomplete.     One  of  the 


140  FUNDAMENTALS  OF  CYTOLOGY 

spores  then  develops  into  the  female  gametophyte  as  the  other  three 
degenerate. 

This  process  shows  many  variations  in  different  genera  of  angiosperms, 
although  the  general  structure  of  the  gametophyte  is  often  essentially 
the  same  after  different  modes  of  development.  Typical  development 
occurs  as  follows.  The  meiotic  divisions  in  the  megasporocyte  are 
accompanied  by  cytokinesis,  the  result  being  a  row  of  megaspores.  The 
innermost  of  these  enlarges  greatly  as  its  nucleus  initiates  a  series  of  three 
mitoses,  yielding  eight  nuclei  lying  in  the  common  cytoplasm  of  the 
embryo  sac.  Membranes  are  formed  about  six  of  the  nuclei,  forming  a 
group  of  three  antipodal  cells  at  one  end  of  the  sac,  and  another  group  of 
three  called  the  egg  apparatus,  consisting  of  an  egg  and  two  synergids,  at 
the  end  near  the  micropyle.  In  the  cytoplasm  of  the  sac  lie  the  two  polar 
7iuclei;  these  are  not  to  be  confused  with  animal  polar  bodies,  which  are 
immediate  products  of  meiosis. 

Certain  other  types  of  development  occasionally  found  may  be  men- 
tioned. (1)  The  female  gametophyte  develops  from  one  of  the  two  cells 
present  after  meiotic  division  I,  two  of  the  nuclei  resulting  from  meiosis 
thus  being  involved  in  the  formation  of  an  eight-nucleate  sac  (Allium 
type)  or  a  four-nucleate  sac  (Podosternon  type).  (2)  A  four-nucleate 
gametophyte  arises  from  a  single  megaspore  {Oenothera  type).  (3)  No 
cytokinesis  accompanies  meiosis,  all  four  nuclei  dividing  once  to  form  an 
eight-nucleate  gametophyte  {Adoxa  type).  (4)  After  no  cytokinesis 
the  four  nuclei  divide  twice,  giving  a  sixteen-nucleate  gametophyte 
{Pcpcromia  t^'pe).  (5)  After  no  cytokinesis  at  meiosis  one  of  the  four 
nuclei  remains  near  the  micropylar  end  while  the  other  three  pass  toward 
the  antipodal  end  of  the  sac.  All  then  undergo  another  mitosis.  In  the 
micropylar  end  this  yields  two  monoploid  nuclei  as  expected,  but  in 
the  antipodal  end  the  three  nuclei  undergo  fusion  just  as  their  division 
begins,  so  that  instead  of  six  resulting  nuclei  there  are  only  two,  each 
with  three  sets  of  chi'omosomes.  The  four  nuclei  in  the  sac,  two  mono- 
ploid and  two  triploid,  now  undergo  another  mitosis,  giving  four  mono- 
ploid and  foiu-  triploid  nuclei.  When  cell  membranes  have  been  formed, 
the  egg,  synergids,  and  one  polar  nucleus  are  monoploid,  while  the  other 
polar  nucleus  and  the  three  antipodal  cells  are  triploid.  This  is  the 
Fritillaria  type  and  occurs  in  several  species  of  Lilium,  long  supposed 
to  develop  like  Adoxa.  Since  both  meiosis  and  nuclear  fusion  may 
result  in  qualitative  differences  among  nuclei,  the  importance  of  these 
phenomena  to  the  geneticist  working  with  such  plants  should  be  obvious. 

Syngamy. — A  necessary  preliminary  to  syngamy  in  seed  plants  is 
pollination.  The  pollen  is  brought  by  insects,  wind,  or  other  agency  to 
the  stigma.  There  it  germinates  by  sending  out  a  pollen  tube  through 
one  of  the  special  germ  pores  in  the  exine.     The  wall  of  the  tube  itself 


CYTOLOGY  OF  REPRODUCTION  IN  ANGIOSPERMS 


141 


is  the  greatly  extended  intine,  and  into  the  tube  move  the  cytoplasm  and 
nucleus  of  the  tube  cell  together  with  the  two  male  gametes.  When 
pollen  is  shed  from  the  anther  in  the  two-cell  stage,  the  division  of  the 
generative  cell  to  form  the  male  gametes  takes  place  as  the  pollen  tube 
grows  down  through  the  style  toward  the  ovary  (Fig.  103).  The  course 
of  the  tube  is  usually  between  the  thin-walled  cells,  the  intercellular  sub- 


FiG.  103. — Development  of  male  gametes  and  syngamy  in  lily  {Lilium  regale).  1. 
germinating  pollen  grain.  2,  generative  cell  and  tube  nucleus  in  pollen  tube.  '3-7,  stages 
in  division  of  generative  cell  and  nucleus  in  pollen  tube.  8,  tip  of  pollen  tube  containing 
tube  nucleus  and  two  male  gametes.  9,  syngamy;  one  male  gamete  near  egg  nucleus,  the 
other  leaving  pollen  tube  (but  not  entering  egg).      {After  D.  C.  Cooper.) 

stance  being  dissolved  by  enzymes  from  the  tube.  If  the  style  has  an 
open  central  canal,  the  tube  grows  along  against  the  cells  lining  it.  When 
the  style  is  very  long,  the  style  and  tube  may  wither  away  at  the  tip 
before  the  growing  end  of  the  tube  containing  the  c.ytoplasm  and  nuclei 
reaches  the  ovule.  Eventually  the  tube  grows  through  the  micropyle  of 
the  ovule  into  the  embryo  sac  where  it  ruptures,  liberating  the  male 
gametes  and  often  the  tube  nucleus. 

Syngamy  now  occurs  (Fig.  104).     The  male  gametes  just  after  enter- 
ing the  sac  have  been  shown  in  numerous  species  to  be  complete  cells, 


142 


FUNDAMENTALS  OF  CYTOLOGY 


with  both  nucleus  and  cytoplasm.  In  other  species  thej^  appear  as 
nuclei  A\dth  no  cytoplasm  distinct  from  that  of  the  sac  or  pollen  tube. 
Often  the  sperm  nuclei  have  a  worm-like  form.     One  of  the  gametes 


«?*Ai% 


r\  N.-^'^D 


t 


Q 


Y^.^//l 


Fig.  104. — Syngamy  in  Crepis  capillaris.  1,  upper  part  of  embryo  sac.  Material  just 
discharged  from  pollen  tube  above  egg  contains  two  sperms  in  one  mass  and  an  irregular 
body.  Polar  fusion  nucleus  below.  2,  two  sperms  at  tip  of  egg;  one  synergid  visible.  .3, 
one  sperm  in  contact  with  egg  nucleus,  the  other  moving  toward  the  polar  fusion  nucleus; 
72  minutes  after  pollination.  4,  sperm  spread  out  on  surface  of  egg  nucleus.  5,  sperm 
spread  on  surface  of  polar  fusion  nucleus.  6,  sperm  beginning  to  transform  inside  egg 
nucleus.  7,  8,  later  stages  in  alteration  of  sperm  in  egg  nucleus.  9,  embryo  sac  about  32 
hours  after  pollination.  The  embryo  is  developing  with  a  diploid  chromosome  comple- 
ment, and  the  endosperm  with  a  triploid  complement.      {After  H.  Gerassimova.) 

applies  itself  to  the  egg  and  fuses  with  it.  The  nucleus  can  be  followed 
through  all  the  stages  leading  to  its  union  with  the  egg  nucleus,  but  the 
sperm's  cytoplasm,  even  if  it  is  recognizable  before  syngamy,  has  not 
been  definitely  proved  by  direct  observation  to  enter  the  egg.     There  is 


CYTOLOGY  OF  REPRODUCTION  IN  ANOIOSPERMS  143 

indirect  genetical  evidence  which  indicates  that  it  does  enter  in  some 
species  (page  232).  As  the  two  gametic  nuclei  unite,  their  aspect  may 
vary  as  it  does  in  animals:  both  may  have  a  thready  structure  much  like 
that  of  the  metabolic  stage ;  the  male  may  be  smaller  and  more  compact ; 
one  or  both  of  them  ma}^  be  in  the  prophase  of  mitosis,  the  maternal 
and  paternal  genomes  being  distinguishable  in  the  next  ensuing  division. 
In  any  case  the  chromosome  complement  of  the  fertilized  egg  consists  of 
two  genomes:  diplosis  has  occurred  in  syngamy.  After  a  cross  in  angio- 
sperms,  therefore,  the  nuclear  material  of  the  zygote  and  the  individual 
which  it  eventually  becomes  is  derived  from  both  parents,  whereas  its 
cytoplasm  may  come  from  the  mother  alone. 

While  the  above  events  are  taking  place,  the  other  male  gamete 
nucleus  takes  up  a  position  near  the  two  polar  nuclei  somewhere  in  the 
sac.  All  three  then  undergo  a  triple  fusion,  formmg  a  primary 
endosperm  nucleus  wth  three  genomes.  Sometimes  the  two  polars 
have  fused  by  the  time  the  male  nucleus  arrives,  the  male  then  being 
added.  All  orders  of  fusion  of  the  three  have  been  observed  and,  as 
in  the  egg,  they  may  be  in  the  metabolic  state  or  in  some  stage  of  the 
prophase.  In  sacs  with  more  than  the  usual  number  of  nuclei,  more 
than  three  may  unite  to  form  the  primary  endosperm  nucleus.  The 
union  of  one  male  nucleus  with  the  egg  nucleus  while  another  unites 
with  the  polar  nuclei  is  called  double  fertilization  and  is  a  process  peculiar 
to  angiosperms. 

The  time  elapsing  between  the  arrival  of  the  pollen  on  the  stigma  and 
the  syngamic  union  varies  greatly,  as  the  following  examples  show: 
rye,  7  hours;  maize,  18  to  24  hours,  in  spite  of  the  length  of  the  style 
(the  silk);  Jimson  weed,  25  hours;  box  elder,  40  to  72  hours;  Indian  pipe 
(Monotropa),  5  days;  pecan,  5  to  7  weeks;  red  oak,  13  to  14  months. 
These  periods  vary,  of  course,  with  temperature. 

Embryogeny  and  Seed  Development. — Pollination  and  syngamy  set  in 
motion  a  number  of  reactions  in  the  ovary  and  ovules  which  result  in 
the  development  of  a  fruit  with  seeds.  This  involves  alterations  in  every 
part,  from  the  fertilized  egg  to  the  tissue  of  the  pistil  and  sometimes  the 
other  floral  organs. 

The  fertilized  egg  develops  into  the  embryo,  which  is  to  become  the 
sporophytic  plant  of  the  next  generation.  It  divides  several  times 
transversely,  forming  a  few  cells  in  a  row  (Fig.  105),  and  then,  beginning 
in  the  terminal  cell  (toward  the  center  of  the  sac),  divisions  in  the  longi- 
tudinal and  other  planes  accompany  its  lateral  growth.  The  stage  of 
embryonic  development  reached  by  the  time  the  ovule  becomes  a  mature 
seed  varies  widely:  in  some  plants  the  embryo  is  a  small  undifferentiated 
mass  of  cells,  most  of  the  seed  being  occupied  by  endosperm  to  b(; 
described  below;  in  others  the  embryo  is  larger,  has  differentiated  its 


144 


FUNDAMENTALS  OF  CYTOLOGY 


cotyledons,  and  has  digested  away  some  of  the  endosperm;  instill  others 
it  fills  the  entire  seed,  having  digested  away  all  the  endosperm  and  even 
some  of  the  surrounding  ovular  tissue.  All  the  mitoses  during  embry- 
ogeny  are  equational,  so  that  every  nucleus  contains  two  genomes,  one 
from  each  parent. 

Even  before  cell  division  begins  in  the  fertilized  egg,  the  development 
of  endosperm  has  usually  commenced  with  the  division  of  the  primary 
endosperm  nucleus.  In  manj^  angiosperms  there  are  several  of  these 
divisions  before  cytokinesis  occurs,  so  that  the  endosperm  has  an  early 

coenocytic  stage.  Cytokinesis  later  converts 
this  into  a  cellular  tissue.  In  other  species 
the  endosperm  is  cellular  from  the  beginning. 
The  endosperm  enlarges  along  with  the  whole 
ovule  and  ma,v  become  stored  with  various 
nutritive  materials.  In  wheat  and  other 
grains  the  outermost  layer  contains  many 
aleurone  granules.  Storage  material  in  some 
plants  is  deposited  in  the  form  of  thick 
cellulose  walls,  the  endosperm  being  hard,  like 
wood  or  even  ivory.  Such  stored  material  is 
utilized  as  the  seed  germinates  and  the  embryo 
develops  into  a  young  plant. 

The  endosperm  is  commonly  triploid  in 
nuclear  constitution,  although  it  often  devel- 
ops with  other  numbers  of  genomes  depending 
upon  the  number  of  nuclei  involved  in  the 
previous  fusion.  Sometimes  unfused  as  well 
as  fused  nuclei  take  part  in  endosperm  devel- 
opment, portions  of  the  tissue  being  mono- 
ploid.  Evidently  it  is  neither  the  derivation 
of  the  nuclei  nor  their  fusion,  but  rather  the 
conditions  under  which  they  develop,  that  de- 
termine the  formation  of  endosperm.  From  the  standpoint  of  compar- 
ative morphology  it  is  most  logical  to  interpret  the  endosperm  as 
gametophytic  tissue  which,  unlike  that  of  gymnosperms,  is  arrested 
in  development  until  after  the  pollen  tube  enters  the  sac;  it  then 
resumes  development  with  or  sometimes  without  fusions  and  the 
incorporation  of  a  male  nucleus.  A  male  nucleus  may,  of  course,  affect 
particular  characters  of  the  endosperm  if  it  comes  from  a  plant  of  different 
genetic  constitution. 

In  most  angiosperms  the  other  cells  present  in  the  embryo  sac  before 
syngamy  disintegrate  early.  The  entering  pollen  tube  often  destroys 
one  synergid,  and  the  other  disappears  soon  after  the  egg  is  fertilized. 


Fig.  105. — Embryo  sac  or 
Crepis  capillaris,  showing  two- 
celled  embryo  with  nuclei  in 
mitosis.  The  endosperm  has 
begun  to  develop.  (From  a 
preparation  by  K.  Koos.) 


CYTOLOGY  OF  REPRODUCTION  IN  ANGI06PERMS  145 

The  antipodal  cells  usually  degenerate  and  disappear  during  endosperm 
development,  but  sometimes  they  enlarge  and  perform  a  nutritive  func- 
tion for  a  time.  In  maize  the  antipodals  undergo  division  and  eventuallj' 
form  in  the  kernel  an  oval  mass  of  monoploid  gametophytic  tissue  h'ing 
next  to  the  endosperm,  which  it  closel}'  resembles  in  cellular  organization 
and  contained  storage  material. 

Alterations  outside  of  the  embryo  sac  may  be  briefly  summarized  here. 
The  tissue  of  the  nucellus  about  the  sac  usually  becomes  less  conspicuous 
as  the  endosperm  enlarges.  In  some  plant  families,  however,  it  may 
become  stored  with  nutritive  substances,  in  which  case  it  is  known  as 
pcrispcrm.  The  tissue  of  the  ovule's  integument  (or  integuments) 
becomes  transformed  into  the  seed  coat.  The  ovarian  tissue  enclosing 
the  ovule  (or  ovules)  is  variously  modified  into  the  fruit  tissue,  or  pericarp. 
This  is  represented  by  the  pod  of  a  bean,  the  hard  covering  of  an  acorn 
or  a  maize  kernel,  the  fleshy  tissue  of  the  tomato,  and  the  fleshy  tissue 
(exocarp)  and  hard  pit  covering  (endocarp)  of  the  cherry.  Accessory 
fruits  incorporate  flower  parts  other  than  the  ovary,  as  in  the  fleshy 
tissue  of  the  strawberry,  which  is  developed  from  the  receptacle,  and  in 
the  plume-like  portion  of  the  dandelion  fruit,  which  is  formed  from  the 
calyx.  In  multiple  fruits,  such  as  the  mulberry  and  pineapple,  the  tissues 
of  several  flowers  are  combined. 

Finally,  it  should  be  pointed  out  that  since  the  pericarp  and  seed  coat 
develop  from  tissues  already  present  in  the  flower  before  pollination,  a 
given  syngamic  union  does  not  alter  the  chromosomal  constitution  of 
these  parts  but  it  does  so  affect  the  fruits  and  seeds  of  the  next  generation. 

Aberrations  of  the  Reproductive  Process. — In  the  angiosperms  there 
are  encountered  a  number  of  modifications  of  the  process  of  sexual 
reproduction  as  described  above.  These  occur  only  occasionally  in 
some  plants,  but  in  certain  species  one  or  more  of  them  have  become 
habitual.  On  the  whole  they  play  a  minor  role  in  nature,  but  they  should 
at  least  be  listed  here  because  they  do  throw  much  light  upon  the  problem 
of  lelationships  within  some  genera  and  often  afford  an  explanation  of 
luiexpected  genetical  behavior  in  the  breeding  plot. 

Several  of  these  aberrations  fall  under  the  heading  of  apomixis,  a 
term  applied  to  asexual  reproductive  processes  substituted  for  the  sexual 
process.  Apomixis  maj^  be  vegetative  when  buds  or  bulblets  of  various 
kinds  appear  in  the  place  of  flowers.  Of  greater  interest,  however, 
are  the  various  types  of  apomixis  involving  the  formation  of  seeds. 
Examples  of  these  are  the  following:  (1)  Reduced  parthenogenesis,  in 
which  an  unfertilized  egg  develops  with  the  gametic  chromosome  number. 
This  phenomenon  has  been  observed  in  experimental  plants  belonging  to  a 
number  of  genera,  but  it  is  not  known  to  have  become  an  established 
habit  anywhere  among  angiosperms  in  nature.      (2)  Unreduced  partheno- 


146 


FUNDAMENTALS  OF  CYTOLOGY 


genesis,  in  which  an  unfertihzed  egg  develops  with  the  diploid  chromosome 
number  (Fig.  106).  This  is  probably  the  commonest  mode  of  apomixis 
involving  seed  formation,  being  known  to  occur  in  a  number  of  genera 
in  nature.  The  female  gametophyte  and  egg  in  this  case  are  diploid 
because  of  a  failure  of  meiosis  at  sporogenesis  in  the  ovule,  or  as  a  result 
of  the  development  of  the  gametophyte  directly  from  a  somatic  cell  of 
the  ovule  without  spore  formation  (apospory).  In  such  cases  there  is  an 
alternation  of  gametophytic  and  sporophytic  generations  in  the  life 
cycle,  but  no  alternation  of  chromosome  numbers.  (3)  Apogamy,  in 
which  an  embryo  arises  from  a  gametophytic  cell  other  than  the  egg. 
This  has  occasionally  been  observed  but  is  not  known  to  occur  regularly 
in  nature.  (4)  Adventitious  emhryony,  in  which 
a  sporophytic  cell  of  the  nucellus  or  the  integu- 
ment buds  into  the  embryo  sac  and  produces  an 
embryo  (Fig.  106).  This  ma}^  occur  without  the 
stimulus  of  pollination  (Euphorbia)  or  only  after 
syngamy  has  taken  place  (orange).  (5)  Polyem- 
hryony,  in  which  more  than  one  embryo  develops 
in  an  embryo  sac.  One  of  these  may  develop 
sexually  in  the  usual  manner,  while  others  arise 
in  one  of  the  ways  mentioned  above.  Another 
rare  mode  is  the  formation  of  several  embryos  by 
proliferation  from  a  single  one  at  an  early  stage. 
The  following  phenomena  not  included  under 
the  heading  of  apomixis  are  also  encountered  at 
times.  (1)  Gynogenesis,  in  which  the  male  nucleus 
enters  the  egg  but  then  disintegrates,  leaving  the 
egg  to  develop  into  an  embryo  with  the  maternal 
nucleus  only.  (2)  Androgenesis,  in  which  the 
maternal  nucleus  presumably  disintegrates,  for  a  monoploid  plant 
develops  with  paternal  characters.  (3)  Parthcnocarpy ,  in  which  fruit 
development  takes  place  without  the  egg  having  been  fertilized  and 
in  different  cases  with  or  without  the  stimulus  of  pollination.  Parthcno- 
carpy occurs  naturally  in  the  banana  and  some  strains  of  grapes  and  citrus 
fruits,  and  it  has  been  found  possible  to  induce  it  with  a  growth  hormone 
in  tomatoes,  pepper,  and  tobacco.  (4)  Metaxenia,  in  which  the  embryo 
or  endosperm  developed  after  a  cross  produces  a  visible  modification  in 
the  character  of  the  maternal  parent  tissue  enclosing  it.  This  has  been 
observed  in  apples  and  the  date  palm.  A  visible  effect  of  the  male 
parent  on  the  character  of  the  endosperm  {xenia)  is  expected  after  some 
crosses,  for  the  male  parent  contributes  nuclear  material  to  the  tissue, 
but  effects  upon  tissues  where  no  such  material  is  present  must  receive 
another  explanation. 


Fig.  106. — Embryo 
sac  of  Alchemilla  with  one 
embryo  developing  by 
unreduced  parthenogene- 
sis (below)  and  another 
by  adventitious  budding 
(above).  {After  S.  Mur- 
heck.) 


CYTOLOGY  OF  REPRODUCTION  IN  ANGIOSPERMS 


147 


Conclusions. — The  foregoing  account  of  the  normal  angiosperm 
reproductive  cycle,  and  of  various  modifications  of  it  which  may  occur 
regularly  or  occasionally  in  some  species,  should  serve  to  emphasize 
the  importance  of  a  thorough  knowledge  of  the  subject  to  one  who  wishes 
to  undertake  genetical  or  cytogenetical  researches  on  this  great  group  of 
plants. 

The  typical  angiosperm  life  cycle  bears  a  certain  resemblance  to  that 
of  animals,  since  each  of  them  includes  a  diploid  body  and  monoploid 
gametes.  In  some  studies  it  may  be  sufficient  to  think  of  them  as  alike, 
but  to  interpret  properly  certain  genetical  behavior  of  the  plant  one  must 
know  that  it  has  a  second  type  of  reproductive  cell,  the  spore,  and  a 
gametophytic  phase,  neither  of  which  is  present  in  the  animal  cycle. 


CHAPTER  XI 

CYTOLOGY  OF  REPRODUCTION  IN  PLANTS  OTHER 
THAN  ANGIOSPERMS 

In  nearly  all  plants  of  the  groups  now  to  be  considered — gymnosperms, 
ferns,  mosses,  liverworts,  algae,  and  fungi— the  life  cycle,  like  that  of 
angiosperms,  includes  two  kinds  of  reproductive  cells:  gametes  and 
spores.  Diplosis  occurs  at  syngamy,  while  haplosis,  except  in  certain 
algae  and  fungi,  takes  place  at  sporogenesis.  The  following  sections 
will  deal  chiefly  with  cytological  features  of  particular  interest  in  each 
group,  although  this  will  entail  some  consideration  of  morphological 
features  described  in  textbooks  of  general  botany. 

Gymnosperms. — The  reproductive  process  in  gymnosperms  is  carried 
out  in  the  cones.  The  staminate  cone  bears  microsporangia  in  which 
microspore  quartets  and  eventually  pollen  grains  are  produced.  The 
male  gametophyte  in  these  grains  has  more  cells  than  in  angiosperms, 
but  the  number  of  male  gametes  produced  is  practically  always  two. 
The  ovulate  cone  bears  the  ovules  on  the  carpels  which  compose  it,  but 
unlike  angiosperm  carpels  these  do  not  form  an  ovary  enclosing  the 
ovules.  This  is  the  most  fundamental  distinction  between  the  gynmo- 
sperm  cone  and  the  angiosperm  flower. 

In  the  ovule  one  megaspore  of  a  quartet  forms  a  coenocytic  and  then 
multicellular  gametophyte  with  archegonia,  each  containing  one  large 
egg  (Fig.  107).  The  female  gametophyte  in  gymnosperms,  which  has 
long  been  termed  endosperm,  thus  develops  far  more  extensively  than 
that  of  angiosperms  before  syngamy  and,  in  addition  to  this,  archegonia 
are  differentiated.  In  the  Gnetales  certain  species  resemble  the  angio- 
sperms in  differentiating  their  eggs  in  the  coenocytic  stage  of  the 
gametophyte,  the  cellular  stage  following  later,  though  with  nothing 
corresponding  to  a  polar  fusion. 

Syngamy  in  gj^mnosperms  occurs  in  two  main  forms  which  differ 
chiefly  because  of  the  character  of  the  male  gametes.  In  conifers  the  male 
cells  are  nonmotile  and  are  delivered  to  the  egg  through  a  pollen  tube 
which  grows  inward  from  the  surface  of  the  nucellus.  In  cycads  (Figs. 
107,  109)  and  the  ginkgo  tree  the  .young  pollen  tube  grows  not  directly 
toward  the  archegonium  but  into  the  tissue  at  the  sides  of  the  ovule,  while 
in  the  pollen  grain  end  of  the  tube  two  very  large  motile  spermatozoids 
are  differentiated.     Disintegration  of  the  nucellus  allows  this  end  of  the 

148 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS 


149 


tube  to  come  into  contact  with  the  gametophyte  near  the  arch(!gonia. 
There  the  tube  Hberates  the  sperms  which  make  their  way  between  the 
archegonial  neck  cells  into  the  egg. 


Fig.  107. — Portion  of  ovuh  ot  i(\i  id  (l)ioon) 
at  the  time  of  fertilization  Pollen  tubes  gi owing 
in  the  nucellar  tissue  are  developing  sperms  and 
discharging  them  into  the  pollen  chamber  above 
the  female  gametophyte.  The  large  archegonium 
at  the  right  is  about  to  be  entered  by  a  sperm.  In 
the  one  to  the  left  the  sperm  has  entered,  leaving  its 
cytoplasmic  sheath  in  the  upper  end  of  the  egg 
while  its  nucleus  has  fused  with  the  egg  nucleus. 
{Reconstructed  from  several  sections  by  C.  J. 
Chamberlain.) 


/ 


1     ^^■ 


\'**fe?-- 


b^' 


J 


o 


^  «  ©  ®  *5,;^5^ 

lUS. — Syngamy  and  early 
embryogeny  in  a  cycad  (Stangeria). 
a,  sperm  nucleus  uniting  with  nucleus 
of  large  egg;  blepharoplast  and  cilia 
in  upper  end  of  egg.  6,  nuclear 
division  without  cytokinesis  in  prog- 
ress in  young  zygote,  c,  cocnocytic 
embryo  becoming  cellular.  {After 
C.  J.  Chamberlain.) 


The  male  gamete  in  conifers  may  enter  the  egg  as  a  complete  cell  or 
only  a  nucleus.  In  cycads  the  whole  sperm  enters  with  its  large  nucleus, 
cytoplasmic  layer,  spirally  coiled  blepharoplast,  and  many  cilia.  The 
nucleus  soon  becomes  free  from  the  cytoplasm  and  motor  apparatus 
and  advances  alone  to  the  egg  nucleus,  the  two  then  fusing  (Fig.  108,  a). 


150 


FUNDAMENTALS  OF  CYTOLOGY 


In  conifers  a  similar  course  may  be  followed  by  the  nucleus,  but  in  some 
cases  the  male  cytoplasm  remains  with  it  and  eventually  surrounds  the 
fusion  nucleus.  As  in  angiosperms,  the  chromosomes  of  the  two  gametic 
nuclei  sometimes  remain  more  or  less  distinct  until  after  the  first  embryo- 
nal mitosis. 

Embryogeny  in  gymnosperms,  like  female  gametophj^te  development, 
includes  an  early  coenocytic  phase  and  a  later  cellular  stage  (Fig.  108,  h,  c). 
The  embryo  grows,  differentiates  its  cotyledons,  and  ceases  development 
before  all  the  endosperm  (female  gametophyte  tissue)  has  been  digested 

■45^ 


^^^'^^- 

^ 


Fig.  109. — Spermatogenesis  in  a  cycad  (Dioon).  1,  microspore.  2,  pollen  grain  with 
tube  nucleus,  generative  cell,  and  prothallial  cell.  3,  germinating  pollen  grain.  4,  genera- 
tive cell  has  divided  to  form  body  cell  and  .stalk  cell.  5,  conspicuous  blepharoplasts  in 
enlarged  body  cell  in  growing  pollen  tube.  6,  two  spermatozoids  formed  by  division  of 
body  cell;  in  each  of  tliem  a  spirally  coiled  blepharopla.st  runs  just  beneath  the  cell  mem- 
brane and  bears  the  cilia.  7,  portion  of  section  through  spermatozoid,  showing  cilia  growing 
from  the  ribbon-shaped  blepharoplast  seen  as  cross  sections  of  two  of  its  coils;  portion  of 
large  nucleus  at  left.      (No.  6  after  H.  J.  Webber;  others  after  C.  J.  Chamberlain.) 


away.  In  many  conifers  a  remarkable  process  known  as  cleavage  poly- 
embryony  occurs.  During  its  period  of  elongation  the  original  embrj-o 
splits  into  a  considerable  number,  one  of  which  finally  surA'ives.  Neigh- 
boring archegonia  in  the  same  gametophyte  may  also  develop  embryos, 
but  the  mature  seed  usually  has  only  one.  About  the  embryo  is  the 
remaining  endosperm;  outside  of  this  are  a  trace  of  the  nucellus  and  a 
well-developed  seed  coat.  Since  the  gymnosperm  o^'ule  is  not  enclosed 
in  an  ovary,  there  is  no  true  pericarp. 

Ferns. — Some  of  the  relatives  of  the  ferns  produce  spore  quartets  of 
two  morphological  kinds  which  differ  in  sexual  tendenc.y,  but  in  tnie 
ferns  themselves  the  spores  are  of  but  one  morphological  type.  They 
are  produced  in  sporangia  on  the  leaves  of  the  sporophyte.     The  meiotic 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS 


151 


fli^^sions  in  the  si^orocytos  proceed  in  the  ordinary  waA',  one  interesting; 
feature  being  the  regular  arrangement  of  the  proplastids  in  layers  close  to 
the  cell  plate  at  each  cytokinesis.  The  chromosome  number  tends  to  be 
high,  so  that  ferns  generally  are  not  favorable  material  for  chromosomal 
studies. 

Upon  germination  the  spore  produces  a  small,  thin  gametophyte,  in 
the  large  cells  of  which  the  chloroplasts  appear  with  admirable  clearness 
(Fig.  18).  Ferns  in  this  stage  have  therefore  been  used  in  significant 
investigations  on  the  genetics  of  plastids  and  theii-  mutability  in  response 
to  irradiation  with  X  rays.  The  gametophytes  develop  sex  organs 
of  two  kinds.  In  the  base  of  each  archegonium  one  large  egg  is  dif- 
ferentiated. Each  antheridium  produces  a  considerable  number  of 
spermatozoids,  and  the  stages  of  this  process  are  of  much  cytological 
interest.     It  is  to  be  remembered  that  no  haplosis  occurs  in  the  formation 

ben 


Fig.  110. — Spermiogenesis  in  a  fern.  1-3,  stages  in  transfoiinatioii  of  the  spermatid;  4, 
spermatozoid;  c,  cytoplasm;  b,  blepharoplast;  n,  nucleus.      (B<istd  on  drawings  by  A.  Yuasa.) 

of  these  gametes,  for  the  gametophj^te  from  which  they  arise  has  itself 
developed  with  the  gametic  chromosome  number. 

Spermiogenesis  occurs  essentially  as  follows  (Fig.  110).  The  sperm- 
atid, or  cell  that  is  to  transform  into  a  spermatozoid,  consists  of  cyto- 
plasm, a  nucleus,  a  blepharoplast,  small  plastids,  and  probably  other 
inclusions.  The  blepharoplast  is  of  special  interest,  for  in  several 
pteridophytes  it  has  been  shown  to  be  the  centrosome  that  functioned 
in  the  preceding  mitosis.  It  is  called  a  blepharoplast  in  the  spermatid 
because  it  bears  the  cilia  of  the  motor  apparatus,  recalling  thus  the  parallel 
behavior  of  the  centrioles  in  animal  spermiogenesis.  It  apparently 
differentiates  into  two  longitudinal  parts;  then  from  one  of  these  the 
numerous  cilia  grow  out  as  the  whole  structure  elongates  spirally  together 
with  the  nucleus,  forming  the  coiled,  compact  body  of  the  sperm.  The 
spermatid  cytoplasm,  often  containing  starch  granules,  is  held  mostly 
as  a  vesicle  in  the  large  posterior  coils. 

When  water  is  present  externally,  the  sperms  are  liberated  from  the 
antheridium  and  swim  about  actively.     If  open  archegonia  with  recently 


152  FUNDAMENTALS  OF  CYTOLOGY 

matured  eggs  are  in  the  vicinity,  the  sperms  tend  to  move  toward  them 
along  a  maUc  acid  gradient.  Many  sperms  may  enter  an  archegoniimi, 
l)iit  ordinarily  only  one  unites  with  the  egg.  The  fate  of  the  components 
of  the  sperm  in  syngamj^  is  apparently  not  precisely  the  same  in  all 
ferns,  but  in  general  their  behavior  is  as  follows.  The  cytoplasmic 
vesicle  is  lost  by  the  swimming  sperm  before  it  reaches  the  egg.  The 
other  components  enter  the  egg  cytoplasm,  although  the  cilia  have  been 
reported  in  at  least  one  species  to  remain  outside.  The  nucleus  then 
separates  from  most  or  all  of  the  other  components  (residual  cytoplasm 
and  portions  of  the  motor  apparatus)  and  alone  imites  with  the  egg 
nucleus. 

The  fertilized  egg  develops  into  an  embryo  sporophyte,  the  early  stages 
being   passed   through  entirely   within   the   archegonium.     In   ordinary 


anth 


B 

Fig.  111. — A,  apogainy  in  a  fern:  young  sporopliytic  tissue  (s)  developing  directly  fi'om 
gametophytie  tissue  (g).  (After  Farmer  and  Dighy.)  B,  apospory  in  a  fern:  gametophyte 
with  antheridiuin  arising  directly  from  sporophytic  tissue  at  base  of  sorus.  sp,  sporangia, 
r,  rhizoid.      {After  F.  0.  Bower.) 

ferns  the  planes  of  the  first  few  divisions  in  the  spherical  cell  form  a 
i-egular  geometrical  pattern:  hemispheres,  quadrants,  and  octants  are 
formed  in  order.  The  embiyonic  parts — stem,  root,  leaf,  and  absorbing 
foot — differentiate  in  regular  positions  with  respect  to  the  early  sub- 
divisions. In  some  ferns  a  considerable  mass  of  embryonic  tissue  is 
formed  before  a  gradual  differentiation  of  parts  becomes  visible.  Thus 
it  appears  that  in  plants,  as  in  animals  (page  129),  cleavage  and  embryonic 
differentiation  may  be  variously  related  in  time  and  that  the  numerical 
equalit}'  of  quadrants  and  embryonic  parts  in  some  ferns  with  early 
differentiation  does  not  mean  that  the  cleavage  divisions  determine  the 
differentiation  of  these  parts. 

The  ferns  have  long  been  favorite  objects  for  the  study  of  apogamy 
and  apospory  (Fig.  111).  These  aberrations  in  the  reproductive  process 
have  been  found  to  occur  more  or  less  constantly  in  several  genera,  and 
they  have  been  induced  by  cultural  conditions  in  certain  others.  In 
apogamy  the  meristematic  region  of  the  gametophyte  gradually  produces 
a  young  sporophyte  directly,  gametes  being  in  no  way  concerned.  In 
apospory  the  meristematic  tissue  at  the  leaf  margin  or  the  base  of  a  sorus 


CYTOLOGY  OF  REFRODLCTION  IN  PLANTS  153 

grows  out  directly  into  a  gametophyte  without  the  intervention  of  spores. 
In  such  cases  both  generations  have  the  same  chromosome  number; 
this  is  the  gametic  number  in  some  instances,  the  zygotic  number  in 
others.  When  one  of  these  aberrations  occurs  regularly  in  successive 
cycles,  compensation  is  made  for  it  by  another  abnormality  elsewhere 
in  the  cycle.  Apogamy,  for  example,  may  be  followed  by  apospory 
or  by  spore  formation  without  haplosis. 

Mosses  and  Liverworts. — The  bryophytes  have  a  life  cycle  essentially 
like  that  of  the  ferns,  with  the  difference  that  the  gametophytic  phase 
is  in  general  more  prominent  than  the  sporophytic  one.  In  mosses  the 
gametophyte  typically  passes  through  an  early  filamentous  stage 
(the  protonema)  and  then  the  more  familiar  leafy  stage.  In  the  liverworts 
it  is  either  leafy  and  moss-like  or  develops  as  a  broad  and  branching 
ribbon.  In  both  groups  the  sporophyte  is  usually  a  stalked  spore 
capsule  standing  on  the  gametophyte  at  the  point  where  it  began  its 
development  from  a  fertilized  egg  in  an  archegonium.  Because  of  their 
fairly  short  life  cycle,  the  prominence  of  their  gametophytic  phase,  their 
adaptability  to  greenhouse  culture,  their  relatively  low  chromosome 
numbers,  and  their  response  to  experimental  treatments,  the  bryophytes 
hold  a  prominent  place  in  cytology  and  have  proved  to  be  especially 
valuable  in  the  genetical  study  of  gametophytic  characters. 

Some  bryophytes  are  homothallic,  both  archegonia  and  antheridia 
being  borne  on  the  same  gametophyte,  while  others  are  heterothallic, 
with  the  two  sexes  in  different  gametophytes.  The  latter  condition  is 
known  to  be  due  to  the  presence  of  two  kinds  of  spore  in  a  quartet,  their 
difference  being  determined  at  the  time  of  meiosis  (page  187).  The 
archegonium  bears  a  single  large  egg  containing  relatively  little  stored 
nutritive  material.  The  antheridium  develops  characteristically  a 
very  large  number  of  small  biciliate  spermatozoids. 

In  spite  of  the  minuteness  of  the  cells  concerned,  spermiogenesis 
has  been  successfully  studied  in  bryophytes,  particularly  in  mosses  (Fig. 
112).  The  spermatid,  like  that  of  ferns,  consists  mainly  of  cytoplasm, 
nucleus,  plastids  in  some  form,  and  a  blepharoplast  which  has  functioned 
as  a  centrosome  during  the  last  mitosis.  The  behavior  of  these  cell 
components  is  of  special  interest  when  it  is  compared  with  that  observed 
in  animal  spermiogenesis  (page  121).  The  nucleus  draws  out  into  the 
form  of  a  curved  body  within  the  cell.  Meanwhile  the  blepharoplast 
also  elongates,  unites  with  what  is  to  be  the  anterior  end  of  the  nucleus, 
and  develops  two  long  cilia.  The  limosphere,  a  large  body  formed  by 
the  plastid  material,  extrudes  a  small  globule  which  moves  to  the  anterior 
end  of  the  nucleus  near  the  blepharoplast  and  develops  into  the  pointed 
apical  body.  The  remnant  of  the  limosphere  takes  up  a  posterior  position 
and  may  eventually-  disappear  along  with  a  portion  of  the  cytoplasm. 


154 


FUNDAMENTALS  OF  CYTOLOGY 


The  mature  sperm,  which  straightens  out  considerably  after  escape  from 
the  antheridium,  commonly  shows  three  main  parts:  (1)  a  long  and  slender 
body  representing  the  condensed  nucleus  chiefly;  (2)  a  small  specialized 
anterior  region  composed  of  the  apical  body,  a  small  amount  of  cytoplasm, 
and  the  blepharoplast  which  bears  the  two  cilia,  often  at  different  points ; 
and  (3)  a  residual  mass  of  cytoplasm  at  the  posterior  end. 


Fig.  112. — Spermiogenesis  in  a  moss  {Polytrichum) .  A—D,  mitosis  in  speiinatogenous 
cells,  showing  aspect  of  plastid  substance  (A;).  E-H,  last  mitosis  in  antheridium,  showing 
behavior  of  centrosome,  which  in  each  spermatid  becomes  a  blepharoplast  (6).  I~L,  trans- 
formation of  spermatid  into  biciliate  spermatozoid:  a,  apical  body;  /,  limosphere;  n,  nucleus. 
{After  C.  E.  Allen.) 

The  details  of  syngamy  in  bryophytes  are  best  known  in  certain  genera 
of  liverworts.  The  behavior  of  the  motor  elements  during  the  process  is 
somewhat  uncertain,  although  it  probably  parallels  that  reported  for 
ferns.  The  nuclear  behavior  has  been  closely  followed,  and  it  may  not 
be  precisely  the  same  in  different  cases.  In  Riccardia  (Fig.  113)  the 
elongate  body  of  the  sperm,  consisting  of  the  nucleus  and  possibly  a 
covering   of   nonnuclear   material,    applies   itself   throughout    its   whole 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS 


155 


length  to  the  egg  membrane  and  appears  to  sink  through  it  laterally 
rather  than  endwise.  This  requu-es  20  to  30  minutes.  The  sperm 
nucleus  lies  without  conspicuous  change  in  the  egg  cytoplasm  for  24  to 
36  hours,  during  which  time  the  egg  enlarges  and  becomes  more  highly 
vacuolate.  Then  one  end  of  the  sperm  nucleus  penetrates  the  membrane 
of  the  egg  nucleus  and  at  once  begins  to  swell.  This  process  continues 
until  the  male  nucleus  is  entirely  within  the  egg  nucleus  and  transformed 
into  a  chromatic  thread}^  mass  ^\dth  a  nucleolus,  these  lying  near  the 
corresponding  elements  of  the  egg  nucleus.  Eventually  the  sperm  and 
egg  chromatin  become  indistinguishable.  The  egg  continues  to  grow 
and  undergoes  the  first  embryonal  division  about  6  to  9  days  after 
syngamy.  In  Pellia  the  male  nucleus  undergoes  marked  alterations  as 
it  lies  in  the  egg  cytoplasm  and  shows  a  structure  more  or  less  like  that 


Fiu.    113. 
prophase 


— Syngamy   in    a   liverwort    {Kiccurdia) .      1,    sperniatozoid.      2-4,    syngamy.      5, 
of  first  mitosis  in  fertilized  egg.      Description  in  text.      (After  A.  M.  Showaltcr.) 


of  the  egg  nucleus  by  the  time  the  two  unite.  Here,  as  in  other  groups 
of  organisms,  there  is  some  variation  in  the  time  relations  of  the  various 
events :  penetration  of  the  egg  membrane  by  the  sperm,  structural  altera- 
tion of  the  sperm  nucleus,  nuclear  union  (karyogamy) ,  and  mitotic 
division  of  the  zygote  nucleus. 

The  young  sporophyte  of  liverworts  contains  in  the  capsule  a  largo 
mass  of  sporogenous  cells,  some  of  which  function  as  sporocytes  while 
others  differentiate  as  hygroscopic  elaters.  The  sporocyte  divisions  in 
some  species  are  noteworthy  because  of  the  four-lobed  form  assumed 
by  the  cell  before  the  meiotic  nuclear  divisions,  which  occur  in  rapid 
succession  at  the  center  shortly  before  cytokinesis  is  carried  to  completion. 
Other  liverworts  and  some  mosses  show  also  a  very  regular  behavior 
of  the  plastids  during  these  stages.  In  Anthoceros  and  Polyfrichum,  for 
example,  the  single  plastid  divides  twice  before  the  nucleus  undergoes 
its  two  divisions,  with  the  result  that  each  spore  at  first  contains  one 
(Fig.  114).  In  Anthoceros  every  gametophytic  and  sporophytic  cell 
contains  one  plastid  owing  to  this  behavior  at  sporogenesis  and  to  the 


156 


FUNDAMENTALS  OF  CYTOLOGY 


further  fact  that  the  egg  contains  one  plastid  while  the  sperm  contributes 
none  at  syngam.y. 

Apospory  and  apogamy  appear  to  be  very  rare  in  bryophytes  in 
nature.  The  ease  with  which  apospory  can  be  induced  in  mosses,  how- 
ever, renders  these  plants  particularly  valuable  in  the  study  of  polyploidy. 
By  placing  small  pieces  of  immature  sporophyte  stalks  under  suitable 
cultural  conditions,  they  can  be  made  to  produce  gametophytes  by 
regeneration.  These  gametophytes  are  diploid,  and  their  diploid 
gametes  unite  to  form  tetraploid  sporophytes.  This  process  can  be 
repeated,  giving  tetraploid  gametophytes  and  octoploid  sporophytes. 
Sterility  in  some  degree  accompanies  these  new  chromosomal  states. 
By  means  of  hybridization,  much  higher  degrees  of  polyploidy  have  been 
obtained.  Chromosome  doubling  can  also  be  induced  by  chilling  moss 
protonemata  and  by  injecting  young  spore  capsules  of  liverworts  with 
certain  chemicals. 


Fig.  114. — Behavior  of  plastids  during  sporogenesis  in  a  moss  {Polytrichum) .  a,  com- 
pletion of  plastid  division  in  sporogenous  cell,  b,  thready  condition  of  plastids  in  sporocyte 
about  to  divide,  c,  nucleus  divided  into  four  (three  visible) ;  immediately  after  cytokinesis 
each  spore  will  contain  one  nucleus  and  one  plastid.  d,  young  spore  with  two  plastids 
formed  by  division.  The  cells  of  the  gametophyte  have  more  plastids.  {After  T.  E. 
Weier.) 

Algae. — The  variety  of  ways  in  which  asexual  reproduction  by  spores 
and  sexual  reproduction  by  gametes  are  correlated  in  the  life  cycles  of 
algae  is  of  great  interest  to  both  cytologists  and  students  of  phylogeny, 
for  it  affords  some  basis  for  speculation  as  to  the  origin  of  the  conditions 
observed  in  other  groups  of  organisms.  In  the  paragraphs  below  we 
shall  therefore  use  the  life  cycle  as  a  general  basis  of  description,  directing 
attention  here  and  there  to  special  cytological  features. 

Considering  first  the  green  algae,  it  is  found  that  some  of  the  most 
familiar  genera,  such  as  Vlothrix,  Oedogonium,  and  Spirogyra,  have  the 
reduced  chromosome  number  throughout  the  life  cycle  except  in  the 
zygote.  They  are  therefore  termed  haplonts  and  show  no  alternation  of 
vegetative  generations.  The  Ulothrix  plant  reproduces  asexually  by 
means  of  motile  zoospores.  Under  appropriate  conditions  motile 
biciliate  gametes  similar  in  form  to  the  zoospores  are  produced,  and 
these  unite  two  by  two  to  form  zygotes.  The  two  which  unite,  although 
morphologically  alike,  are  "plus"  and  "minus"  (female  and  male?)  in 
reaction  and  come  from  different  filaments:  the  plants  are  heterothallic. 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS 


157 


The  zygote  upon  germination  produces  four  nonmotile  or  motile  spores 
with  the  reduced  chromosome  number.  These  in  turn  develop  into 
new  plants. 

Oedogoniiim  produces  motile  asexual  zoospores  and  male  gametes 
which  resemble  them  in  having  a  crown  of  many  cilia  (Fig.  115).  The 
female  gamete,  how^ever,  is  a  large  nonmotile  egg  with  a  visibly  differ- 
entiated "receptive  spot"  at  the  point  where  the 
sperm  is  to  enter.  The  nucleus  lies  near  this  spot. 
The  sperm  passes  through  a  pore  in  the  wall  of  the 
cell  bearing  the  egg,  and  syngamy  takes  place. 
The  resulting  zygote  ripens  into  a  resting  oospore, 
and  as  it  does  so  meiosis  occurs.  Upon  germina- 
tion of  the  oospore  four  zoospores,  each  with  the 
reduced  chromosome  number,  are  produced,  and 
these  develop  into  new  plants.  Some  species  are 
heterothallic,  a  segregation  of  the  two  sex  ten- 
dencies occurring  at  meiosis. 

The  motor  apparatus  of  the  motile  cells  of 
Oedogonium  is  noteworthy  (Fig.  116).  The 
nucleus  of  the  cell  which  is  to  transform  into  a 
zoospore  comes  in  contact  with  the  cell  mem- 
brane, which  there  forms  a  convex  thickening. 
A  ring  of  granules  appears  in  this  region,  and  as 
the  nucleus  moves  away  the  ring  becomes  double, 
a  crown  of  cilia  then  growing  out  from  the  outer 
half  of  the  ring.  Such  cilia-bearing  organs  formed 
apparently  by  the  cell  membrane  are  called 
plasmodermal  blepharoplasts  to  distinguish  them 
from  the  centrosomal  blepharoplasts  of  ferns  and 
mosses.  It  is  of  interest  to  observe  that  the 
zoospore  resembles  the  motile  gamete  morphologi- 
cally^ in  Ulothrix  and  Oedogonium,  although  the 
morphology  differs  in  the  two  genera.  This  sug- 
gests a  common  origin  of  the  two  types  of  reproductive  cell. 

Syirogyra  differs  sharply  from  the  genera  described  above  in  having 
neither  zoospores  nor  other  ciliate  cells.  At  the  time  of  sexual  reproduc- 
tion two  cells  become  joined  by  a  conjugating  tube  (Fig.  117).  Their 
two  protoplasts  with  their  remarkable  chloroplasts  then  become  slightly 
modified  in  appearance  and  behave  as  gametes.  One  of  them  may 
pass  through  the  conjugating  tube  to  the  other,  with  which  it  then 
unites,  or  both  protoplasts  may  move  into  the  tube  and  unite  there. 
Contractile  vacuoles  have  been  found  to  play  a  role  in  the  movement  of 
these  gametes  ])y  withdrawing  water  from  the  central  sap  vacuole  and 


Fiu.  115. — Sexual  re- 
production in  Oedogo- 
nium. One  cell  of  the 
filament  has  enlarged  as 
an  oogonium  and  contains 
a  large  egg.  A  sperm  is 
about  to  enter  the  oogo- 
nium by  way  of  a  pore 
with  an  extruded  slime 
papilla.  {Modified  from 
H.  Khhahn.) 


158 


FUNDAMENTALS  OF  CYTOLOGY 


discharging  it  between  the  protoplast  and  the  cell  wall.  The  nuclei 
of  the  two  gametes  fuse,  but  their  plastids  do  not.  It  appears  that  in 
some  species  where  only  one  of  the  gametes  migrates  the  plastids  of  this 
gamete  degenerate  in  the  fusion  cell,  leaving  those  of  the  other  gamete 
to  continue  into  the  next  generation.  This  is  more  easily  demonstrated 
in  the  related  genus  Zygnema.  As  the  zygote  matures  and  becomes  a 
thick-walled  resting  zygospore,  the  fusion  nucleus  divides  meiotically, 
three  of  the  four  resulting  nuclei  then  degenerating  (Fig.  118).  Upon 
germination  the  zygospore  develops  into  a  new  plant.  Of  frequent 
occurrence  in  these  genera  is  the  formation  of  parthenospores,  outwardly 
resembling  the  zygospores,  by  single  cells  without  fusion. 

Chlamydomonas,  a  unicellular  green  alga,  is  also  a  haplont.     The 
motile  vegetative  cells  under  certain  conditions  unite  two  by  t^^•o  as 


^-::^i'w^^2£l- 


^.,^£^EP*\ 


Fig.  116. — Development  of  blepharoplast  in  zoospore  of  Ocdogonium.      (After  H.  Krctschmer.) 

gametes.  The  resulting  zygote  is  diploid,  and  when  it  germinates  meiosis 
occurs,  each  of  the  four  new  motile  cells  arising  from  it  having  the  reduced 
chromosome  number.  These  organisms,  which  can  be  grown  in  large 
numbers  in  a  small  space,  have  been  the  subject  of  a  number  of  genetical 
researches.  The  inheritance  of  characters  exhibited  by  the  vegetative 
cells  and  zygotes  are  as  should  be  expected  in  an  organism  with  this  type 
of  life  cycle. 

A  second  general  tj^pe  of  life  cycle  is  represented  in  certain  species  of 
Cladophora  and  other  genera.  In  C.  suhriana,  for  example,  there  are  two 
kinds  of  plants  that  look  but  do  not  behave  alike.  The  plants  of  one 
type  arise  from  zoospores,  have  the  monoploid  chromosome  number, 
and  produce  biciliate  gametes.  This  species  is  heterothallic.  After 
the  union  of  two  gametes,  the  resulting  zygote  grows  into  a  diploid 
plant  which  bears  monoploid  zoospores.  Hence  this  cycle  shows  a  well- 
marked  alternation  of  generations,  and  species  having  it  are  termed 
diplohaplonts. 

A  third  general  type  of  cycle  is  found  in  Cudium  and  certain  other 
genera  of  the  coenocytic  Siphonales.  These  plants  show  no  alternation 
of  generations  and  are  diplonls,  the  nuclei  being  diploid  throughout  the 


CYTOLOGY  OF  REFRODUCTION  IN  PLANTS 


159 


whole  cycle  except  in  the  gametes.     Meiosis  occurs  in  the  male  and 
female  gametangia  when  the  nuclei  of  the  biciliate  gametes  are  formed. 


Developing  Conjugation  Tube 


Conjugation  Tube 


Male  Gamete 


Male  Gamete 


Female  Gamete 


Female  Gam£te 
Zygote 


Fi(i.  117. — Stages  in  the  union  of  gametes  and  the  formation  of  the  zygote  in  the  green 
alga  Spirogyra.  Explanation  in  text.  {From  Smith,  Overton,  et  al.:  A  Textbook  of  General 
Botany,  Ath  ed..  The  Mactnillan  Com^pany.) 


Fig.  1 
in  text. 
Company. 


18 
{Fro 

.) 


Stages  in  tlie  history  of  a  zygote  of  the  green  alga  Spirogyra.      Explanation 
m  Smith,  Overton,  et  al.:  A  Textbook  of  General  Botany,  -Ith  ed..  The  Macmillnn 


Such  gametic  meiosis  is  very  rare  in  plants.  The  female  gamete  contains 
numerous  plastids,  while  the  male  is  much  smaller  and  contains  only  one. 
After  syngamic  union  the  resulting  zygote  develops  at  once  into  a  new 


160  FUNDAMENTALS  OF  CYTOLOGY 

plant.  Cladophora  glomerata  is  also  reported  to  be  a  diplont,  but  it 
differs  from  C odium  in  having  zoospores:  these  occur  throughout  the 
year,  while  gametes  are  formed  only  in  the  spring. 

The  hrown  algae  also  exhibit  life  cycles  of  the  three  main  types 
described  above.  Ectocarpus  virescens  is  a  haplont.  The  plant  body 
produces  biciliate  gametes  which  fuse  in  pairs,  and  the  resulting  zygotes 
undergo  meiosis  as  they  germinate  to  form  new  plants.  Other  types  of 
cj^cle  are  shown  by  certain  other  species  of  the  genus.  Evidentl}^  the 
same  species  may  differ  in  this  respect  in  the  North  Atlantic  Ocean  and 
the  Mediterranean  Sea. 

Dicfyota  dichotoma  is  a  diplohaplont.  There  are  three  kinds  of  plant 
.similar  in  external  appearance.  Monoploid  female  plants  produce 
large  nonmotile  eggs  which  are  liberated  into  the  sea  water.  These 
are  fertilized  by  small  uniciliate  sperms  from  monoploid  male  plants. 
The  zygotes  germinate  in  a  few  hours  and  develop  into  diploid  plants 
producing  tetraspores.  These  tetraspores,  which  are  products  of 
meiotic  divisions,  develop  into  new  sexual  plants.  In  Lami7iaria  the 
gametophytic  phase  is  very  minute  and  consists  of  onl}^  a  few  cells.  The 
very  large  body  (kelp)  is  the  sporophytic  phase. 

Fucus  and  species  of  certain  other  genera  are  diplonts.  Here  the 
plant  bod}^  is  diploid  and  produces  large  motile  eggs  and  very  small 
laterally  biciliate  sperms.  Some  species  are  dioecious.  The  zygotes 
very  soon  germinate  and  develop  into  new  diploid  bodies.  Since  meiosis 
occurs  in  the  divisions  of  the  gametogenous  cells  in  the  sex  organs,  there 
are  no  monoploid  vegetative  cells  in  the  cj^cle.  There  are  no  zoospores. 
It  is  thought  that  this  form  of  cycle,  which  is  very  rare  in  plants,  may 
have  arisen  through  the  assumption  of  gametic  functions  by  spores. 
This  should  not  be  surprising  in  view  of  the  fact  that  the  spores  in  many 
plant  groups  show  a  differentiation  into  two  types  correlated  with  the 
sex  of  the  plants  they  produce.  It  is  of  further  interest  to  note  that  in 
brown  algae  (e.g.,  Cutleria)  having  zoospores  these  cells  and  the  male 
gametes  are  both  laterally  biciliate. 

Of  the  several  cycle  types  observed  among  the  red  algae  only  two 
will  be  mentioned  here.  In  Polysiphonia  violacea  there  are,  as  in  the 
brown  alga  Dictyota  dichotoma,  morphologically  similar  plants  of  three 
kinds:  monoploid  females,  monoploid  males,  and  diploid  tetraspore- 
bearing  plants.  The  female  gamete  is  at  the  base  of  a  flask-shaped  cell 
with  a  long  hair-like  extension,  the  trichogyne.  Small  nonmotile  male 
gametes  (spermatia)  from  male  plants  become  attached  to  the  trichogynes. 
A  spermatium  nucleus  enters  the  trichogyne  and  moves  through  it  to 
the  female  nucleus,  with  which  it  unites.  The  diploid  nucleus  then 
divides,  and  the  cell  sends  out  short  filaments  from  the  ends  of  which 
diploid  carpospores  are  budded  off.     About  the  mass  of  carpospores  the 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS 


161 


vegetative  cells  form  a  cystocarp.  The  carposporcs  later  develop  into 
diploid  plants  which  bear  monoploid  tetraspores.  These  in  turn  develop 
into  monoploid  male  and  female  plants.  Polysiphonia  thus  has  a  regular 
alternation  of  sporophytic  and  heterothallic  gametophytic  generations 
in  the  cycle,  and  in  addition  it  has  a  diploid  carposporic  phase  between 
S3'ngamy  and  the  initiation  of  sporophj'te  development.  This  extra 
phase  is  more  prominent  in  certain  other  genera. 

Nemalion  and  Scinaia  are  haplonts.  The  only  vegetative  plants  are 
sexual,  and  after  syngamy  meiosis  occurs  in  the  zygote.  Carpospores 
soon  formed  by  proliferation  from  the  zygote  are  monoploid  and  produce 
new  sexual  plants. 


Fig. 


5  6  7  8 

119. — Diagram  of  nuclear  behavior  during  conjugation  in   a  diatom    (Rhopalodia) . 


Explanation  in  text.      {Based  on  drawings  by  H.  Klebahn.) 

Among  the  diatoms  the  cytology  of  reproduction  is  best  known  in  the 
bilaterally  symmetrical  Pennales.  When  ordinary  vegetative  multiplica- 
tion by  cell  division  occurs,  the  two-valved  shell  is  pushed  apart  and  each 
of  the  two  daughter  cells  secretes  a  new  valve  fitting  inside  the  old  one; 
hence  in  successive  asexual  generations  the  average  size  of  the  cells  tends 
to  diminish.  At  the  time  of  sexual  reproduction,  e.g.,  in  Rhoyalodia 
(Fig.  119),  two  individuals  conjugate  in  a  common  mass  of  secreted  jelly. 
In  each  individual  the  nucleus  undergoes  two  successive  divisions  which 
are  meiotic  in  character.  Two  of  the  resulting  nuclei  become  large  and 
two  small.  Cytokinesis  occurs  in  a  transverse  plane,  giving  two  cells 
each  with  a  large  and  a  small  nucleus.  Then  these  cells  fuse  with 
two  similar  ones  formed  in  the  same  manner  by  the  other  individual. 
The  large  nuclei  unite,  while  the  small  ones  disappear.  The  two  zygotes 
then  grow  and  become  large  mixospores  which  secrete  a  pectic  covering 
and  eventually  the  siliceous  wall  characteristic  of  the  species.     In  this 


162  FUNDAMENTALS  OF  CYTOLOGY 

process  the  maximum  cell  size  is  restored.  Diatoms  with  this  type  of 
cycle  are  diplonts.  The  radially  symmetrical  Centrales  have  been 
supposed  to  be  haplonts,  but  recent  investigations  have  shown  that  at 
least  some  of  them  are  diplonts. 

In  the  hlue-greeti  algae  no  sexual  reproductive  cycle  has  ever  been 
discovered.  The  cell  in  this  group  multiplies  by  division  and  in  some 
genera,  e.g.,  Oscillatoria,  multicellular  filaments  are  formed.  These 
filaments  multiply  by  simple  fragmentation.  At  the  time  of  cell  division 
the  problematical  central  body,  which  varies  widely  in  distinctness  and 
resembles  a  nucleus  to  the  extent  of  containing  chromatin,  may  also 
divide,  but  its  exact  nature  and  its  function  are  not  yet  understood. 

Fungi. — The  fungi,  like  the  algae,  show  a  rather  bewildering  array 
of  life-cycle  types.  This  section  must  therefore  be  limited  to  a  few  of  the 
cytologicall}^  better  known  cases.  These  will  illustrate  the  characteristic 
reproductive  behavior  observed  in  the  main  subdivisions  of  this  great 
plant  phylum. 

In  the  phycomycetes  the  m^xelium  is  coenocytic.  In  the  order 
Mucorales  abundant  asexual  spores  are  produced  in  sporangia  on  the 
mycelium.  The  sexual  process  involves  the  fusion  of  the  multinucleate 
contents  of  two  gametangia.  The  fusion  product  becomes  a  thick-walled 
resting  zygospore.  This  germinates  to  form  a  mycelium  or  a  "germ 
sporangium,"  the  spores  from  which  produce  new  mycelia.  The  nuclear 
behavior  during  this  process  is  exceedingly  difficult  to  follow,  but  recent 
studies  indicate  several  variations  in  the  different  species  examined: 
(1)  the  nuclei  fuse  in  pairs  and  then  undergo  mciosis  before  the  zygospore 
matures  (Mucor  genevensis  and  others) ;  (2)  some  of  the  nuclei  fuse,  the 
others  degenerate,  and  meiosis  is  delayed  until  zygospore  germination 
{e.g.,  Rhizopus  nigricans);  (3)  the  nuclei  associate  in  groups  in  the  zygo- 
spore, and  some  of  them  fuse  in  pairs  just  before  germination,  meiosis 
following  in  the  developing  germ  sporangium  {e.g.,  Phycomyces  Blakes- 
leeanus);  (-1)  there  are  no  nuclear  fusions  at  any  stage  {e.g.,  Sporodinia 
grandis).  In  heterothallic  species  of  Mucorales  the  plus  and  minus 
tendencies   are   segregated  in  the  spores  from  the   germ   sporangium. 

In  the  Saprolegniales  there  are  motile  asexual  zoospores.  There  are 
also  well-differentiated  eggs  produced  singl}^  or  in  groups  in  oogonia. 
An  antheridium  applies  itself  to  the  surface  of  an  oogonium  and  sends 
in  a  tube  which  delivers  a  male  nucleus  to  the  single  egg  {Pythium). 
When  there  are  several  eggs  in  the  oogonium  {Saprolegnia) ,  the  antheridial 
tube  branches  and  delivers  a  nucleus  to  each  of  them.  The  zygote 
takes  the  form  of  a  resting  oospore.  When  it  germinates,  meiosis  occurs 
(demonstrated  in  Achlya),  indicating  that  these  plants  are  haplonts. 

In  the  Blastocladiales  there  is  a  genus  {AUomyces)  in  which  several 
strains  have  been  shown  to  be  diplohaplonts.     The  sexual  plants  bear 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS  163 

iiniciliate  male  and  female  gametes  differing  in  size.  The  plants  devel- 
oped from  the  zygotes  bear  zoospores  which  j^roduce  the  next  generation 
of  sexual  plants. 

The  most  familiar  basidiomycetcs  are  the  rusts,  which  do  so  much 
damage  to  grain  crops,  and  the  mushrooms.  The  latter  will  furnish 
our  example  of  nuclear  cytology  in  this  group  of  fungi  (Fig.  120).  Th(> 
spores  discharged  from  the  gills  or  pores  of  the  mushroom  germinate  and 
produce  a  septate  primary  mycelium,  usually  \\dth  one  nucleus  per  cell 
but  sometimes  with  more.  IMost  species  are  heterothallic,  the  spores 
being  of  two  kinds  and  producing  plus  and  minus  mycelia,  respectively. 
A  primary  mycelium  may  produce  small  asexual  reproductive  bodies 
known  as  oidia,  but  usually  it  does  not  develop  the  familiar  sporophores, 
or  mushrooms.  When  plus  and  minus  mycelia  come  in  contact,  their 
hyphae  unite  at  one  or  more  points  where  openings  are  formed  in  the 
intervening  walls.  At  each  point  of  union  the  plus  nucleus  divides,  one 
of  the  daughter  nuclei  then  passing  into  the  minus  hypha.  At  the  same 
time,  the  minus  nucleus  divides,  one  of  its  daughter  nuclei  passing  into 
the  plus  hypha.  In  this  way  each  primary  mycelium  comes  to  have  a 
binucleate  cell.  In  the  plus  hypha  the  introduced  minus  nucleus  divides, 
one  of  the  products  passing  through  a  pore  in  the  w^all  into  an  adjacent 
plus  cell,  rendering  it  binucleate  also.  This  process  is  repeated  cell  by 
cell  until  much  of  the  primary  plus  mycelium  becomes  diploidized.  The 
binucleate  mycelium  so  formed  is  called  a  secondary  mycelium.  Mean- 
while the  same  process  may  be  carried  out  by  the  plus  nucleus  delivered 
to  the  minus  mycelium  so  that  it,  too,  becomes  diploidized.  The  two 
mycelia  are  thus  mutually  diploidized.  As  the  binucleate  secondary 
mj'celium  continues  its  growth,  the  nuclei,  now  in  pairs,  divide  in  unison 
(conjugately).  Each  pair  of  nuclei  is  called  a  dikaryon,  and  this  second- 
ary mycelium,  which  is  the  stage  commonly  observed  in  nature,  is  accord- 
ingly known  as  the  dikai-yophase.  It  is  often  said  to  be  diploid,  even 
though  the  nuclei  are  individually  monoploid,  for  the  protoplasmic 
activity  is  now  influenced  by  two  genomes  differing  in  constitution. 

Numerous  hyphae  of  the  secondary  type  become  much  intertwined 
and  differentiated  as  thick  strands  (rhizomorphs)  which  develop  the 
sporophores.  At  the  surface  of  the  gills,  or  in  some  species  the  pores,  of 
the  sporophore  the  ends  of  some  of  the  binucleate  hj'phae  enlarge  as 
basidia.  In  each  young  basidium  are  two  nuclei  which  are  descendants 
of  the  two  brought  together  when  primary  hyphal  union  and  diploidiza- 
tion  occurred.  The  two  nuclei  now  fuse  (karyogamy),  doubling  the 
chromosome  number.  Then  the  diploid  nucleus  undergoes  two  meiotic 
divisions,  and  the  four  resulting  nuclei  pass  into  the  four  basidiospores 
which  are  budded  off  from  the  basidium.  Wh(^n  these  spores  germinate, 
two  of  them  produce  plus  while  two  produce  minus  mycelia.     This  can 


1G4 


FUNDAMENTALS  OF  CYTOLOGY 


be  determined  by  removing  the  quartet  of  spores  from  a  basidium  and 
cultivating  them  separately. 

In  this  reproductive  cycle,  which  is  paralleled  in  structures  of  very 
different  appearance  in  the  rusts,  the  stage  of  greatest  cytological  interest 
is  the  dikaryophase.  The  significance  of  this  peculiar  nuclear  state  will 
be  pointed  out  below. 

The  ascomycetes,  like  the  basidiomycetes,  include  forms  with  cycles 
including  a  dikaryophase.     The  spores,  which  are  borne  in  sacs,  or  asci, 


Fig.  120. — Diagram  of  nuclear  history  in  the  Ufe  cycle  of  a  heterothallic  basidiomycete. 
At  the  point  of  hyphal  union  the  plus  and  minus  nuclei  are  about  to  undergo  division. 
One  of  the  products  of  each  division  will  pass  to  the  other  hypha  (see  text).  One  primary 
mycelium  is  shown  partially  diploidized  as  it  would  be  at  a  later  stage;  the  other  is  com- 
monly diploidized  also.  Diploidization  has  been  followed  by  the  formation  of  a  sporophore. 
The  arrangement  of  successive  stages  in  the  basidia  is  arbitrary.  Natural  proportions  are 
not  represented,  and  nuclei  are  not  drawn  in  all  cells. 

produce  septate  uninucleate  mycelia,  which  in  heterothallic  species  are 
of  two  kinds,  plus  and  minus.  The  uninucleate  mycelium  in  forms  like 
Peziza  or  Pyronema  develops  the  familiar  apothecia,  or  open  cup-shaped 
fruit  bodies  (Fig.  121).  At  a  very  early  stage  in  the  development  of  the 
apothecium  there  differentiates  in  its  midst  a  multinucleate  female  organ, 
the  ascogonium,  and  a  multinucleate  antheridium.  The  nuclei  of  the 
latter  are  discharged  through  a  trichogyne  into  the  ascogonium,  where 
they  mingle  with  the  nuclei  of  the  ascogonium.  The  ascogonium  then 
sends  out  a  number  of  ascogenous  hyphae,  into  which  pairs  of  nuclei 


CYTOLiXiY  OF  REPRODUCTION  IN  PLANTS 


105 


((likaiya)  migrate.  As  these  hyphae  grow,  the  paired  iiiiclei  (li\'i(ie 
conjugatoly.  Meanwhile  the  surrounding  uninucleate  hyi)lia('  continue 
the  development  of  the  apothecium. 

Eventually  an  ascus  is  developed  from  the  subterminal  ctdl  of  each 
ascogenous  hypha.  The  two  nuclei  in  this  cell  unite,  and  very  soon 
the  resulting  diploid  nucleus  undergoes  three  successive  divisions,  of 
which  the  first  two  are  meiotic  in  character.     Spores  are  then  formed 


Fig.  121. — Diagram  of  nuclear  history  in  the  life  cycle  of  a  heterothallic  ascomycete. 
Early  and  late  stages  of  the  cycle  are  shown,  the  early  stages  being  in  the  lower  portion  of 
the  diagram  and  later  stages  in  the  upper  portion.  Long  before  the  apothecium  and  the 
spores  are  mature  the  sex  organs  have  disappeared.  The  apothecium  is  composed  of 
uninucleate  hyphae  and  binucleate  ascogenous  hyphae.  The  arrangement  of  successive 
stages  in  the  asci  is  arbitrary.  In  the  ascus  at  the  extreme  left  the  last  conjugate  division 
is  being  completed.      Natural  proportions  are  not  represented. 

about  the  eight  nuclei  as  centers.  This  involves  a  curious  cytokinetic 
activity  of  the  astral  rays  remaining  from  the  third  nuclear  division. 
They  curve  around  from  each  centrosome  and  cut  out  a  portion  of  the 
cytoplasm  about  each  nucleus,  leaving  the  residual  cytoplasm  to  dis- 
integrate. The  eight  ascospores  sometimes  lie  in  a  row,  and  in  some 
species  the  positions  of  the  mitotic  figures  in  the  three  divisions  that 
produced  spore  nuclei  have  been  so  regular  that  it  can  be  readily  deter- 
mined which  portions  of  the  chromatic  matter  now  constituting  the  eight 
nuclei  were  separated  at  each  of  the  divisions.  This  has  made  it  possible 
in  certain  species  to  show  by  genetical  evidence  that  disjunction  of 


166  FUNDAMENTALS  OF  CYTOLOGY 

li()iii()l()jj;(nis  chromosomal  elements  occurs  in  the  first  two  divisions  but 
not  in  the  third.  This  is  of  special  interest  in  connection  with  a  conten- 
tion that  in  some  species  there  are  two  nuclear  fusions  in  the  cycle,  one 
in  the  ascogonium  and  another  in  the  ascus,  and  that  a  double  reduction 
compensates  for  this  in  the  three  ascus  divisions.  It  now  seems  evident 
that  if  such  a  process  occurs  it  is  very  rare. 

An  interesting  feature  of  nuclear  division  in  asci  is  the  intranuclear 
character  of  the  achromatic  figure.  As  the  nucleus  enlarges  in  the  pro- 
phase, the  spindle  develops  in  the  karyolymph  with  its  poles  at  the  nuclear 
membrane.  Asters,  when  present,  lie  in  the  cytoplasm.  The  membrane 
may  remain  intact  until  anaphase  or  even  throughout  the  entire  nuclear 
cycle,  constricting  between  the  daughter  chromosome  groups  at  the  close 
of  division.  Such  figures  are  also  present  in  certain  other  plant  and 
animal  cells. 

Conclusions. — The  series  of  three  chapters  now  being  concluded 
should  serve  several  purposes. 

1.  It  should  furnish  cytological  pictures,  albeit  sketchy  ones,  of  the 
reproduction  and  life  cycles  of  organisms  representing  many  natural 
groups.  These  constitute  an  essential  part  of  the  working  cytologist's 
background. 

2.  It  should  indicate  the  great  variet}^  of  materials  available  for 
research  projects  of  various  kinds.  The  success  of  a  project  may  depend 
largely  or  entirely  upon  a  wise  selection  of  an  organism  or  tissue  as  a 
basis  of  investigation,  and  there  are  many  of  these  to  choose  from  in 
nature.  From  what  has  been  set  forth  in  these  chapters  it  should  be 
obvious  that  one  can  find  materials  peculiarly  suited  to  the  study  of 
such  problems  as  the  role  of  asters  in  cytokinesis,  the  causes  and  effects 
of  parthenogenesis,  the  role  of  cells  as  units  in  tissues,  the  relative  effects 
of  monoploidy  and  diploidy,  the  process  of  secretion  in  cells,  the  role  of 
the  nucleus  in  development  and  heredity,  and  so  on. 

A  single  striking  illustration  of  this  is  afforded  by  certain  fungi 
described  in  the  preceding  section.  Cytologists  and  geneticists  have 
long  wished  to  know  if  or  in  what  manner  the  effect  upon  development 
exerted  by  a  fusion  nucleus  after  syngamy  differs  from  that  of  the  nuclei 
before  the  fusion;  in  other  words,  does  the  association  of  two  genomes 
within  a  common  nuclear  membrane  result  in  activity  differing  from 
that  of  two  unfused  monoploid  nuclei  lying  in  the  common  cytoplasm? 
Material  nicely  suited  to  the  solution  of  this  problem  has  been  found  in 
the  peculiar  dikaryophase  of  basidiomycetes  and  ascomycetes.  In 
Penio-phora  and  Neurospora,  representing  these  two  groups  respectively, 
evidence  is  accumulating  that  will  yield  an  answer  to  this  important 
question.  At  the  present  time  it  appears  that  at  least  some  of  the 
activities  of  a  fusion  nucleus  are  duplicated  by  the  dikaryon,  or  unfused 


CYTOLOGY  OF  REPRODUCTION  IN  PLANTS  167 

pair  of  nuoloi.  H(>rice  this  peculiar  nuclear  condition  in  a  pnvi  of  the  hfe 
cycle,  the  possibility  of  determining  the  capacities  of  each  sjjore  of  a 
given  quartet  or  octet,  and  the  convenience  and  rapidity  with  which 
the  plants  maj^  be  cultured  and  subjected  to  biochemical  analysis  (page 
202),  have  combined  to  bring  these  fungi  from  obscurity  to  a  prominent 
position  in  biological  research. 

3.  These  chapters  show  in  what  a  great  variety  of  ways  a  given 
process  may  be  carried  out  or  a  given  result  attained.  A  recognition  of 
the  unity  underlying  all  this  diversity  should  enable  one  to  distinguish 
more  surely  what  is  fundamental  from  what  is  accessory  or  only  inci- 
dental. Concepts  based  on  only  one  or  two  typical  cases  often  require 
revision  when  viewed  in  the  light  of  what  occurs  in  organisms  of  many 
kinds.  Far  too  many  definitions  of  biological  phenomena  are  merely 
descriptions  of  single  examples  of  a  class  and  fail  to  indicate  what  is 
significant  in  all  cases.  The  mastery  of  many  definitions  is  a  poor 
substitute  for  the  possession  of  a  few  broadly  based  concepts. 

Finally,  an  acquaintance  with  the  cytological  diversities  of  plants  and 
animals  should  contribute  something  to  the  value  of  one's  speculations 
on  the  origin  of  the  cytological  constitutions  and  reproductive  processes 
characterizing  the  various  groups.  This  is  a  part  of  the  great  problem 
of  phylogeny,  which,  because  of  its  complexity  and  the  significance  of 
its  conclusions,  must  be  investigated  with  thoroughness  and  long  suspen- 
sion of  judgment.  In  no  other  field  of  inquiry  is  it  truer  that  ''it  is 
easy  to  use  simple  logic  when  the  facts  are  few." 


CHAPTER  XII 
CYTOLOGY  AND  MENDELIAN  HEREDITY 

For  two  thousand  years  and  more,  men  have  speculated  on  the  causes 
of  Hkeness  and  difference  between  parent  and  offspring.  The  fact  that 
some  parental  characters  are  transmitted  to  the  immediate  offspring 
while  others  are  not,  although  the  latter  may  reappear  several  generations 
later,  exemplifies  the  puzzles  that  could  not  have  been  solved  until 
recent  times.  Actuall}^  most  of  the  characters  exhibited  by  organisms 
are  not  literally  transmitted  at  all:  they  are  developed  anew  in  each 
generation.  The  protoplasmic  system  which  performs  this  development 
is  itself  a  direct  inheritance  from  the  previous  generation;  hence,  what  is 
transmitted  is  a  mass  of  protoplasm  capable  of  developing  the  characters 
under  the  appropriate  environmental  conditions.  Since  each  generation 
begins  as  a  bit  of  protoplasm  derived  from  the  previous  generation, 
similarities  between  the  two  are  expected,  while  differences  must  be  due 
either  to  the  environmental  conditions  or  to  an  actual  constitutional 
difference  between  the  protoplasms  with  which  the  two  generations  begin 
their  development.  Under  uniform  environmental  conditions  the 
similarities  and  differences  have  been  found  to  appear  according  to 
definite  rules.  The  inference  from  this  is  that  there  is  in  the  protoplasm 
an  organized  system  of  some  kind  that  persists  through  successive 
generations,  yet  undergoes  minor  orderly  alterations  affecting  the  char- 
acters developed.  The  problem  of  modern  cytogenetics  is  that  of 
describing  this  system,  its  transmission,  its  alterations,  and  its  mode  of 
action  in  ontogeny. 

Before  an  adequate  cytological  theory  of  heredity  could  be  devised, 
certain  prerequisites  had  to  be  furnished.  (1)  There  was  needed  a  more 
precise  formulation  of  the  specific  facts  to  be  explained.  This  was 
supplied  by  the  famous  nineteenth-century  researches  of  Mendel,  who 
made  mathematical  analyses  of  the  manner  in  which  individual  char- 
acters were  inherited  after  carefully  controlled  crosses.  From  these  he 
derived  the  laws  that  bear  his  name.  (2)  There  was  needed  a  more 
detailed  description  of  the  organism's  life  cycle,  especially  through 
the  reproductive  phases,  in  terms  of  visible  structural  units — cells, 
nuclei,  and  chromosomes.  This  physical  framework  for  a  theory  was 
also  largely  furnished  in  discoveries  made  during  the  nineteenth  centurj-: 
the  genetic  continuity  of  protoplasm;  the  regular  presence  of  the  nucleus; 

168 


CYTOLOGY  AND  MEN  DELI  AN  HEREDITY  109 

the  multiplication  of  cells  and  nuclei  by  division;  the  presence  of  chromo- 
somes and  their  equational  division;  the  disjunction  of  homologous 
pairs  of  chromosomes  at  meiosis;  the  fusion  of  parental  nuclei  in  syngam>-; 
the  fact  that  one  of  the  fusing  gametes  may  be  only  a  nucleus;  the 
presence  of  a  set  of  chromosomes  from  each  parent  in  all  the  offspring's 
nuclei. 

The  rise  of  cytogenetics  as  a  modern  branch  of  biological  science  began 
with  the  opening  of  the  twentieth  century,  when  it  was  realized  that  the 
phenomena  of  inheritance  described  by  Mendel  in  1865  and  redisco^  ered 
in  1900  could  probably  be  explained  on  the  basis  of  chromosome  behavior. 
That  nuclear  elements  had  a  special  role  of  this  sort  was  advocated  before 
1900  by  Weismann  and  others.  Mendel's  clear  analysis  of  his  data 
and  his  interpretation  of  the  results  in  terms  of  representative  factors, 
combined  with  definite  findings  regarding  the  chromosome  cycle,  made  it 
possible  in  this  century  for  the  first  time  to  proceed  effectively  with  an 
experimental  investigation  of  the  whole  problem.  How  intimate  the 
fusion  of  cytology  and  genetics  has  become  will  be  evident  in  the  pages 
to  follow. 

Examples  of  Mendelian  Heredity. — We  may  begin  with  one  of 
Mendel's  own  classic  experiments  with  garden  peas  (Fig.  122).  Plants 
of  a  pure-bred  race  of  tall  peas  (6  to  7  feet  in  height)  and  plants  of  a 
pure-bred  dwarf  race  (^/^  to  13^  feet  in  height)  were  selected  for  parents 
(Pi).  When  the  two  types  were  crossed,  all  the  plants  of  the  first  filial 
generation  (Fi)  were  tall  like  one  of  their  parents.  When  these  tall 
hybrids  were  self-pollinated  (or  bred  to  one  another),  it  was  found  that 
the  second  generation  (Fo)  comprised  individuals  of  the  two  grand- 
parental  types,  tall  and  dwarf,  in  the  ratio  of  3 : 1 .  It  was  further  found 
that  the  tall  individuals  of  this  generation,  though  alike  in  visible  char- 
acters, were  unlike  in  genetical  constitution:  one-third  of  them,  if  bred 
among  themselves  for  another  generation,  produced  nothing  but  tall 
offspring,  showing  that  they  were  pure  for  the  character  of  tallness; 
whereas,  the  other  two-thirds,  if  similarly  bred,  produced  again  in  the 
next  generation  both  tall  and  dwarf  plants  in  the  ratio  of  3:1,  showing 
that  they  were  hybrids  with  respect  to  tallness  and  dwarfness.  The 
dwarf  plants  of  the  second  generation  (Fo)  produced  nothing  but  dwarfs 
Avhen  selfed:  thej^  were  pure  for  dwarfness.  From  these  facts  it  was 
evident  that  the  plants  of  the  F2  generation,  although  they  formed 
only  two  visibly  distinct  classes,  were  in  reality  of  three  kinds:  pure  tall 
individuals,  tall  hybrids,  and  pure  dwarfs,  these  kinds  occurring  in  the 
ratio  of  1:2:1. 

The  explanation  offered  by  Mendel  for  these  phenomena  may  be 
stated  briefly  as  follows.  The  germ  cells  produced  by  the  pure  tall 
plant  carry  something  (now  termed  a  factor,  or  gene,  represented  in  Fig. 


170 


FUNDAMENTALS  OF  CYTOLOGY 


122  by  T)  which  tends  to  make  the  resulting  plant  tall.  The  germ  cells 
of  the  dwarf  carry  something  {t)  causing  the  dwarf  condition.  In  the 
first  hybrid  generation  {h\)  both  factors  are  present,  T  coming  from  one 
parent  and  t  from  the  other,  but  T  dominates  and  prevents  the  expression 
of  the  recessive  i  so  that  the  plants  of  this  generation  are  all  tall.  When 
the  hybrid  (Fi)  produces  germ  cells,  the  two  factors  for  tallness  and 
dwarfness  segregate,  half  the  gametes  receiving  T  and  the  other  half 
t.  Each  gamete  therefore  carries  either  one  or  the  other  of  the  two 
factors  in  question  but  never  both;  it  is  pure  either  for  T  or  for  t.  This 
segregation  in  the  germ  cells  of  factors  associated  throughout  the  soma 
is  the  central  feature  of  the  entire  series  of  Mendelian  phenomena  and 
is  the  basis  of  Mendel's  first  law.  Since  the  gametes,  both  male  and 
female,  produced  by  the  hybrid  plants  of  the  Fi  generation  are  of  two 
kinds  (half  of  them  bearing  T  and  half  bearing  t),  four  combinations  are 


Parents 

I 


PureTall 
TT 


Pure  Dwarf  • 


F. 


Toll  Hybrid 
Ti 


Tall  Hybrid 
Tt 


Gameies  Fj 

I  1 

PureTall       TT  -  Breeds  true 
Tall  Hybrid  Tt    -  3>  ■  \ 

Tall  Hybrid  Tt   -  3    \ 

Pure  Dwarf    tt    -  Breeds  true 


Fig.  122. — A  typical  case  of  Mendelian  liei'edity  in  the  garden  pea. 

now  possible:  a  T-sperm  with  a  T-egg,  a  T-sperm  with  a  /-egg,  a  /-sperm 
with  a  T'-egg,  and  a  /-sperm  with  a  /-egg.  These  four  combinations  result, 
respectively,  in  a  tall  plant  (pure  dominant,  T  T),  two  tall  hybrids  (T  t), 
and  a  dwarf  plant  (pure  recessive,  /  /) .  It  is  obvious  that  in  the  long  run 
these  three  types  will  tend  to  occur  in  the  ratio  of  1:2:1. 

The  pure  tall  individuals  and  the  tall  hybrids  in  F2  are  ordinarily 
distinguished  from  each  other  by  the  testcross,  or  backcross  test.  It  will 
be  readily  seen  that  when  a  pure  tall  plant  {T  T)  is  crossed  with  the  pure 
recessive  type  (/  /)  all  the  offspring  will  be  tall  (T  t);  whereas,  when  a  tall 
hybrid  (7"  /)  is  crossed  with  /  /,  half  the  offspring  will  be  tall  {T  /)  and 
half  will  be  dwarf  (/  /). 

The  ]\Iendelian  proportion  of  hybrids  and  pure  types  is  perhaps  better 
illustrated  by  characters  in  which  dominance  is  imperfect  or  lacking. 
In  four-o'clocks,  for  example,  certain  hybrids  are  more  or  less  inter- 
mediate with  respect  to  flower  color  and  are  easily  distinguishable  from 
the  pure  parental  types.  When  plants  bearing  pure  crimson  flowers  are 
crossed  with  those  bearing  pure  white  flowers,  the  hybrid  plants  of  the 
Fi  generation  have  magenta  flowers.     When  these  hybrids  are  bred  among 


CYTOLOGY  AND  MENDELIAN  HEREDITY  171 

themselves,  the  nvsultin^  /''•_.  K^'neratioTi  comprises  phints  of  three  visilily 
different  types:  i)ure  floiniiuiiits  with  crimson  flo\v(M-s,  hyl)ri{ls  with 
magenta  flowers,  and  f)ure  recessives  with  white  flowei-s;  these  types 
tend  to  occur  in  the  ratio  of  1:2:1. 

Two  points  should  be  emphasized  before  proceeding  further.  (1)  It 
should  be  understood  that  the  above  ratios  indicate  only  the  degree  of 
probability  of  obtaining  the  various  types  through  random  combination 
of  gametes.  When  the  number  of  individuals  is  sufficiently  large,  the 
ratios  tend  to  be  approached  rather  closely;  sometimes  they  are  equalled 
exactly,  even  in  small  populations.  The  ratios,  then,  represent  statistical 
expectations.  (2)  One  factor,  or  gene,  is  not  wholly  responsible  for  the 
production  of  a  given  character.  When  one  gene  is  singled  out  as  the 
gene  for  a  character,  it  is  either  because  it  is  the  most  influential  one 
known  to  affect  that  character,  or  because  it  is  the  only  one  of  the  influ- 
ential genes  that  exists  in  both  the  dominant  and  recessive  states  in 
the  material  studied.  Thus  it  is  the  "differential"  in  a  system  of  factors 
otherwise  uniform  throughout  the  material,  the  character  varying  with 
this  one  gene.  In  other  strains  of  mateiial  some  other  gene  might  be 
the  differential  one.  When  two  or  more  gene  pairs  are  acting  differ- 
entially at  the  same  time,  the  characters  appear  in  ratios  other  than  those 
stated  above.  From  such  ratios  the  geneticist  is  able  to  infer  the  number 
of  genes  concerned. 

Terminology. — The  genotype  is  the  entire  assemblage  of  genetic 
factors,  or  genes,  which  the  organism  actually  possesses  in  its  constitu- 
tion, irrespective  of  how  many  of  these  may  be  expressed  in  externally 
visible  characters;  or,  it  is  a  class  of  individuals  with  the  same  genetic 
constitution.  The  phenotype  is  the  aggregate  of  externally  visible 
characters,  irrespective  of  any  other  factors,  unexpressed  in  characters, 
which  ma}'  be  present  in  the  organism;  or,  it  is  a  class  of  outwardly 
similar  individuals.  For  illustration:  In  the  case  of  the  tall  and  dwarf 
peas  there  are  in  the  second  generation  (F-i)  three  genotypes  (with 
respect  to  the  single  character  pair  discussed) :  T  T,  T  t,  and  /  /,  repre- 
sented, respectively,  by  pure  tall  plants,  tall  hybrids,  and  dwarfs;  but 
there  are  only  two  phenotypes:  tall  and  dwarf,  because  the  complete 
dominance  of  tallness  over  dwarfness  renders  the  hybrids  externally 
indistinguishable  from  the  pure  tall  individuals.  Thus  one  phenotype 
(tall  plants)  here  includes  individuals  with  two  genotypic  constitutions, 
and  the  two  can  be  distinguished  only  by  a  study  of  their  progeny.  In 
the  four-o'clocks  described  above,  there  are  represented  in  the  F^  genera- 
tion not  only  three  genotypes,  but  also  three  phenotypes,  since  incomplete 
dominance  rendei-s  the  hybrids  externally  unlike  either  of  the  pure  forms. 
Practically,  a  phenotype  is  a  class  of  individuals  that  look  alike,  and  a 
genotype  is  a  class  of  individuals  that  breed  alike  (Castle). 


172  FUNDAMENTALS  OF  CYTOLOGY 

Two  contrasting  characters  such  as  tallness  and  dwarfness  are  said 
to  be  allelomo7'phs  or  alleles.  The  same  terms  are  also  used  for  the  pair 
of  differential  genes  influencing  them.  The  corresponding  adjectives 
are  allelomorphic  and  allelic. 

An  individual  is  said  to  be  homozygous  for  a  given  allelic  factor  pair 
if  it  has  received  the  same  type  of  factor  from  the  two  parents — a  pea, 
for  example,  with  the  constitution  T  T  or  t  t.  If  it  has  unlike  members 
in  the  pair,  such  as  T  t,  it  is  said  to  be  heterozygous.  It  may  be  homozy- 
gous for  some  pairs  and  heterozygous  for  others,  or  it  may  conceivably 
be  either  homozygous  or  heterozygous  for  all  its  factors.  Thus  an 
organism  with  the  genotypic  constitution  A  A  B  b  c  c  is  homozygous  for 
the  factors  A  A  and  cc  and  heterozygous  for  Bb.  It  is  a  pure  dominant 
with  respect  to  A  and  a,  a  pure  recessive  with  respect  to  C  and  c,  and  a 
hybrid  with  respect  to  B  and  b.  The  phenotypic  appearance  of  the 
organism  is  here  determined  by  the  dominant  factors  A  and  B  and  the 
recessive  c.  It  is  a  common  practice  to  represent  dominant  factors  by 
capital  letters  and  their  recessive  alleles  by  the  corresponding  small 
letters. 

Explanation  in  Terms  of  Chromosomes. — The  basic  reason  for  the 
manner  in  which  an  allelic  pair  of  characters  is  inherited  becomes  evident 
when  factor  distribution  is  compared  with  chromosome  distribution. 
Each  parent  furnishes  the  offspring  with  one  chromosome  set,  or  genome. 
Referring  to  Fig.  75,  it  is  seen  that  in  meiosis  the  two  members  of  an}' 
homologous  chromosome  pair,  e.g.,  the  ones  marked  A  and  a,  are  so 
distributed  that  a  descendant  of  A  lies  in  half  the  spores  or  gametes, 
while  a  descendant  of  a  lies  in  the  rest  of  the  spores  or  gametes.  If 
such  an  organism  is  self-fertilized  or  crossed  to  a  similar  one,  both  A 
gametes  and  a  gametes  being  functional  in  each  sex,  random  unions  will 
result  in  combinations  of  three  kinds  (A  A,  A  a,  a  a)  in  the  ratio  of  1:2:1. 
Hence  the  inference  is  that  a  given  allelic  pair  of  genes  is  located  in  a 
homologous  pair  of  chromosomes.  If  the  genes  T  and  t  are  so  located 
in  a  chromosome  pair  of  the  garden  pea,  Mendel's  results  with  tallness 
and  dwarfness  are  accounted  for.  His  first  law  was  a  result  of  chromo- 
some disjunction  at  meiosis,  although  at  the  time  of  its  formulation 
almost  nothing  was  known  about  chromosome  behavior. 

Mendel^lso  studied  six  other  pairs  of  heritable  characters  in  peas.  He 
observed  that  all  seven  pairs  (including  tallness  and  dwarfness)  were 
inherited  independently,  i.e.,  while  each  character  gave  the  usual  ratio 
with  its  allele,  there  was  no  tendency  on  the  part  of  any  character  to 
appear  more  often  with  a  given  nonallelic  character  than  with  any  other 
after  a  testcross.  This  independence  of  character  pairs  was  stated  in 
Mendel's  second  law.  A  case  of  this  kind  is  illustrated  in  the  left-hand 
part  of  Fig.  123,  which  shows  also  the  cytological  basis  for  it.     The  two 


CYTOLOGY  AND  MENDELIAN  HEREDITY 


173 


character  pairs  are  tall  vs.  dwarf,  and  green  pod  vs.  yellow  pod,  the 
first-named  character  in  each  pair  being  the  dominant  one.  A  plant 
dominant  and  homozygous  for  both  differential  gene  pairs  (7"  T  G-p  Gp) 
is  crossed  to  one  that  is  recessive  and  homozygous  for  both  pairs  (/  /  gp  gp). 
The  Fi  plants  are  heterozygous  for  both  pairs  (T  t  Gp  gp).  When  these 
plants  are  testcrossed  to  a,  t  t  gp  gp  plant,  plants  of  four  kinds  appear  in 
equal  numbers  in  Fo:  tall-green,  dwarf -yellow,  tall-yellow,  and  dwarf- 


T     AND      Gp     INDEPENDENT 


/\        and 
Somatic   nucleus  MefaphoseX    i 


1^ 


t  t  gp  gp  rTGpjftgpj       (Tgp)(tGp) 

Mgp),^cr".  Gametes 


1^ 


1^ 


T  t  Gp  gp 


Tt  gpgp  t  t  Gp 


IF    T    AND    Gp    WERE    LINKED 


T  t  Gp  gp 


t  t  gp  gp 


Fig.  123. — Diagram  illustrating  the  cytological  basis  of  independent  and  of  linked 
inheritance  of  characters.  Vertical  lines  represent  plants;  green  pods  stippled;  yellow  pods 
unshaded.  Actually,  these  particular  characters  are  independent,  not  linked.  Further 
explanation  in  text. 


green.  This  shows  that  the  Fi  plants  must  have  produced  spores  and 
gametes  of  four  kinds  in  equal  numbers:  T  Gp,  t  gp,  T  gp,  and  t  Gp.  The 
explanation  for  this  lies  in  the  fact  that  the  two  gene  pairs  concerned 
lie  in  different  chromosome  pairs  which  have  two  possible  relative  orienta- 
tions at  metaphase  /  in  the  sporocytes  of  the  Fi  plants.  Hence  Mendel's 
second  law  expressed  the  results  of  random  assortment  of  chromo.some 
l^airs  at  meiosis. 

An  important  qualification  has  had  to  be  made  in  Mendel's  second 
law,  now  that  more  character  pairs  have  been  studied.     Organisms  ha\-e 


174  FUNDAMENTALS  OF  CYTOLOGY 

large  numbers  of  gene  pairs,  but  relatively  few  chromosome  pairs;  hence, 
each  chromosome  must  carry  many  genes.  The  inheritance  of  two  char- 
acter pairs  dependent  upon  differential  genes  in  the  same  chromosome 
pair  would  be  unlike  that  described  above,  for  random  assortment  of 
chromosomes  would  play  no  part  in  determining  their  combinations. 
This  is  illustrated  in  the  right-hand  part  of  Fig.  123.  If  the  two  gene 
pairs  in  the  Fi  plant  were  located  in  one  chromosome  pair  as  shown, 
there  would  be  formed  only  two  kinds  of  spores  and  gametes  instead  of 
four,  and  the  F^  generation  would  show  only  the  two  parental  combina- 
tions, tall-green  and  dwarf-yellow.  The  two  characters  contributed 
together  by  each  grandparent  are  still  associated.  This  phenomenon 
is  known  as  linkage.  As  would  be  expected,  there  are  as  many  groups 
of  linked  genes  as  there  are  chromosome  pairs;  there  are,  for  example,  4  in 
Drosophila  and  10  in  maize.  Independent  inheritance  is  exhibited  only 
by  characters  having  their  differential  genes  in  different  chromosome 
pairs.  From  a  mathematical  point  of  view  it  is  remarkable  that  Mendel 
happened  to  select  for  special  study  seven  independent  character  pairs, 
for  the  garden  pea  has  since  been  found  to  have  just  seven  pairs  of 
chromosomes. 

Assignment  of  Genes  to  a  Chromosome. — How  is  it  ascertained  in 
what  chromosome  of  a  genome  a  given  gene  and  those  linked  with  it  are 
located?  In  plants  one  of  the  convenient  methods  involves  the  use  of 
individuals  occasionally  appearing  in  the  breeding  plot  with  one  extra 
chromosome.  This  condition  arises  as  a  result  of  nondisjunction, 
commonly  at  sporogenesis.  Two  members  of  a  pair  that  should  disjoin 
in  meiosis  fail  to  do  so,  a  spore  and  later  a  gamete  therefore  arising 
with  one  member  of  its  genome  in  duplicate.  Union  of  this  gamete  with 
a  normal  one  yields  a  plant  with  one  of  its  chromosomes  in  triplicate,  all 
the  other  chromosomes  being  in  duplicate  as  usual.  At  meiosis  the  three 
members  of  the  "trisome"  usually  disjoin  two  from  one,  so  that  some  of 
the  spores  and  gametes  carry  an  extra  chromosome.  It  can  readily  be 
calculated  that  Mendeiian  ratios  in  plants  obtained  when  this  trisomic 
plant  is  testcrossed  will  not  be  the  same  for  characters  dependent  upon 
genes  in  the  trisome  as  for  characters  due  to  genes  in  the  other  chromo- 
somes, provided  the  trisomic  plant  is  heterozygous  for  genes  in  the 
trisome.  For  example,  when  a  plant  carries  the  genes  ^  ^  a  in  the 
trisome  and  the  factors  B  b  in  &  normal  chromosome  pair,  the  population 
obtained  after  a  testcross  to  a  plant  with  a  a  h  h  is  expected  to  show  a 
phenotypic  ratio  of  5 : 1  for  a  character  due  to  A  and  the  normal  1 : 1 
ratio  for  a  character  due  to  B.  Hence,  by  observing  what  characters 
appear  in  trisomic  ratios,,  and  by  examining  the  plants  cytologically  to 
see  which  chromosome  of  the  genome  is  present  in  triplicate,  the  con- 
clusion can  be  drawn  that  the  genes  responsible  for  the  abnormal  ratios 


CYTOLOGY  AND  MENDELIAN  HEREDITY  175 

are  located  in  that  chromosome.  The  expected  trisomic  ratios  are 
ordinaril}'  obtained  only  when  the  trisomic  plant  is  used  as  a  female 
parent,  for  it  has  been  found  that  pollen  grains  carrying  the  extra  chromo- 
some usually  do  not  function  well  in  competition  with  normal  pollen. 
Moreover,  the  ratios  may  be  disturbed  by  the  tendency  of  the  extra 
chromosome  to  lag  in  the  meiotic  anaphase  and  thus  sometimes  fail  to  be 
included  in  a  spore  nucleus. 

The  first  character  to  be  assigned  mainly  to  genes  in  a  given  chromo- 
some was  sex.  This  was  possible  because  in  many  organisms  there  is  a 
chromosome  pair  that  differs  visibly  from  the  other  pairs  and  differs 
also  in  males  and  females.  As  will  be  described  in  a  later  section,  the 
behavior  of  this  pair  is  such  as  to  yield  two  visibly  distinct  chromosome 
complements  in  equal  numbers  in  each  generation,  and  these  are  found, 
respectiveh^,  in  the  cells  of  the  two  sexes.  Nonsexual  characters  depend- 
ent upon  genes  in  the  sex  chromosomes  are  said  to  be  sex-linked,  and 
they  show  a  mode  of  inheritance  differing  somewhat  from  characters  not 
so  linked.  Unlike  sex-limited  characters,  they  may  appear  in  either 
sex,  as  will  be  explained  further  on  (page  190). 

By  these  and  other  methods,  genes  have  been  assigned  to  their  proper 
chromosomes  in  a  considerable  number  of  organisms.  In  some  of  those 
which  have  been  most  intensively  investigated,  this  has  been  done  for 
every  chromosome  of  the  genome. 

Recombination  and  Crossing  Over. — We  have  seen  that  when  two 
pairs  of  differential  genes  are  located  in  one  chromosome  pair  the 
characters  dependent  upon  them  are  linked,  i.e.,  the  character  combina- 
tions present  in  the  two  individuals  originally'  crossed  tend  to  reappear 
in  the  Fo  generation  following  a  testcross.  In  Fig.  123,  right-hand 
part,  the  F^  comprises  individuals  of  only  two  classes,  and  these  show 
the  original  combinations.  As  a  general  rule,  however,  there  are  four 
classes  in  F^:  the  individuals  of  the  two  additional  classes  show  recom- 
binations, i.e.,  the  two  pairs  of  characters  have,  as  it  were,  exchanged 
partners.  The  percentage  of  F^  individuals  showing  such  recombination 
tends  to  maintain  a  characteristic  average  in  repeated  tests  involving 
the  same  characters,  but  for  different  character  combinations  it  varies 
all  the  way  from  0  to  50.  When  the  value  lies  near  50,  it  becomes 
impossible  without  other  indirect  tests  to  distinguish  the  results  from 
those  of  random  chromosome  assortment  (left  half  of  Fig.  123).  The 
mechanism  involved,  however  is  a  quite  different  one,  since  only  one 
chromosome  pair  is  involved.  The  nature  of  this  mechanism  is  brought 
out  in  the  following  example  (Fig.  124). 

Two  well-known  characters  in  Drosophila  cultures  are  black  body  and 
vestigial  wings.  Each  of  these  is  a  recessive  character,  appearing  in  a 
fly  only  when  the  gene  has  been  received  from  both  parents  in  the  reces- 


176 


FUNDAMENTALS  OF  CYTOLOGY 


sive  condition  b  or  v.  In  the  dominant  conditions  B  and  V  these  genes 
produce,  respectively,  normal  gray  body  and  normal  long  wings.  When  a 
fly,  homozygous  for  both  dominant  factors,  is  mated  to  one  with  all 
the  corresponding  factors  recessive,  the  offspring  all  have  normal  body 
and  wings,  because  of  the  dominance  of  B  and  V  over  h  and  v,  respectively. 
If  the  females  of  this  Fi  generation  are  backcrossed  to  the  homozygous 
recessive,  flies  of  four  types  appear  in  the  next  generation:  gray-long, 
black-vestigial,  gray-vestigial,  and  black-long.  Those  flies  mth  the 
original  combinations  (gray-long  and  black-vestigial)  together  comprise 
83  per  cent  of  the  total  number;  only  17  per  cent  are  of  the  new  t^^pes 


B"'  V 


V      V  -» 


b 


Back-cross 


b       b 


V         V 


% 


Fig.  124. — Linkage  in  Drosophila.  One  pair  of  chromosomes  in  each  fly  represented  by 
vertical  lines;  chromosomes  in  gametes,  by  diagonal  lines.  Explanation  in  text.  {Adajited 
from  T.  H.  Morgan  et  al.) 

(gray- vestigia,!  and  black-long).  It  thus  appears  that  if  the  two  char- 
acters, gray  body  and  long  wings,  are  contributed  to  the  offspring  b^^ 
the  same  parent,  they  tend  to  appear  together  in  the  majority  of  the 
individuals  resulting  from  the  backcross;  in  other  words,  they  are 
linked.  This  is  explained  by  the  fact  that  the  differential  genes  con- 
cerned are  located  in  one  chromosome  of  a  pair.  The  same  is  obviously 
true  of  the  allelic  characters,  black  body  and  vestigial  wings,  for  their 
genes  are  carried  in  the  other  chromosome  of  the  pair.  Hence  in  the  Fi 
fly  one  chromosome  of  a  homologous  pair  carries  B  V,  while  the  other 
carries  h  v,  and  they  tend  strongly  to  continue  thus  into  the  next  genera- 
tion. Were  the  two  pairs  of  genes  in  question,  B  b  and  V  v,  carried  by 
different  pairs  of  chromosomes  instead  of  by  the  same  pair,  there  would 


CYTOLOGY  AND  MENDELIAN  HEREDITY 


177 


be  no  linkage:  the  two  character.s,  gray  and  long,  and  likewise  the  two 
characters,  black  and  vestigial,  would  then  be  exhibited  together  in  the 
next  generation  by  about  50  per  cent  of  the  flies,  the  chance  frequency 
based  on  random  assortment,  rather  than  83  per  cent. 

We  have  next  to  inquire  into  the  origin  of  the  new  combination  appear- 
ing in  17  per  cent  of  the  flies  after  the  backcross.  In  the  original  female 
both  chromosomes  carry  B  V;  hence  every  egg  has  this  combination. 
The  male  has  b  v  in  both  chromosomes  of  the  pair;  hence  every  sperm  has 
b  V.  All  flies  in  Fi  will  therefore  have  B  V  in  one  chromosome  of  the  pair 
and  6  y  in  the  other;  they  are  heterozygous  for  both  pairs  of  genes. 
When  the  females  of  the  Fi  generation  mature  their  eggs,  the  two  chro- 
mosomes disjoin  in  meiosis  so  that  half  of  the  eggs  carry  one  and  half 
the  other.  If  the  chromosomes  are  passed  along  unaltered,  no  new 
combinations  appear  in  the  next  generation. 


■b 


Fig.  125. 


-Diagram  of  crossing  over  between  Bb  and  Vv  responsible  for  lecoinbinaticns  in 
case  illustrated  in  Fig.  124. 


Now  let  it  be  supposed  that  in  some  of  the  ooc.ytes  two  nonsister 
chromatids  exchange  portions  at  some  point  between  the  two  pairs  of 
genes  in  question  (Fig.  125).  Some  eggs  will  then  carry  unaltered 
chromosomes  {B  V)  {b  v),  while  others  will  carry  altered  ones  (B  v) 
(b  V).  Fertilization  of  these  four  classes  of  eggs  bj^  sperms  carrying 
b  V  will  obviously  result  in  flies  of  four  classes,  two  of  which  are  of  new 
kinds.  The  percentage  of  recombinations  appearing  depends  upon  the 
proportion  of  the  oocytes  in  which  chromatid  exchange  (crossing  over) 
occurs  between  the  two  pairs  of  genes.  If  it  occurred  in  every  oocyte, 
only  50  per  cent  of  the  resulting  flies  would  show  the  recombinations, 
since  two  normal  as  well  as  two  altered  chromatids  would  result  in  each 
oocyte.  From  this  it  can  readily  be  seen  that  the  frequency  (17  per  cent) 
of  recombination  in  the  present  example  is  due  to  the  fact  that  the  proper 
chromatid  exchange  occurred  in  34  per  cent  of  the  oocytes.  Such  crossing 
over  between  pairs  of  linked  genes  is  a  phenomenon  occurring  generally 
in  plants  and  animals,  although  in  some  cases,  notably  in  the  males  of 
Drosophila,  it  is  absent. 

Position  of  Genes  in  the  Chromosome. — One  of  the  theories  of 
inheritance  propounded  late  in  the  nineteenth  century  stated  that 
"ancestral  germ  plasms"  are  arranged  in  a  linear  series  in  the  chromo- 


178  FUNDAMENTALS  OF  CYTOLOGY 

somal  thread  and  that  the  significance  of  longitudinal  chromosome 
division  lies  in  the  division  of  these  numerous  units.  The  confirmation 
of  this  view  during  the  twentieth  century  has  involved  the  answering  of 
two  questions:  "In  what  particular  order  are  the  various  genes  arranged?" 
and  "What  is  the  precise  location  of  each  gene  in  the  chromosome?" 

The  determination  of  the  serial  order  of  the  genes  has  been  made 
possible  mainl}^  through  studies  on  recombination.  Soon  after  Morgan 
and  his  coworkers  had  their  intensive  investigations  of  inheritance  in 
Drosophila  well  under  wa}^,  certain  cases  of  recombination  of  linked 
characters  were  observed  in  the  flies.  It  was  then  (1911)  suggested 
that  this  might  be  a  result  of  chromatid  exchange  (crossing  over)  such 
as  had  been  reported  for  salamander  chromosomes  by  Janssens  (1909). 
This  hypothesis  was  employed  with  notable  success  in  the  further  develop- 
ment of  cytogenetics,  although  the  complete  proof  of  its  correctness  was 
not  available  until  1931.  The  determination  of  serial  order  was  based 
upon  the  fact  that  different  couples  of  allelic  character  pairs  yield  recom- 
l)inations  in  different  percentages.  The  hypothesis  was  advanced  that 
the  frequency  of  crossing  over  simply  varies  with  the  distance  separating 
the  two  gene  pairs  concerned,  exchanges  occurring  with  uniform  frequency 
throughout  the  length  of  the  tetrad.  For  convenience  it  was  assumed 
that  when  recombinations  appear  in  1  per  cent  of  the  individuals  in  F^ 
after  a  testcross  the  two  gene  pairs  are  separated  by  one  "crossover 
unit"  in  the  threads.  On  this  basis  a  diagram,  or  "linkage  map,"  was 
gradually  built  up  showing  the  arrangement  of  the  various  genes  as 
indicated  by  recombination  percentages.  The  same  procedure  has  since 
been  followed  in  the  case  of  a  number  of  other  animals  and  plants.  The 
method  used  in  constructing  such  maps  is  illustrated  in  the  following 
example  from  maize. 

Among  the  mutant  characters  known  to  be  linked  and  assignable  to 
chromosome  4  in  maize  are  the  following:  sugary  endosperm  (sui),  which 
is  recessive  to  the  normal  starchy  endosperm  (Siii) ;  tunicate  ear  (each 
kernel  with  a  pod)  (Tu),  dominant  to  normal  ear  (tu);  glossy  leaf  (gh), 
recessive  to  normal  leaf  surface  (Gh).  When  plants  heterozj^gous  for 
all  three  pairs  are  testcrossed  to  plants  homozygous  and  recessive  for  all 
three,  the  results  are  as  follows  (Fig.  126).  The  recombination  per- 
centage of  sugary  and  tunicate  is  29;  hence  these  two  genes  are  placed 
29  units  apart  on  a  line  representing  the  chromosome.  Between 
tunicate  and  glossy  the  recombination  percentage  is  11;  hence  gh  is 
1 1  units  from  Tu,  but  on  which  side?  This  is  decided  by  the  recombina- 
tion percentage  given  by  sugarj^  and  glossy,  which  is  34:  gh  must 
therefore  be  located  to  the  right  of  Tu.  In  this  way  the  relative  order 
of  these  three  genes  in  the  chromosome  is  determined.  Recombinations 
between  further  characters  likewise  indicate  the  positions  of  other  genes. 


CYTOLOGY  AND  MENDELIAN  HEREDITY  179 

It  will  be  noted  that  the  recombination  percentage  for  sui-gh  is  somewhat 
less  than  the  sum  of  the  percentages  for  sui-Tu  and  Tu-gU.  This  is 
because  genes,  when  rather  widely  s(^parated  in  the  chromosome,  may 
ha\'(!  two  crossovers  between  them,  leaving  them  still  in  the  same  chro- 
matid. This  makes  the  observed  recombination  percentage  lower  than 
it  would  have  been  if  no  such  double  crossing  over  had  occurred.  Linkage 
maps,  lik(!  those  for  Zea  and  Drosophila  (Figs.  127,  128),  are  built  up 
l)y  plotting  ]:)Ositions  only  on  the  basis  of  closely  linked  genes,  thus 
without  taking  double  crossing  over  into  consideration.  This  means 
that  map  distance  (number  of  units)  between  two  genes  represents 
recombination  percentage  only  when  these  genes  are  rather  closely 
linked — within  about  10  units  in  Drosophila  and  maize.  Thus  the  map 
may  exceed   100  units  in  length,  whereas  the  upper  limit  of  observed 


sui  Tu  gl. 

Fig.  126. — Diagram  illustrating  the  method  of  determining  the  serial  order  of  linked 
genes  by  comparing  recombination  percentages.  The  four  parallel  lines  represent  the 
chromatids  in  the  tetrad  at  pachytene  in  the  plant  to  be  testcrossed.  Further  explanation 
in  text. 

recoml)i nation  is  50  per  cent  owing  to  the  fact  that  each  crossover  alters 
only  two  of  the  four  chromatids.  At  the  present  time  the  most  fully 
developed  linkage  maps  for  insects,  vertebrates,  and  plants  are  those  of 
Drosophila  melanogaster,  the  common  fowl,  and  maize,  respectively. 
The  map  as  just  described  is  a  convenient  record  of  the  linkage 
relationships  and  serial  order  of  the  genes  in  the  chromosome,  but  there 
are  limits  to  its  reliability  as  a  picture  of  the  chromosome  itself.  This  is 
because  such  a  map,  especially  when  only  a  few  genes  have  been  placed 
upon  it,  does  not  necessarily  show  the  actual  position  of  the  genes  in  the 
chromosome.  If,  after  numerous  linkage  studies,  no  gene  has  to  be  given 
a  place  beyond  the  one  already  at  the  end  of  the  map  being  constructed, 
it  is  a  reasonable  inference  that  this  gene  is  actually  at  the  end  of  the 
chromosome,  but  it  might  be  that  the  chromosome  beyond  this  point 
is  inert  or  occupied  only  by  unmutated  genes.  A  gene  is  detectable 
in  normal  material  only  when  it  is  present  in  the  mutated  as  well  as  the 
unmutated  form,  thus  giving  a  character  contrasting  with  the  normal 
one  and  revealing  the  fact  that  a  corresponding  normal  gene  exists. 
Another  method,  therefore,  must  be  used  to  ascertain  actual  gene  position. 


180  FUNDAMENTALS  OF  CYTOLOGY 

sr         msl7  ts2  P  zl  as  br   f  on  gs  bmZ 


25'  27  28~"30  -53  80  85 


ws3       ig  9I2  B    sk        ft   ts^     v4  Ch 


H 1 1 1 — l-O 


49   56        68  74         83  128 


Rg        ts4  ba 


18  40       47  64        75  103 


de  Go  ts5        sp  ^u ,del6  .zb6    tu  JZ  gl3 

-\ 1 1 — Q I   (  C 1 i-H — I — 

0  35  56  66   71   74     ^84    lo'o  105  III 


aS  bm    bt y3_bv        pr     ys  vg 


6 


0     6       7     10  12        31      40 


po               Y                        PI       sm     py 
— K'") 1 1 1 ^ 


13 


in  v5  ra  gt    Tp    ij  Bn  bd 


\ — O"^"* — ' — ' •■ 

0  4  18  22     32  38  56 


8 


ms8 


28 


knob   yg2    C  sh          bp            »x 
1-4 ?-i 1 ^-0~ 


0 


Rp  Og        sp2  ti    18  g 

—\ 1— «''>-4+- 


0 


16    "'    Zi  ~~28  38  43        57 

Fig.  127.-^Linkage  map  for  Zea  viaya.  The  kiiietochores  are  assigned  tentatively  to 
positions  with  respect  to  the  genes;  those  shown  with  broken  lines  are  less  definitely  placed 
than  the  others.      (After  L.  F.  Randolph.) 


CYTOLOGY  AND  MENDELIAN  HEREDITY 


181 


lo.t 
03 
-\  V0.6 

4-'.  'I. 

1.5 

\h 

4.5 

5.5 

-■\\6.9 

.  -  75 

-  -  M6.± 
--  ^I± 

20. 

21. 

27.5 
T  27.7 


m 


IV 


yellow  CB) 
Hairy  wing  (W) 
scute  (H) 
lethal-7' 
broad  (W) 
prune  CE) 
whiteCE) 
fcce+CE) 
Ho+ch  CE) 
Abnormal  CB) 
echinus  Cf) 
bifid  CW) 
ruby  CE) 
crossveinless  (W) 
clubCW) 
deltex  CW) 
cui  CW) 
singed  CH) 

+01  n  CB) 
lozenge  CE) 


telegraph  (VV) 

Star  CE) 
arista  less  CB) 

expoindedCw) 

Gull  Cw) 
Truncate  (W) 
doichsous  CB) 
Streak  CB) 


( 


0.       rougboid  CE) 


33. 

36.1 
=  =  36.2 
--   58.± 

-  -  45. 
-.  444 


54.2 

-  54.5 
56.5 

'-  57. 

-  585 
■-   59. 

. .  59.6 
62. 

-  65. 


vermillionCE) 
miniature  Cw) 
dusky  CW) 

furrowed  CE) 

sable  CB) 
garnet  CE) 


-  -31 


35. 


-41. 


dachs  Cb) 
Ski-E  Cw) 


--    20.     divergent  Cw) 


26.     sepia  CE) 
26  5    hairy  CB) 


small  wing 
rudimentary  CW)-  •  °^-- 
forked  CH) 
Bar  CE) 
small  eye 
fused  0^) 
Beadex  Cw; 
Minute-n  Ch) 
cleft  Cw) 


Jammed  Cw)      -I-    35.     rose  (t) 

36.2  cream-nr  CE) 

Minute-e  Ch)  40.1  Minute-h  CH) 

black  CB)  ■-■■  40.2  tilt  Cw) 

jaunty  Cw)  40.4  Dichae+e  CH) 

-  ■  42.2  thread  CB) 

-  ■   44.     scarlet  CE) 


purple  Ce) 
cinnabarCE) 
safroinin  CE) 


vestigial  Cw) 
telescope  CW) 


48.     pink  Ce) 
-  -    49.7   maroon  CE) 
I50.t   dwoirf  Cb) 
150.     curled  CW) 
64.±     pink-wingCEW)  f    54.8   Hairy  win^supr 


—  1  70.    bobbedCH) 


rrr 


--72.       LobeCE) 

.±     gapCwJ 
.5     curved  Cw) 


■•83.5     fringed  Cw) 


-90.       humpy  Cb) 


99.5    arc  Cw) 
00.5    plexus  (W) 
102.+  Tethal-Ha 
(105.     brown  CE) 
•il05.±  blistered  Cw) 
106.     purpleoid  CE) 
t107.±  morula  QE.) 
1)07.     speck  Cb) 
107.5  bc3i1loonCW) 


68.2   S■^ubble  CH) 

:    58.5   spinelessCH) 

■,  587    bi+horaxCB) 

-  -  "59.5  bithorax-b 

-  \  62.     stripe  CB) 
. .  ~~63.!    gloiss  CE) 

66.2    Delta  CW) 

■  -    69.5   hairless  Ch) 

■  -   707   ebony  Cb) 
•  •    72.    band  CB) 

75.7   cardinal  Ce) 
■    76.2  white  ocelli  Ce) 


91.1    rough  Ce) 
93.     crumpled  Cw) 
93.8   Beaded  Cw) 
94.1    Painted  Cw) 

1007    claret  Ce) 
101.     Minute  CH) 


106.2  Minute-9  (H) 


bent  Cw) 
shaven  CB) 
eyeless  Ce) 
rotated  Cb) 
Minute-IY  Cm) 


male  fertility 


Long  bristled 


male  fertility 


Fig.  128. — Linkage  map  for  Drosophila  melanogaster,  showing  .serial  order  of  many  of  the 
known  genes  as  determined  by  genetical  methods.  Letters  in  parentheses  indicate  portions 
of  fly  in  which  characters  appear:  B,  body;  E,  eye;  H,  hairs;  W,  wings,  .\rrows  indicate 
kinetochore  position.  Genes  in  chromosome  IV  are  very  closely  linked.  Positions  of 
genes  in  F-chromosome  not  well  determined.  {Adapted  from  T.  H.  Morgan,  A.  H.  Sturte- 
vant,  and  C.  B.  Bridges  and  from  C.  Stern.) 


182  FUNDAMENTALS  OF  CYTOLOGY 

The  solution  of  the  problem  of  gene  position,  like  that  of  numerous 
other  cytogenetical  questions,  is  being  greatly  facilitated  by  the  use  of 
abnormalities  in  chromosome  behavior.  In  the  next  chapter,  abnormali- 
ties of  various  types  vnW  be  described.  Our  present  purpose  will  be 
served  by  one  of  them,  deletion.  Occasionally  there  appears  spon- 
taneously or  in  experimentally  treated  material  a  chromosome  with  a 
portion  missing.  This  portion  may  have  been  deleted  from  the  end 
(terminal  deletion)  or  from  some  other  region  (intercalary  deletion). 
Most  deletions  render  a  monoploid  cell  inviable.  The  same  is  true  of  a 
diploid  cell  if  both  chromosomes  of  the  pair  carry  the  deletion,  but  if 
only  one  member  of  the  pair  carries  it  the  cell  is  often  functional.  By 
observing  the  characters  affected  by  the  absence  of  a  chromosome 
portion,  it  can  be  inferred  what  genes  were  lost  at  the  time  of  deletion. 
Obviously  the  most  useful  deletions  are  those  which  are  small  and  which 
do  not  prevent  the  development  of  the  cells  concerned.  Two  examples 
illustrating  the  procedure  will  now  be  described. 

In  maize  there  is  a  mutant  gene  {yg^}  in  the  linked  group  occupying 
chromosome  9.  Plants  carrying  this  gene  in  the  homozygous  recessive 
condition  {ijg^  yg^)  are  yellow-green,  while  homozygous  dominant 
{Ygi  Yg^  and  heterozygous  {Yg^  yg^)  plants  have  the  normal  green  color. 
In  some  strains  of  maize,  chromosome  9  has  a  small  terminal  knob,  while 
in  other  strains  the  knob  is  large;  hence  the  chromosome  can  be  easilj' 
distinguished  in  certain  stages  of  the  nuclear  cj^cle.  The  position 
of  the  yellow-green  gene  in  the  map  already  made  on  the  basis  of  linkage 
relationships  was  near  one  end.  That  it  was  also  near  the  end  of  the 
actual  chromosome  was  demonstrated  in  the  following  cross  (Fig.  129). 

A  yellow-green  plant  homozygous  for  yg^  and  a  small  knob  was  used 
as  a  pistillate  parent,  whereas  the  staminate  parent  was  normal  green 
and  homozygous  for  Yg2  and  a  large  knob.  Pollen  from  the  latter  plant 
was  given  X-ray  treatment,  which  is  known  to  induce  chromosomal 
aberrations,  and  then  placed  upon  the  silks  of  the  pistillate  parent. 
When  the  resulting  kernels  were  sown,  they  produced  a  population  com- 
prising plants  of  several  classes.  Most  of  them  were  normal  green  in 
color,  as  expected  in  heterozygotes,  and  cytological  examination  showed 
the  members  of  their  ninth  pair  of  chromosomes  to  have  been  unchanged 
by  the  irradiation.  Plants  of  a  second  class  were  yellow-green  and  had 
the  large-knobbed  chromosome  (from  the  pollen  parent)  considerably 
shortened,  evidently  by  the  deletion  of  a  region  near  the  knob.  A  third 
class,  also  yellow-green,  had  the  large  knob  reduced  in  size,  indicating 
a  deletion  including  a  portion  of  the  knob.  In  all  cases  the  maternal 
chromosome  remained  unaltered.  These  cytological  and  genetical  data 
showed  that  the  deletions  had  removed  the  dominant  normal  gene, 
Yg2,  from  chromosome  9  in  some  of  the  pollen  and  also  that  the  gene 


CYTOLOGY  AND  MENDELIAN  HEREDITY 


183 


when  present  lies  very  close  to  the  terminal  knob.  In  addition  to  the 
above  three  classes  there  was  a  single  individual  that  was  partly  normal 
green  and  partly  yellow-green.  In  the  normal-green  portion  of  the  plant 
the  ninth  chromosome  pair  was  unaltered,  as  in  the  first  class,  whereas 
in  the  yellow-green  portion  the  paternal  chromosome  was  shortened  and 
had  no  knob.     Evidently,  therefore,  the  deletion  in  this  case  was  truly 


yg^' 

1     1 

yg^ 

Yg. 

Yg. 

Yellow -green 


i         t 

Eggs 


Normal   green 


Sperms 
(irradiated! 


,        I 


yg. 


Yg, 


y<3t 


1 

1 

1 

yg* 

Yg. 

< 
ygi 

Normal   green 


Yellow-green 
I 


Majority  of  — . n     ,^    .    .      . 

*w         \\  J- Deleted 

the   plants  — 


Yellow  -green 
Deleted 


-K 


Partly    normal 
and   partly 
yel  low-greerv 

Terminal  deletion 
in  embryo 


Fig.   129. — Diagram  illustrating  the  method  of  determining  gene  location  by  the  use  of 
deletions.     Explanation  in  text.      {Based  on  work  of  H.  B.  Creighton.) 

terminal,  a  rare  condition;  moreover,  it  must  have  occurred  in  the 
embryonic  stage  of  the  plant,  since  only  a  portion  of  the  plant  was 
affected.  This  plant  was  of  further  interest  from  the  standpoint  of  the 
physiology  of  genie  action,  for  the  particular  effect  of  each  of  the  two 
genotypes  was  restricted  to  the  portion  of  the  plant  containing  it. 
Relatively  few  exceptions  to  this  type  of  genie  action  are  known. 

Gene  location  by  the  deletion  method  is  most  precisely  determinable 
in  organisms  having  chromosomes  that  are  large  and  distinguishable  on 


184 


FUNDAMENTALS  OF  CYTOLOGY 


the  basis  of  characteristic  morphology.  In  the  above  example  tlu^ 
condition  of  the  chromosomes  was  determined  chiefly  in  the  micro- 
sporocytes,  for  although  the  chromosomes  in  the  somatic  cells  of  maize 
are  not  large,  they  are  beautifully  displayed  in  a  greatly  extended  form 
at  the  pachytene  stage  in  microsporocytes  (Fig.  79).  Not  only  do  they 
show  well  their  minute  structural  features  there  (page  92),  but  they  are 
present  as  synapsed  pairs,  which  makes  it  possible  to  compare  minutely 
the  two  parental  members  part  for  part. 


£1^ 


eyes  white 


r\. 


eyes  white 


W 
eyes  red 


Fig.   130. — Diagram,  illustrating  the  method  of  locating  genes  by  means  of  deletions  in 
saUvary-gland  chromosomes.     Explanation  in  text.      (Based  on  drawings  by  0.  Mackenseji.) 

A  second  example  illustrating  this  method  of  locating  genes  is  taken 
from  the  literature  on  Drosophila.  The  somatic  chromosomes  in  the 
fly  are  very  small.  Gross  alterations  in  them  can  be  detected,  and  the 
approximate  positions  of  certain  genes  have  been  determined  by  such 
alterations.  The  rediscovery  of  the  giant  chromosomes  in  the  salivary 
glands  of  the  larva  (page  94)  has  now  made  it  possible  to  carry  on  such 
researches  with  far  greater  speed  and  precision.  The  method  used  in  the 
case  about  to  be  described  parallels  that  used  in  the  case  of  maize.  Male 
flies  carrying  dominant  genes  including  W  (normal  red  eyes)  in  their 
chromosome  I  were  X-rayed  with  the  purpose  of  inducing  deletions  or 
other  aberrations  in  this  chromosome.  These  flies  were  then  mated  to 
females  carrying  recessive  genes,  including  iv  (white  eyes,  recessive), 
in  their  chromosomes  I   (the  X-chromosomes).     Fi  females  were  then 


CYTOLOGY  AND  MEN  DELI  AN  HEREDITY 


185 


selected  for  study  because  the}^  contained 
two  A'-chromosomes,  one  from  each 
parent.  Some  of  them  had  white  eyes, 
indicating  a  loss  or  mutation  of  the  domi- 
nant W  originally  present  in  the  father's 
A"-chromosome.  When  these  and  other 
females  were  tested  by  further  crosses,  the 
chromosomes  in  the  salivary  glands  re- 
vealed the  conditions  shown  in  Fig.  130. 
These  chromosomes,  it  will  be  recalled. 
(Fig.  67),  are  synapsed  pairs,  each  of  them 
representing  two  parental  homologues  in 
intimate  lateral  union.  Since  the  union 
is  so  precise,  band  for  band,  dissimilarities 
in  the  two  can  be  clearly  analyzed. 

The  three  cases  in  the  diagram  includ(> 
deletions  in  the  same  general  region  of  the 
paternal  A'-chromosomes,  and  by  compar- 
ing these  with  the  normal  maternal  chro- 
mosomes with  which  they  are  in  synapsis 
it  can  be  seen  that  where  the  band  indi- 
cated by  an  arrow  is  present  in  the  lower 
(paternal)  longitudinal  half  of  the  chromo- 
some pair  the  eyes  of  the  fly  are  red,  in- 
dicating the  presence  of  W,  whereas  the 
eyes  are  white  when  this  band  is  lacking 
in  this  half.  From  this  it  is  concluded 
that  the  gene  W  was  removed  in  the  por- 
tion deleted  in  two  of  the  three  cases, 
leaving  the  recessive  w  to  function  alone 
and  produce  white  eyes.  This  gene  affect- 
ing eye  color  is  accordingly  assigned  to  a 
small  region  occupied  by  this  band.  This 
region  represents  considerably  less  than  1 
per  cent  of  the  length  of  the  chromosome. 
In  this  manner  a  large  number  of  genes 
have  now  been  located  with  various  de- 
grees of  accuracy  in  the  chromosomes  of 
Drosophila. 

Fig.  131. — Diagram  comparing  the  linkage  map 
of  the  X-chromosome  of  Drosophila  melanogaster 
with  the  salivary-gland  chromosome  map  for  some 
of  the  genes  that  have  been  located.  Explanation 
in  text.  {Adapted  from  T.  S.  Painter  and  (). 
Mackensen.) 


SI. 5   sd 


33.0   V 


27.7   Iz 


20.0  ct 


13.7  cv      _ 


186  FUNDAMENTALS  OF  CYTOLOGY 

When  the  chromosome  map  constructed  on  the  basis  of  such  data  is 
compared  mth  the  Unkage  map  for  the  same  chromosome,  it  is  found 
that  the  two  agree  with  respect  to  the  serial  order  of  the  genes  (Fig.  131). 
This  is  gratifying  proof  of  the  value  of  the  crossover  method  long  used 
for  the  determination  of  such  order.  The  spacing  of  the  genes,  however, 
differs  in  some  regions,  showing  that  the  occurrence  of  crossing  over 
is  not  uniform  in  frequenc.y  throughout  the  length  of  the  chromosome 
as  was  originally  assumed  in  constructing  the  linkage  map.  A  lower 
frequency  in  certain  chromosomal  regions,  notably  near  the  kinetochore, 
yields  recombination  percentages  lower  than  the  average;  hence  the  genes 
in  such  regions  appear  closer  together  in  the  linkage  map  than  they  do 
in  the  chromosome  map  showing  the  true  positions.  The  successful 
construction  of  such  chromosome  maps  accurately  summarizing  large 
bodies  of  observational  data  is  surely  one  of  biology's  major  achievements. 
Their  usefulness  and  appearance  suggested  an  earlier  remark  (page  97) 
that  the  salivary-gland  chromosome  is  a  sort  of  ''biological  spectrum" 
indicating  the  genetical  composition  of  the  organism. 

The  Special  Case  of  Sex. — Sex,  like  other  characters  of  the  organism, 
has  a  genie  basis.  Its  inheritance  in  dioecious  organisms,  however, 
differs  from  that  of  other  characters  because  of  a  special  type  of  chromo- 
somal mechanism.  This  specialization  is  manifested  in  the  differentiation 
of  one  chromosome  pair  from  the  others  in  its  influence  upon  sex  and 
often  in  its  visible  morphology.  The  members  of  this  pair  are  therefore 
known  as  sex  chromosomes,  although  the  other  chromosomes,  called 
autosomes,  may  also  have  some  share  in  the  determination  of  sex.  In 
addition,  there  is  a  further  differentiation  of  the  sex  chromosomes  into 
two  kinds,  usually  designated  as  X  and  Y  (Figs.  132-136).  The  indi- 
viduals of  one  sex,  nearly  always  the  female,  have  two  similar  chromo- 
somes {XX),  while  those  of  the  other  sex  have  two  unlike  ones  (XY). 
In  a  few  organisms,  notably  birds  and  lepidoptera,  the  females  have  the 
XY  pair.  In  some  species  there  is  no  Y,  one  sex  therefore  having  only 
one  sex  chromosome.  Again,  the  X  or  the  Y  may  comprise  two  or  more 
elements.  In  certain  monoploid  organisms,  e.g.,  bryophyte  gametophytes 
and  some  algae,  the  single  genome  includes  an  X  in  the  female  and  a  Y 
in  the  male,  both  X  and  Y  being  present  in  the  zygote  and  the  asexual 
sporophyte. 

In  all  the  above  cases  the  behavior  of  the  sex  chromosomes  in  meiosis 
and  syngamy  normally  results  in  an  approximate  numerical  equality  of 
the  two  sexes.     This  is  shown  in  the  following  examples. 

The  XY  type  referred  to  above  is  the  one  of  most  frequent  occurrence, 
being  found  in  plants  and  animals  of  many  natural  groups.  In  Fig.  132 
it  is  seen  that  the  disjunction  of  the  two  X-chromosomes  in  the  female 
animal  yields  gametes  of  but  one  class:  every  egg  carries  an  X.     Dis- 


CYTOLOGY  AND  MENDELIAN  HEREDITY 


187 


junction  in  the  male,  however,  yields  gametes  of  two  classes:  half  the 
sperms  carry  A'  and  half  carry  )'.  The  autosomes  are  the  same  in  all 
gametes,  excepting  of  course  any  differences  due  to  ordinary  hetero- 
zygosity in  the  individuals  producing  them.  Syngamic  unions  of  two 
kinds  are  now  possible,  A'  with  X  and  A'  ^nth  Y.  These  yield,  respec- 
tively, offspring  of  two  sexes:  females  (A" A')  and  males  (AF).  This 
type  of  mechanism  is  found  in  man  (Fig.  133),  although  the  large  number 


Animal 
XY 


mm 


Female 

Animal 

XX 


M«iosi9 


Mai* 

Animal 

XY 


Egg  and 
Polocytej 


Female 

Animal 

XX 


Slaminaf  e 
Plant 
XY 


Male 

GameJophyte 

Y 


Pistillate 

Plant 

XX 


Male 

Gametophyte 

X 


Female 

Gametoptiyte 

X 


Megosporei 


Staminate 

Plant 

XY 


Pistillate 

Plant 

XX 


Sporophyte 
XY 


Spores 


Male 

Gametophyte 

Y 


Female 

Gametophyte 

X 


Sporophyte 
XY 


Fig.  132. — Diagram  illustrating  the  relation  of  the  -Y-  and  F-chroraosonies  to  sex  in  the 
life  cycles  of  an  animal,  an  angiosperm,  and  a  bryophyte. 

of  chromosome  pairs  (24)  and  their  small  size  have  made  it  difficult 
to  identify  the  sex  pair,  particularly  the  F. 

Many  dioecious  angiosperms  have  the  same  mechanism  (Figs.  134, 
135).  Here  the  pistillate  plant  has  AA  and  the  staminate  plant  A}'. 
All  megaspores,  female  gametophytes,  and  eggs  are  alike  in  carrying  A. 
Half  the  microspores,  pollen  grains,  and  male  gametes  carry  A',  while  the 
other  half  carry  F.  Here  again  two  sorts  of  combination  are  possible: 
A  with  A,  giving  pistillate  offspring,  and  A'  with  F,  giving  staminate 
offspring. 

An  example  of  the  operation  of  the  AF  mechanism  in  monoploid 
organisms  is  afforded  by  the  liverwort  Sphaerocarpos,   the  first   plant 


FUNDAMENTALS  OF  CYTOLOGY 


known  to  have  s(>x  chromosomes  (Fig,  136).  Hero  the  female  plants 
(gametophytes)  ha\'e  in  their  single  genome  one  large  X,  while  the  male 
plants  (gametophytes)  have  a  very  small  Y.  After  syngamy  the  zygote 
with  XY  develops  into  the  asexual  sporophj^te. 
When  meiosis  occurs  at  sporogenesis,  disjunction  of 
the  X  and  Y  results  in  the  presence  of  two  spores 
with  X  and  two  with  Y  in  each  quartet.  These  de- 
velop, respectively,  into  new  female  and  male  game- 
tophytes. It  is  of  interest  to  compare  this  case  with 
that  of  the  angiosperm  in  the  preceding  paragraph. 
In  the  liverwort  X  and  Y  are  correlated,  respectively, 

J         9k        h        mth  femaleness   and   maleness   in  different   gameto- 
I  f        phytes  and  gametes,  with  XY  in  the  asexual  phase 

9  •       of   the   cycle;  whereas,   in  the  angiosperm  the  male 

gametophytes  and  gametes  have  either  X  or  F,  this 
difference  determining  in  turn  which  of  the  sporo- 
phytes  shall  be  female  (XX)  and  which  male  (XT). 
A  characteristic  feature  of  sex  chromosomes  is 
their  degree  of  heteropyknosis  (page  86).  About 
half  the  X-chromosome  in  Drosophila  is  heterochro- 
matic,  the  numerous  genes  that  exert  an  influence 
toward  femaleness  being  arranged  all  along  the  euchromatic  portion.  In 
some  organisms  practically  the  entire  sex  chromosome  is  heterochromatic 
and  is  visililc  during  the  metabolic  stage  as  a  dense,  stainable  body  in 
the  midst  of  the  chromonemata  of  the  other  chromosomes.  Experiments 
have  shown  that  in  Drosophila  the  Y  exerts  practically  no  influence  upon 


Fig.  133.— A' 1- 
chromosoine  pairs 
disjoining  in  first 
meiotic  divi.sion.  a, 
in  spermatocyte  of 
man.  b,  XY  pairs  in 
opossum,  monkey, 
and  man.  (After  T. 
S.  Painter.) 


Fiu.  134. — ooiuatic  chromosome  comple- 
ments from  staminate  (a)  and  pistillate  (b) 
individuals  of  a  dioecious  angiosperm  {Melan- 
drium  album).      (After  M.  Westergaard.) 


Fiu.  13.").      Mctaphaso  of  first 
division    in    microsporocyte    of 
drium.     XY     pair     about     to 
(Photograph  by  H.  E.  Warmke.) 


meiotic 
Melaiv- 
disjoin. 


the  kind  of  sex  developed,  although  it  does  influence  fertility.  In 
Melandrium,  a  genus  of  angiosperms,  on  the  other  hand,  the  large  )' 
seems  clearly  to  exercise  a  strong  influence  toward  maleness.     Another 


CYTOLOGY  AND  MEN  DELI  AN  HEREDITY  189 

notable  feature  is  the  frequent  absence  of  crossing  over  between  the 
X  and  Y. 

The  significance  of  all  these  facts  is  by  no  means  well  understood,  but 
there  is  a  tendenc}^  to  associate  them  with  the  advantages  of  dioecism. 
It  has  been  pointed  out  above  that  the  sex  chromosome  mechanism 
results  in  the  production  of  males  and  females  in  equal  numbers,  even 
though  this  ratio  may  be  disturbed  by  other  causes.  The  1 : 1  ratio  is 
expected  not  only  for  sex  but  for  all  allelic  character  pairs  in  each  genera- 
tion in  monoploid  organisms.  In  diploid  organisms  the  sexes  also 
appear  1 : 1  in  each  generation,  even  though  most  of  the  other  character 
pairs  show  a  1 :0  ratio  in  the  F^  generation  and  a  3: 1  ratio  in  F2.  This 
numerical  equality  of  the  sexes  in  spite  of  inequality  in  other  characters 
is  a  significant  result  of  the  differentiation  of  a  special  sex  chromosome 
pair  with  unlike  members.     If  X  and  Y  exert  different  influences  upon  the 


^^<9. 


Fig.  136. — Sex  chromosomes  in  a  liverwort  (Sphaerocarpos  Donneliii) .  a,  genome  in 
female  gametophyte.  b,  genome  in  male  gametophyte.  c,  diploid  complement  in  sporo- 
phyte.     d,  first  meiotic  division  in  sporocyte.      (After  G.  Lorhccr.) 

type  of  sex  developed,  it  can  be  realized  that  crossing  over  could  impair  the 
distinctness  of  the  two  sex  differentiating  processes  and  lead  to  deleterious 
sexual  states;  hence  the  value  of  the  lack  of  crossing  over  between  these 
chromosomes,  at  least  in  some  organisms.  Also  suggestive  is  the  further 
fact  that  heteropyknosis  evidently  interferes  Avith  or  prevents  crossing 
over. 

It  is  to  be  emphasized  that  the  chromosomes  are  not  alone  responsible 
for  the  type  of  sex  developed.  As  in  the  case  of  all  other  characters,  the 
sex  expressed  is  a  result  of  the  interaction  of  genetical  and  environmental 
factors.  The  significance  of  the  sex  chromosomes  lies  in  the  fact  that 
they  automatically  maintain  two  genotypes  in  the  species,  these  resulting 
in  the  development  of  two  types  of  organism,  male  and  female,  under 
the  range  of  environmental  conditions  normally  encountered.  This  is 
all  that  nature  requires  for  the  successful  operation  of  the  mechanism: 
absolute  distinctness  between  males  and  females  in  all  cases  and  under 
all  possible  conditions  is  not  to  be  expected.  The  fact  that  abnormal 
environments  may  sometimes  alter  the  sex  is  accordingly  no  argument 
against  the  sex-determining  role  of  the  chromosomes  as  properly  con- 
ceived.    Under  normal  conditions  the  genotype  is  the  sufficiently  decisive 


190 


FUNDAMENTALS  OF  CYTOLOGY 


differential  factor;  under  other  conditions  the  environmental  factor  may 
be  differential;  under  all  conditions  both  factors  share  in  determining 

what  the  organism  does.  It  should  be  e\'ident 
that  the  same  principle  holds  for  inorganic  sj's- 
tems.  Thus  the  behavior  (curved  flight)  of  a 
pitched  baseball  depends  upon  the  combined 
influences  of  a  constitutional  factor  (rotation) 
and  an  environmental  factor  (air  resistance). 
The  decisive  role  of  differential  factors  in  the 
chromosomes  is  strikingly  shown  in  gynan- 
dromorphic  insects,  in  which  portions  of  the  body 
differing  in  the  number  of  A"-chromosomes  differ 
also  in  sex  and  sex-linked  characters  (Fig.  137). 
Sex-linkage. — An  interesting  example  of  the 
inheritance  of  a  nonsexual  character  depending 
upon  differential  genes  located  in  the  sex  chro- 
mosomes is  that  of  Daltonism,  a  type  of  color- 
blindness that  prevents  some  people  from 
distinguishing  properly  red  from  green.  The  defect  occurs  in  relatively 
few  individuals  in  affected  families  and  appears  less  frequently  in  women 
than  in  men  because  it  is  both  recessive  and  sex-linked.  The  chromo- 
somal basis  for  its  inheritance  is  illustrated  in  Fig.  138. 


Fig.  137. — A  Drosophila 
gynandromorph.  The  left 
side  is  female  and  has  two 
X-chromosomes  in  its  nu- 
clei. The  right  .side  is  male 
and  has  only  one  X-chro- 
mosome  as  a  result  of  the 
loss  of  the  other  X  in  an 
early  embryonal  mitosis. 
{After  T.  H.  Morgan  et  al.) 


Fig.  138. — Diagram  illustrating  the  inheritance  of  red-green  color  blindness,  when  (A) 
the  father,  or  (B)  the  mother,  or  (C)  both  parents  manifest  the  defect.  9,  female. 
cf ,  male.  Recessive  (defective)  gene  indicated  by  dot  above  X-chromosome  carrying  it. 
Individuals  manifesting  the  defect  indicated  by  triple  outlines. 

The  gene  responsible  for  the  defect  when  in  the  recessive  state  is 
located  in  the  X-chromosome ;  hence  females  may  have  either  one  or  two 


CYTOLOGY  AND  MENDELIAN  HEREDITY  191 

such  recessives,  while  males  can  have  only  one.  The  F-chromosome 
has  no  influence  on  the  character.  When  a  male  has  the  recessive  gene, 
he  is  color-blind,  for  no  normal  allele  is  present  to  dominate  it  (first  part 
of  diagram).  His  offspring  by  a  normal  female  all  have  normal  vision: 
the  daughters  are  heterozygous,  carrying  one  recessive  gene  dominated 
by  a  normal  one,  while  the  sons  have  onlj^  a  normal  gene.  If  one  of  the 
daughters  mates  -with  any  normal  male,  the  probabilities  are  that  half  of 
her  daughters  ^^ill  resemble  her  in  carrying  the  hidden  defect,  while  half 
of  her  sons  will  be  color-bUnd. 

A  female  is  color-blind  only  when  she  carries  the  recessive  genes  in 
both  of  her  A^-chromosomes  (second  part  of  diagram) .  When  mated  to  a 
normal  male,  her  daughters  all  have  normal  vision  but  carry  the  hidden 
defect,  while  all  her  sons  are  color-blind.  When  one  of  these  heterozygous 
daughters  mates  Avith  a  color-blind  male,  the  probabihties  are  that  half 
of  the  offspring  of  both  sexes  will  be  color-bUnd.  What  happens  when  a 
normal  male  is  involved  has  been  shown  in  the  preceding  paragraph. 

When  a  color-blind  female  mates  with  a  color-blind  male,  the  results 
are  even  more  serious  (third  part  of  diagi-am).  In  such  a  case  every 
A-chromosome  carries  the  recessive  gene,  and  all  the  offspring  are 
color-bHnd.  This  is  the  fifth  of  six  possible  crosses,  the  sixth  being  normal 
b}^  normal.     Fortunately  the  fifth  type  occurs  very  rarely. 

Among  organisms  that  have  been  extensively  studied  cytogenetically 
many  such  sex-linked  characters  have  been  identified. 

Summary  and  Conclusions. — The  characters  exhibited  by  an  organism 
are  determined  by  the  constitution  of  the  protoplasm  and  by  the  environ- 
mental conditions,  external  and  internal,  in  cooperation  with  which  the 
protoplasm  undergoes  development.  Character  relationships  of  succes- 
sive generations,  when  attributable  to  protoplasmic  composition,  con- 
stitute organic  heredity. 

The  data  on  the  inheritance  of  many  different  characters  show  the 
dependence  of  those  characters  upon  constitutional  factors  located  in  the 
nucleus,  and  they  suggest  that  the  factors,  or  genes,  are  individualized 
organic  units  or  semi-independent  portions  of  the  chromosomes.  That 
a  given  gene  is  present  is  indicated  when  it  exists  in  two  forms  producing 
two  effects.  When  both  forms  are  present  together,  one  commonly 
dominates  the  other  in  character  production.  A  given  gene  affects 
several  characters,  while  a  given  character  results  from  the  action  of 
numerous  genes  acting  at  the  same  or  different  stages  of  ontogeny. 

The  Mendelian  rules  according  to  which  characters  are  inherited  have 
their  cause  in  the  distribution  of  the  chromosomes  through  successive 
life  cycles.  The  association  of  chromosomes  beginning  at  syngamy 
results  in  a  combination  of  parental  characters  in  the  offspring.  The 
segregation  of  their  controlling  genes  pair  by  pair  when  si)ores  or  gametes 


192  FUNDAMENTALS  OF  CYTOLOGY 

are  formed  is  due  to  synapsis  and  disjunction  of  homologous  chromosomes. 
The  independence  of  many  character  pairs  in  inheritance  has  its  basis 
in  the  random  assortment  of  the  various  pairs  of  chromosomes  in  meiosis. 
The  linked  inheritance  of  many  characters  is  a  result  of  the  location  of 
their  differential  genes  in  the  same  chromosome  pair.  Recombinations 
of  the  characters  composing  two  hnked  pairs  are  due  to  crossing  over  of 
chromatids  somewhere  between  the  two  differential  gene  pairs. 

Sex  inheritance  in  dioecious  plants  and  animals  has  its  basis  in  the 
behavior  of  a  chromosome  pair  specially  differentiated  \\ith  respect  to 
genes  influencing  sex  and  often  in  visible  morphology.  Nonsexual 
characters  are  linked  with  sex,  though  not  restricted  to  one  sex,  when 
their  controlling  factors  are  located  in  the  X  chromosome. 

Which  chromosome  of  a  genome  carries  a  given  gene  or  linked  group 
of  genes  can  be  ascertained  by  using  trisomic  strains.  The  chromosome 
observed  to  be  present  in  triplicate  is  responsible  for  characters  showing 
special  trisomic  ratios  in  inheritance.  Other  methods  involving  further 
chromosomal  aberrations  also  are  available. 

The  serial  order  of  the  linked  genes  in  a  single  chromosome  pair  is 
found  by  comparing  the  recombination  percentages  obtained  after  crosses 
involving  various  couples  of  character  pairs. 

The  actual  positions  of  the  genes  in  the  chromosome  are  determined 
by  relating  abnormahties  of  characters  depending  on  these  genes  to 
visible  abnormalities  in  chromosome  structure,  such  as  deletions. 

As  a  result  of  the  normal  operation  of  the  chromosomal  mechanism, 
members  of  a  human  family  resemble  each  other  and  their  ancestors 
because  they  bear  many  similar  genes  in  chromosomes  derived  from 
common  sources.  They  differ  from  one  another  in  particular  ways 
because  of  the  orderly  reshuffling  of  the  chromosomes  at  each  meiosis 
and  the  various  combinations  resulting  from  syngamy.  The  human 
race  is  so  highly  heterozygous  that  complete  similarity  is  to  be  expected 
nowhere  except  in  identical  tmns,  which  arise  from  the  same  fertilized 
egg  and  therefore  carry  identical  genie  outfits,  barring  new  mutations. 
Extensive  studies  on  such  twin  pairs  reared  together  and  apart  have 
strongly  emphasized  the  fundamental  role  of  genetical  constitution  in 
determining  the  results  of  development.  Unlike  environments  may 
influence  the  development  of  different  capacities,  but  the  experimental 
evidence  indicates  that  the  initial  potentialities  in  identical  twins  are 
similar  to  a  degree  rarely  approached  in  other  pairs  of  individuals.  The 
naivete  of  attempts  to  explain  the  development  of  characters  without 
reference  to  the  genetical  nature  of  the  material  should  be  obvious. 


CHAPTER  XIII 
CHROMOSOMAL  ABERRATIONS 

Exceptions  to  the  normal  mode  of  chromosome  behavior  are  frequently 
observed,  particularly  when  large  numbers  of  individuals  are  being 
examined  in  experimental  work.  Often  one  is  led  to  discover  them  by 
suggestive  abnormalities  in  the  characters  of  one  or  more  individuals. 
Some  types  of  aberration  occur  only  rarely,  while  others  appear  much 
more  frequenth^;  and  it  has  been  found  that  by  various  experimental 
treatments,  such  as  irradiation  with  X  rays  or  ultraviolet  light,  the 
frequency  of  occurrence  can  be  markedly  increased.  Some  aberrations 
involve  whole  genomes  and  result  in  polyploidy  (Chap.  XIV).  Others 
affect  a  single  whole  chromosome,  as  in  the  nondisjunction  leading  to 
the  trisomic  condition  found  so  useful  in  assigning  genes  to  their  proper 
chromosomes  (page  174).  Still  others  produce  alterations  involving 
breakage  of  the  chromosome.  It  is  this  last  group  of  alterations  that 
will  concern  us  in  this  chapter. 

AbnormaUties  of  several  kinds  have  proved  their  value  in  cytogenetical 
research,  for  not  only  do  they  furnish  material  for  tests  of  hypotheses 
founded  upon  normal  chromosomal  and  genie  behavior,  but  to  certain 
questions  they  often  yield  answers  not  obtainable  in  normal  material. 
They  also  throw  a  most  interesting  light  upon  the  fundamental  problem 
of  the  nature  of  the  gene.  Finally,  they  afford  important  additional 
clues  to  the  role  of  intrachromosomal  changes  in  the  evolution  of  diversity 
among  organisms  in  nature. 

Deletion. — A  deletion  is  the  removal  of  a  portion  of  a  chromosome, 
the  remaining  portion  with  the  kinetochore  then  continuing  as  a  deficient 
chromosome  (Fig.  139).  A  deleted  segment,  if  it  does  not  include  the 
kinetochore,  cannot  function  in  mitosis  and  is  soon  lost.  Just  how  this 
alteration  is  accomplished  is  not  known,  but  the  evidence  indiiiates 
that  while  two  portions  of  a  chromonema  lie  very  close  together  two 
breaks  may  occur  there,  the  four  freshly  broken  ends  then  reuniting 
two  by  two  in  a  new  way.  The  loss  of  a  portion  usually  leads  to 
the  inviability  of  cells  having  no  corresponding  normal  chromosome, 
although  there  are  some  small  deletions  that  are  not  lethal  even  in 
monoploid  cells.  Deletions  are  nearly  always  intercalary,  as  shown  in 
the  diagram;  terminal  deletions  are  relatively  rare. 

193 


19-i 


FUNDAMENTALS  OF  CYTOLOGY 


When  a  deficient  chromosome  meets  a  nonnal  one  in  synapsis, 
homologous  regions  pair  closely,  leaving  a  region  of  the  normal  chromo- 
some extending  as  an  unpaired  loop.  Some  degree  of  sterihty  is  expected 
among  the  resulting  spores  or  gametes.  The  great  value  of  deletions  in 
determining  gene  location  has  already  been  emphasized  (page  182). 
Of  special  interest  is  the  fact  that  they  also  reveal  the  presence  and 
position  of  unmutated  genes  which  would  not  be  detected  in  crosses  of 


Fig.  139. — Diagram  illustrating  the  production  of  a  deletion  (of  the  cd  region)  and  the 
synapsis  of  the  resulting  deficient  chromosome  with  a  normal  chromosome.  If  the  ring  had 
included  the  kinetochore  (small  circle),  it  would  have  remained  functional  and  the  ends  of 
the  chromosome  would  have  been  lost.  Such  ring-shaped  chromosomes  disappear  eventu- 
ally becaxise  of  difficulties  in  mitosis. 

normal  individuals,  for  in  normal  material  both  the  normal  gene  and 
its  mutant  allele  must  be  present  before  the  presence  of  either  is  suspected. 
Inversion. — An  inversion  is  a  reversal  in  the  position  of  a  portion  of  a 
chromosome.  Most  inversions  are  intercalary  (Fig.  140);  a  few  are 
terminal.  The  mode  of  formation  is  apparently  like  that  of  deletions, 
except  that  the  four  broken  ends  recombine  in  a  different  pattern.  The 
l^ehavior  of  a  chromosome  carrying  an  inversion  is  normal  in  mitosis. 


r  /■ 


Fig.  140. 


-Diagram  illustrating  the  production  of  an  inversion  (of  the  cd  region)  and  the 
synapsis  of  the  chromosome  carrying  it  with  a  normal  chromosome. 


Its  genetical  effects  are  like  those  of  an  uninverted  chromosome,  except 
for  differences  in  linkage  relations  and,  in  some  cases,  a  modified  effect 
upon  characters  due  to  the  altered  relative  positions  of  certain  genes 
{position  effect).  Inversions  can  be  used  for  the  purpose  of  locating 
genes  when  linkage  relations  \\dthin  the  chromosome  are  well  known. 

At  the  time  of  synapsis  in  individuals  heterozygous  for  the  inversion, 
the  association  of  homologous  regions  results  in  the  looped  configuration 


CHROMOSOMAL  ABERRATIONS 


195 


shown  in  the  diagram.     Meiosis  may  be  carried  through,  yielding  spores 

or  gametes  with  and  without  the  inversion.     Frequently,  however,  there 

is  sterihty,  sometimes  in  a  high  degree.     This  is  due  in  part    to  the 

disturbing  effects  of  crossing  over  in  the  region  of  the 

inversion,  for  this  may  produce  a  chromatid  with  two 

kinetochores    and    a    chromatid    with    none.     The 

akinetic  chromatid  is  lost,    while  the  dikinetic  one 

either  forms  a  "bridge"  between  the  two  groups  at 

anaphase  (Fig.  141)  or  goes  in  its  entirety  to  one  pole. 

In  either  case  an  abnormal  complement  and  sterility 

result. 

Such  sterihty  is  of  special  interest  in  connection 
with  the  origin  of  diveree  types  in  nature,  for  it  has 
l)een  found  that  certain  geographical  races  of  Droso- 
phila  differ  in  being  homozygous  for  different  inver- 
sions. These  races  are  meiotically  regular  and  fertile, 
but  heterozygotes  formed  by  crossing  two  of  them  are 
not;  hence,  two  such  races  are  kept  distinct  bj^  this 
internal  mechanism  and  can  proceed  independently 
with  their  differentiation  into  more  widely  divergent 
types. 

Translocation. — The  transfer  of  a  portion  of  one 
chromosome  to  another  except  by  normal  crossing  over 
is  known  as  translocation.  It  may  involve  homologous 
or  nonhomologous  chromosomes.  In  nearly  all  cases 
the  translocation  is  reciprocal,  i.e.,  parts  of  two  chromosomes  are 
exchanged  (Fig.  142).  These  parts  may  be  of  any  relative  length, 
for  breaks  and  recombinations  evidently  can  occur  at  many  places  in 
the  chromosomes.     Sometimes  one  of  the  resulting  chromosomes  has 


Fig.  141.— Ana- 
phase /  in  inioro- 
.spororyte  of  maize 
showing  a  chromatid 
bridge  due  to  cross- 
ing over  in  the  region 
of  an  inversion. 
The  crossover  re- 
sulted in  a  clu-omatid 
with  two  kineto- 
chores and  one  with 
none.  The  latter  is 
seen  as  a  small  frag- 
ment near  the  bridge. 
{Courtesy  of  B.  Mc- 
Clintock.) 


Fig.  142. — Diagram  illustrating  reciprocal  translocation  between  nonhomologous  chromo- 
somes and  the  synapsis  of  the  resulting  chromosomes  with  their  normal  homologues. 

two  kinetochores  while  the  other  has  none,  but  in  the  successful  cases 
each  translocated  chromosome  has  one  only  and  behaves  normally  in 
mitosis.  The  simple  translocation  of  one  piece  without  exchange  seems 
to  occur  very  rarely. 


196 


FUNDAMENTALS  OF  CYTOLOGY 


The  meiotic  behavior  typical  of  plants  in  which  reciprocal  transloca- 
tion of  nonhomologous  chromosomes  has  occurred  is  shown  in  Figs. 
143  145.  An  individual  carrying  the  two  translocated  chromosomes  and 
the  two  normal  chromosomes  with  which  they  are  homologous  is  called  a 
structural  hybrid:  it  is  "heterozygous  for  the  translocation."  In  its 
sporocytes  these  chromosomes  form  a  cross-shaped  configuration  by  the 
synapsis  of  homologous  parts  and  then  open  out  into  a  ring-of-4  at  dia- 
kinesis.     Such  ring  or  chain  formation  is  known  as  catenation.     While 


Alternate  distributionj  give 
functional  spores  and  gametes 


Adjacent  distributions   give 
non -functional  spores  and  gametes 


Unions  of  functional   gametes  give  individuals  of   three  types: 


Stanolarcl   type 


Structural   hybrid 


Modified   type 


Fig.  143. — Diagram  sliowing  some  of  the  effects  of  reciprocal  translocation  between 
nonhomologous  chromosomes  (distinguislied  by  heavy  and  light  lines  and  unUke  knobs.) 

the  other  chromosomes  of  the  complement  form  bivalents  and  disjoin 
as  usual,  the  chromosomes  in  the  ring  disjoin  in  two  ways.  In  some 
sporocytes,  alternate  members  go  to  the  same  pole,  giving  spores  and 
later  gametes  of  two  kinds.  Since  both  kinds  contain  all  the  chromo- 
somal elements,  although  in  unlike  arrangements,  they  are  both  func- 
tional. In  other  sporocytes,  adjacent  members  of  the  ring  go  to  the  same 
pole,  yielding  spores  and  gametes  all  of  which  lack  certain  elements  of  the 
genome  and  are  nonfunctional.  Hence  such  plants  are  said  to  be  semi- 
sterile,  although  the  percentage  of  sterility  differs  considerably  in  different 
cases.  When  such  a  plant  is  selfed,  male  gametes  of  the  two  functional 
kinds  meet  female  gametes  of  the  same  two  kinds  and  j^roduce  offspring 


CHROMOSOMAL  ABERRA  TIONS 


197 


of  three  classes:  (1)  standard  plants  with  normal  chromosomes  in  dupli- 
cate, (2)  structural  hj^brids,  and  (3)  modified  plants  with  the  translocated 


Fig.  144.  —Synaptic  complex  forined  in  inicrosporocyte  of  maize  plant  heterozytious  for  a 
reciprocal  translocation.      {After  B.  McCIintock.) 


1 

2 

1^ 

4 

5 

6 

Fig.  115. — First  meiotic  division  in  microsporocytes  of  a  .strain  of  wheat  {Triticum) 
heterozygous  for  a  reciprocal  translocation.  1,  cross  configuration  in  prophase.  2,  ring- 
of-4  at  diakinesis.  3,  metaphase;  members  of  ring  about  to  disjoin  alternately.  4,  5, 
adjacent  disjunction;  metaplia.se  and  early  anapliasc.      G,  anaphase.      (After  L.  Smith.) 

chromosomes  in  duplicate.     These  three  types  tend  to  occur  in  the  ratio 
of  1:2:1. 

Among  plants,  both  types  of  homozygote,  standard  and  modified, 
commonly  show  a  regular  formation  and  disjunction  of  bivalent  chromo- 


198 


FUNDAMENTALS  OF  CYTOLOOY 


somes  and  breed  true.  They  differ,  of  course,  in  the  linkage  relations 
of  certain  characters  controlled  by  genes  in  the  chromosomes  involved 
in  the  translocation.  In  Drosophila  the  "modifieds"  are  for  some  reason 
sterile.  Modified  lines  with  all  the  chromosomes  of  the  genome  altered 
by  translocations  have  been  established  in  Crepis  tectorum  by  crossing 
strains  carrying  different  translocations,  and  these  new  lines  are  kept 
distinct  by  the  sterility  of  hybrids  formed  between  them  and  the  original 
standard  line. 

A  second  reciprocal  translocation  may  follow  a  first  in  the  same  line, 
or  two  translocations  may  be  brought  together  from  different  lines.     In 


t 

A 

1 

1 

3 

3 

t 

B 

1 

y 

1 

Fig.  146. — Diagram  illustrating  formation  of  ring-of-6  by  two  reciprocal  translocations. 
In  line  A,  chromosomes  1  and  2  are  translocated.  In  line  B,  chromosomes  2  and  3  are 
translocated.  Union  of  gametes  from  these  two  lines  may  give  a  group  of  six  chromosomes 
forming  a  double-cross  configuration  at  synapsis  and  a  ring-of-6  at  diakinesis.  If  the  two 
translocations  had  occurred  at  corresponding  levels  in  the  chromosomes,  the  synaptic  com- 
plex would  have  been  a  six-rayed  star.  Chromcsomes  are  marked  with  knobs  to  di.s- 
tinguish  ends. 


either  manner,  rings  of  more  than  four  chromosomes  may  be  built  up 
(Fig.  146).  In  extreme  cases  all  the  chromosomes  of  the  complement  are 
combined  in  one  large  ring  or  chain  in  the  meiotic  prophase  (Fig.  147,  h). 
Fertility  in  such  plants  tends  to  vary  with  the  percentage  of  alternate 
chromosome  distribution  in  anaphase  /. 

Reciprocal  translocation  and  its  effects  have  been  found  in  a  con- 
siderable number  of  plant  genera  in  nature.  In  a  species  of  peony 
(Paeonia  calif ornica),  which  has  10  somatic  chromosomes,  there  have 
been  found  seven  types  showing  respectively  in  sporocytes  five  inde- 
pendent bivalent  pairs,  a  ring-of-4  and  3  pairs,  a  ring-of-6  and  2  pairs, 
a  ring-of-8  and  1  pair,  a  ring-of-10,  2  rings-of-4  and  1  pair,  and  a  ring-of-4 
with  a  ring-of-6.  Near  the  center  of  the  range  of  the  species  in  California 
the  types  with  free  pairs  and  small  rings  are  more  abundant,  whereas 


CHROMOSOMAL  ABERRA TIONS 


199 


near  its  periphery  the  types  with  larger  rings  are  more  numerous.     It 
seems  hkclv  that  such  translocation  has  been  a  factor  in  the  differentiation 


i 


Fig.  147. — a,  Kings  and  cliains-of-4  in  microsporocyte  o£  Tradescantia.  (Some  of  the 
chromosomes  have  been  pressed  out  of  the  coll  in  making  the  preparation.)  b,  ring-of-12 
in  microsporocyte  of  Rhoco.     Each  chromosome  shows  its  two  arms.      {Courtesy  of  K.  Sax.) 

of  races  and  species,  although  its  importance  in  this  respect  cannot  yet 
be  measured.  It  is  held  to  be  a  major  factor  in  the 
genus  Crepis.  In  Datura  it  has  been  found  in  investi- 
gations extending  over  many  years  that  the  same 
fundamental  genome  of  12  chromosomes  has  its  parts 
arranged  in  numerous  ways  in  the  various  species  from 
different  parts  of  the  world.  They  appear  to  be  homo- 
zygous translocants,  or  modifieds,  derived  from  one  or 
more  main  chromosomal  types.  Within  the  species 
stramonium  there  are  several  such  natural  races,  or  prime 
types.  The  arrangement  of  chromosome  parts  in  the 
genome  of  a  given  race  is  determined  by  observing  the 
meiotic  configurations  (rings,  etc.)  in  crosses  between 
this  race  and  one  or  more  others  in  w^hich  the  arrange- 
ment is  know-n  in  terms  of  an  arbitrarily  chosen  stand- 
ard. Since  prime  tj^pes  in  the  same  species  are  so 
closely  similar  morphologically,  it  seems  best  to  regard 
reciprocal  translocation  as  supplementary  to  mutation, 
hybridization,  and  isolation  as  a  factor  in  speciation  in 
such  genera. 

The  genus  Oenothera  is  of  unusual  interest  in  this  con- 
nection because  of  its  relation  to  the  mutation  theory 
propounded  many  years  ago  by  Hugo  de  Vries,  the  great 
Dutch  botanist.  Oenothera  lamarckiana,  a  truebreeding 
type,  was  observed  to  produce  occasional  offspring  unlike 
itself.  This  was  interpreted  as  the  production  of  new 
species  by  sudden  large  steps,  or  mutations,  from  a  pure 
parent  species.  Since  that  time  cytogeneticists  have  developed  another 
interpretation  of  the  phenomenon. 


Fig.  148.- 
Catenated  chro- 
mosomes in  an 
evening  prim- 
rose, Oenothera 
franciscana  sul- 
phur ea.  a,  late 
meiotic  prophase, 
with  a  ring-of-12 
and  one  free  pair. 
b,  first  meiotic 
anaphase,  show- 
ing alternate  dis- 
tribution  of 
members  of  ring. 
(After  R.  E . 
Cleland.) 


200 


FUNDAMENTALS  OF  CYTOLOGY 


The  principal  cytological  element  in  the  new  explanation  is  the  fact 
that  12  of  the  14  chromosomes  occupy  constant  positions  in  a  ring  at 
meiosis  and  show  a  regularly  alternate  mode  of  disjunction  in  a  high 
percentage  of  the  sporocytes  (Fig.  148).  The  main  genetical  element 
in  the  explanation  is  the  evidence  that  there  are  two  groups  of  genes, 
known  as  Renner  complexes,  that  segregate  in  sporogenesis  and  that  each 
complex  carries  a  gene  which  prevents  development  when  in  the  homo- 
zygous state  (Fig.  149).  If  the  two  Renner  complexes  (designated  as 
gaudens  and  velans  in  this  species)  are  located,  respectively,  in  the  two 
groups  of  seven  chromosomes  regularly  passing  to  opposite  poles  in  meiosis 
(six  from  the  ring,  plus  one  member  of  the  free  pair  carrying  similar 
Renner  complex  factors),  it  is  possible  to  account  for  the  production  of 


Fig.  149. — Diagram  of  cytogenetic  constitution  of  an  evening  primrose,  Oenothera 
lamarckiana.  A,  ring-of-12  and  one  free  pair  at  diakinesis.  Factors  of  Renner  complex 
gaudens  (G)  represented  by  large  dots,  those  of  velans  complex  (F)  by  circles,  those  common 
to  both  complexes  by  shaded  circles.  Factors  ordinarily  recombining  by  crossing  over 
represented  by  small  dots.  B,  segregation  of  two  Renner  complexes  by  alternate  disjunc- 
tion of  chromosomes  in  ring  at  anaphase  /.  C,  effect  of  lethal  factors  in  Renner  complexes: 
of  three  possible  combinations  only  the  heterozygotes  (GF)  survive. 

spores  and  gametes  of  two  main  classes :  those  carrying  the  gaudens  com- 
plex and  those  carrying  the  velans  complex.  Random  combinations  of 
male  and  female  gametes  of  these  two  kinds  produce  zygotes  of  three 
kinds,  but  since  the  development  of  two  of  these  is  prevented  by  homo- 
zygosity of  lethal  genes,  the  only  plants  appearing  are  of  the  original 
heterozygous  0.  lamarckiana  type.  Thus  the  type  breeds  true  not 
because  it  is  a  pure  species,  but  because  it  is  a  hybrid  whose  homozygous 
offspring  do  not  survive. 

The  mutations  which  occasionally  appear  in  0.  lamarckiana  have 
been  found  to  have  several  causes:  (1)  gene  mutation  of  the  ordinary 
kind;  (2)  the  removal  of  a  lethal  gene  from  a  Renner  complex  by  crossing 
over;  (3)  the  occasional  nonlethal  action  of  a  lethal  gene;  (4)  nondis- 
junction, giving  trisomic  and  other  types;  (5)  chromosome  doubling. 

Other  species  of  Oenothera  also  exhibit  the  results  of  reciprocal  translo- 
cation, and  relationships  are  strongly  suggested  by  the  ways  in  which  the 


CHROMOSOMAL  ABEIiRA  TIONS 


201 


\arious  f];cnomes  and  Renner  complexes  present  in  the  genus  interact 
wlien  brought  together  by  hybridization. 

Duplication. — Occasionally  a  chromosome  comes  to  have  a  certain 
portion  represented  two  or  more  times  instead  of  once.  This  is  known  as 
duplication.     The  extra  portion  or  portions  may  lie  next  to  the  one 


'ffg^ll'-:-'^ 


-<?^^%" 


Fig.  15U. — Diagram  illustrating  the  duplication  (of  the  cd  region)  and  the  synapsis  of  the 
chromosome  cari-ying  it  with  a  normal  chromosome. 

normally  present  or  at  some  distance  from  it.  Their  orientation  may  be 
normal  or  inverted,  depending  upon  the  manner  in  which  they  were 
added.  Evidently  they  originate  by  one  or  more  translocations  (Fig. 
150).  The  presence  of  duplications  has  in  some  cases  been  revealed  by 
genetical  data,  and  in  salivary-gland  chromosomes  they  can  be  readily 
seen  and  correlated  with  abnormalities  in 
characters  and  breeding  behavior.  A  sig- 
nificant point  regarding  duplication  is  that 
it  may  produce  a  genetical  effect  like  that 
of  gene  mutation.  The  dominant  mutation 
known  as  bar  eye  in  Drosophila  has  turned 
out  to  be  a  result  of  duplication  (Fig.  151). 
Aberrations  and  the  Nature  of  the 
Gene.  —The  chromosomal  aberrations  de- 
scribed briefly  in  these  pages  are  leading 
toward  a  needed  improvement  in  our  under- 
standing of  the  gene.  For  many  years  the 
gene  concept  has  been  of  inestimable  value 
in  the  task  of  reducing  to  order  the  multi- 
farious data  of  genetics.  The  theory  that 
the  organism  has  within  it  discrete  units 
with  a  special  role  in  the  inheritance  and 
development  of  characters  rests  upon  the 
independent  "Mendelizing"  of  various  small  character  differences  in 
sexually  reproducing  organisms,  and  upon  the  further  fact  that  different 
characters  can  be  correlated  with  the  activity  of  definitely  localized  small 
regions  in  the  chromosomes.  Evidence  for  the  occasional  mutation  of 
the  units  is  found  in  the  sudden  alterations  of  characters  ascribed  to  them. 


BAR 


NORMAL 

Fig.  151. — Portion  of  salivary- 
gland  chromosome  of  Drosophila, 
showing  the  duplication  respon- 
sible for  the  bar-eye  mutation. 
{After  T.  S.  Painter.) 


202  FUNDAMENTALS  OF  CYTOLOGY 

The  gene  has  been  generally  regarded  as  a  niinute  body  with  a  con- 
siderable degree  of  structural  and  physiological  independence,  perhaps 
the  last  member  of  the  series  organism-cell-nucleus-chromosome-chromo- 
mere-gene,  the  gene  itself  being  possibly  a  single  protein  molecule  or  small 
group  of  molecules.  There  have  been  various  conjectures  regarding 
the  nature  of  its  chemical  and  physical  activity,  and  attempts  have 
even  been  made  to  estimate  its  size.  Most  geneticists  have,  however, 
been  content  to  employ  the  gene  concept  mainly  as  a  useful  tool  in 
research  and  to  define  it,  if  at  all,  in  terms  of  its  effects,  leaving  the  future 
to  furnish  an  adequate  description  of  it  in  physicochemical  terms.  This 
is  a  situation  in  biology  comparable  with  that  in  physics  and  chemistry, 
where  the  concept  of  the  atom  was  long  employed  \\'ith  conspicuous 
success  when  far  less  was  known  about  its  actual  nature  than  is  known 
today.  The  hope  that  our  knowledge  of  the  gene  is  to  become  more 
intimate  is  encouraged  by  researches  now  in  progress  on  the  biochemical 
aspects  of  genie  action.  In  the  ascomycetes,  for  example  (page  167), 
are  nuclear  conditions  that  are  making  it  possible  to  associate  particular 
chemical  reactions  with  certain  genes,  and  from  the  nature  of  these 
reactions  it  is  expected  that  much  can  be  learned  concerning  the  physico- 
chemical  constitution  of  the  genes  involved.  This  association  of  cyto- 
genetics with  biochemistry  promises  to  be  as  useful  in  leading  us  toward 
a  solution  of  the  problem  of  the  role  of  genes  in  ontogeny  as  the  union 
of  genetics  and  cytology  40  years  ago  has  been  in  elucidating  their  role 
in  heredity. 

Discussion  of  the  nature  of  the  gene  has  been  stimulated  anew  by  the 
discovery  that  certain  aberrations  in  the  visible  structure  of  the  chromo- 
some produce  effects  similar  in  many  respects  to  those  of  gene  mutations. 
Three  illustrative  cases  are  the  following.  A  color  character,  brown 
midrib,  which  had  been  ascribed  to  6m  i,  a  recessive  gene  located  near 
the  kinetochore  in  chromosome  5  of  maize,  has  been  found  to  develop 
even  when  the  region  carrying  this  gene  is  removed  altogether  by  a  small 
deletion.  In  Droso'phila  the  character  roughest-3  mutated  when  the 
small  region  carrying  its  differential  gene  in  the  X-chromosome  was 
inverted;  moreover,  when  the  former  alignment  was  reestablished  by  a 
reinversion,  the  normal  character  was  restored.  The  dominant  mutant 
character,  bar  eye,  in  Droso'phila  is  now  known  to  be  due  to  a  duplication. 
This  character  may  increase  in  intensity  when  a  second  duplication  adds 
still  another  like  portion,  and  it  may  revert  to  normal  when  the  extra 
portion  or  portions  are  removed.  An  additional  observation  of  impor- 
tance is  that  in  some  X-rayed  cells  both  gene  mutations  and  chromosomal 
deficiencies  show  the  same  response  to  variations  in  irradiation  dosage, 
the  frequency  of  both  varying  as  a  linear  function  of  the  total  energy 


CHROMOSOMAL  ABERRATIONS  203 

applied.  The  two  processes  are  also  similarly  affected  by  dormancy 
as  opposed  to  activity  in  tissues  at  the  time  of  irradiation. 

Such  phenomena  have  led  to  a  current  theory  that  a  gene  is  any  small 
portion  of  a  chromosome  having  an  effect  upon  character  development 
differing  from  that  of  neighboring  portions,  so  that  when  this  portion  is 
unlike  in  the  two  chromosomes  of  a  pair  Mendelian  behavior  is  exhibited 
by  characters  influenced  by  it.  \ny  change  in  this  portion  affecting  its 
influence,  whether  the  change  be  a  loss,  gain,  or  rearrangeinent,  represents 
a  mutation.  In  other  words,  genes  are  not  all  elements  of  the  same 
nature,  even  though  their  effects  are  generally  comparable.  The  extreme 
form  of  this  view  is  that  genes  as  discrete  biological  units  do  not  exist 
and  that  the  only  real  genetical  unit  is  the  chromosome,  in  particular 
the  chromonema.  This  may  be  regarded  as  a  sort  of  gigantic  chain 
molecule  whose  various  parts  alter  the  action  of  the  whole  in  some  man- 
ner whenever  they  are  sufficiently  modified.  Opponents  of  this  extreme 
view  have  cited  phenomena  in  normal  and  aberrant  material  which 
would  not  be  expected  in  a  single  molecule.  For  example,  the  positions 
of  crossovers  and  induced  breaks  and  the  relatively  small  amounts  of 
energy  required  to  produce  them  indicate  the  presence  of  numerous 
distinct  units  not  bound  together  by  strong  intramolecular  chemical 
bonds.  They  regard  the  evidence  as  indicative  of  a  process  of  gene 
mutation  distinct  from  chromosomal  aberrations,  although  both  may 
share  in  producing  the  phenomena  studied  by  the  geneticist. 

In  any  event,  genes  are  localized  constitutional  conditions  that  can  be 
treated  as  units  in  genetical  research.  Such  conditions  are  the  physical 
basis  of  Mendelian  phenomena,  and  any  stable  modification  of  their 
character-influencing  power  may  be  regarded  as  a  mutation.  We 
continue  to  look  to  the  future  for  a  determination  of  the  precise  physico- 
chemical  nature  of  these  conditions  and  the  manner  of  their  association 
in  the  chromosome.  In  this  search  for  further  light  on  the  nature  of  the 
gene,  few  developments  are  more  suggestive  than  the  increasingly  close 
association  of  genetics,  protein  chemistry,  and  virus  research.  It  seems 
that  genes,  proteins,  and  viruses  have  much  in  common,  and  the  clarifica- 
tion of  their  relationships,  when  achieved,  should  be  of  immense  value 
in  many  branches  of  biological  science. 


CHAPTER  XIV 
CHROMOSOME  NUMBERS  AND  THEIR  ALTERATION 

In  earlier  chapters  it  has  been  shown  that  in  the  hfe  cycle  of  sexually 
reproducing  organisms  there  is  an  alternation  of  two  chromosome  num- 
bers, one  of  them  being  double  the  other.  In  typical  cases  the  numeri- 
cally smaller  chromosome  group  consists  of  one  genome,  while  the  larger 
consists  of  two.  It  has  also  been  mentioned  that  higher  numbers  of 
genomes  are  sometimes  present.  For  reasons  not  well  understood  this 
condition  is  very  rare  among  animals,  but  it  is  of  frequent  occurrence 
in  plants,  especially  among  angiosperms.  In  many  genera  of  this  group 
it  characterizes  certain  species  of  a  genus  or  even  all  of  them.  Sometimes 
a  single  altered  individual  appears  in  a  population  of  diploid  plants  in 
the  field  or  breeding  plot.  An  individual  may  show  the  increase  in 
number  only  in  a  portion  of  the  body,  this  portion  constituting  a  sector 
or  sometimes  a  layer  of  tissue  overlying  the  normal  portion. 

Plants  with  increased  numbers  of  certain  chromosomes  or  of  whole 
genomes  are  valuable  in  many  ways.  They  furnish  material  for  the  study 
of  the  action  of  individual  chromosomes,  since  they  may  have  the  various 
members  of  a  genome  present  in  different  numerical  relations.  Again, 
differences  in  chromosome  number  and  the  characters  sometimes  cor- 
related with  them  are  often  useful  in  classifying  related  species  and  in 
determining  their  probable  origin  (Chap.  XVII).  Finally,  the  change 
in  chromosome  number  is  sometimes  accompanied  by  morphological  or 
physiological  alterations  that  render  the  j^lant  more  valuable  com- 
mercially. This  point  has  increased  in  interest  since  the  discovery  that 
such  chromosomal  changes  can  be  induced  by  experimental  means. 

Terminology. — A  nucleus  with  some  number  other  than  the  true 
monoploid  or  diploid  number  of  chromosomes  is  said  to  be  heteroploid . 
This  term  and  others  given  below  are  also  applied  to  cells,  tissues, 
individuals,  races,  or  species  with  such  nuclei.  When  the  number  is  an 
exact  multiple  of  the  monoploid,  the  nucleus  (or  tissue,  etc.)  is  ewploid. 
The  terms  designating  the  multiples  up  to  10,  beginning  with  the  triple 
number,  are  as  follows:  triploid,  tetraploid,  pentaploid,  hexaploid,  hep- 
taploid,  octoploid,  enneaploid,  decaploid.  The  higher  multiples,  which 
are  of  rarer  occurrence,  are  usually  designated  as  eleven-ploid,  twelve- 
ploid,  etc.  Euploid  types  are  often  said  to  be  polyploid.  In  such 
spocies  the  zygotic  and  gametic  chromosome  numbers  are,  for  example, 

204 


I 


CHROMOSOME  NUMBERS  AND  THEIR  ALTERATION  205 

hexaploid  and  triploid,  or  tetraploid  and  diploid,  rather  than  diploid  and 
monoploid  as  in  the  types  selected  for  discussion  in  foregoing  chapters. 

A  nucleus  (or  tissue,  etc.)  with  some  number  other  than  an  exact 
multiple  of  the  monoploid  number  is  anewploid.  When  the  number  is  a 
little  lower  than  some  multiple,  it  is  hypoploid;  when  it  is  a  little  higher, 
it  is  hyperploid.  Obviously,  a  number  falling  between  the  diploid  and 
triploid  numbers,  for  example,  may  be  called  either  hjqDerdiploid  or 
hypotriploid. 

A  chromosome  complement  in  which  heteroploidy  is  due  to  the 
multiplication  of  a  single  kind  of  genome  (or  of  some  of  its  members) 
is  said  to  be  autoheteroploid ;  whereas  one  in  which  specifically  different 
genomes  or  members  are  combined,  as  in  an  interspecific  hybrid,  is 
aUoheter opioid.  Although  this  distinction  cannot  always  be  sharply- 
drawn,  it  is  of  considerable  importance,  as  will  appear  later.  This 
chapter  deals  with  the  first  type  of  heteroploidy.  The  second  type  is 
discussed  in  the  following  chapter. 

Unfortunately,  authors  have  not  agreed  in  their  use  of  symbols 
denoting  chromosome  numbers.  At  present  it  seems  best  to  let  x  and 
2x  stand,  respectively,  for  the  gametic  and  zygotic  nvimbers  in  the  life 
cycle,  regardless  of  whether  the  organism  is  heteroploid  or  not.  The 
syml)ols  n  and  2n  have  often  been  used  in  the  same  sense,  but  the  present 
tendency,  which  should  be  followed  in  the  interest  of  uniformity,  is  to 
use  n  for  the  true  monoploid  number  (one  genome) ,  2n  for  the  true  diploid 
number  (two  genomes),  3w  for  the  true  triploid  number  (three  genomes), 
etc.  Obviously,  there  are  cases  in  which  one  cannot  determine  without 
special  study  whether  a  gametic  complement  of  x  chromosomes  is  made 
up  of  one,  two,  or  more  genomes.  In  some  of  the  literature  the  symbol  h 
])enotes  the  basic  number,  or  true  monoploid  number. 

Tetraploidy, — Next  to  diploid  plants,  tetraploids  constitute  the  com- 
monest chromosomal  type  in  natvu'e.  The  doubling  of  the  chromosome 
number  occurs  in  two  principal  ways.  The  first  of  these  is  by  somatic 
doubling,  in  which  the  chromosomal  division  cycle  and  the  spindle 
mechanism  lack  their  normal  correlation,  so  that  the  divided  chromosomes 
at  the  close  of  the  resulting  aberrant  mitosis  are  enclosed  in  one  nucleus 
instead  of  two.  If  this  aberration  occurs  in  a  very  young  embryo,  the 
whole  plant  into  which  it  develops  has  the  tetraploid  number,  whereas 
its  occurrence  at  a  later  stage  results  in  a  plant  with  tetraploidy  in  one 
or  more  branches  or  other  portions.  The  second  way  is  by  ameiosis, 
in  which  failure  of  haplosis  leads  to  the  formation  of  diploid  spores  and 
gametes,  a  union  of  two  such  gametes  then  giving  a  tetraploid  plant. 

The  principal  methods  used  for  inducing  tetraploidy  artificiall}'-  are 
temperature  treatment,  decapitation,  and  treatment  with  colcliicine. 
When,  for  example,  young  maize  ears  are  kept  unusually  warm  during 


206 


FUNDAMENTALS  OF  CYTOLOGY 


the  stages  when  the  embryos  are  beginning  their  development,  some  of 
these  embryos  may  develop  into  tetraploid  plants  instead  of  diploids. 
Abnormal  temperature,  either  high  or  low,  may  induce  ameiosis  in  flower 
buds,  so  that  diploid  spores  and  gametes  become  available  for  crossing. 
When  some  plants,  notably  tomatoes,  are  decapitated,  the  shoots  arising 
from  the  callus  tissue  are  often  tetraploid.  At  present  the  most  popular 
method  for  inducing  tetraploidy  and  higher  stages  of  heteroploidy  is 
the  one  employing  colchicine.     This  substance,   which  is  an  alkaloid 

/  / 


Fig.  152. — Induced  autopolyploidy  in  maize.  Left:  normal  diploid  plant,  an  ear  from 
such  a  plant,  and  diploid  chromosome  complement  in  root  tip.  Right:  tetraploid  plant 
from  kernel  on  ear  subjected  to  heat-treatment  during  early  stages  of  embryo  development; 
also  mature  ear  and  tetraploid  chromosome  complement  in  root  tip.  {Courtesy  of  L.  F. 
Randolph.) 

derived  from  the  autumn  crocus,  may  be  applied  by  painting  very  young 
buds  with  a  lanolin  emulsion  containing  it,  or  by  standing  cut  shoots  or 
roots  in  an  aqueous  solution  for  brief  periods,  or  by  spraying  young 
plant  parts  with  such  a  solution,  or  by  treating  seeds  before  planting. 
Many  useless  malformations  may  result  from  such  treatments,  particu- 
larly when  the  dosage  is  too  high,  but  when  the  technique  is  sufficiently 
refined  tetraploid  shoots  may  appear.  Colchicine  produces  its  effect  by 
preventing  the  formation  of  the  mitotic  spindle.  The  chromosomes  pass 
through  their  division  cycle  as  usual,  but  since  no  spindle  is  developed 
their  halves  undergo  no  anaphasic  separation  and  reorganize  as  a  single 


CHROMOSOME  NUMBERS  AND  THEIR  ALTERATION 


20- 


tetraploid  nucleus.  When  the  influence  of  the  drug  has  declined  suffi- 
ciently for  normal  nuclear  and  cell  divisions  to  occur,  development  of  the 
tissues  continues  with  the  altered  chromosome  number. 

The  characters  exhibited  by  tetraploid  plants  derived  by  doubling 
as  described  above  often  serve  to  distinguish  them  from  their  diploid 
relatives  (Figs.  152,  155).     In  many  instances  they  are  stockier  in  habit. 


wXwi 


Fig.   153. — Stoniates  and  epidermal  cells  of  diploid,  tetraploid,  and  octoploid  Nicotiana 
hybrids.      {After  W.  H.  Greenleaf.) 

darker  green  in  color,  bear  larger  flowers  and  seeds,  and  have  larger  nuclei 
and  cells.  With  a  hand  lens  one  can  often  tell  the  polyploids  from  the 
diploids  by  the  size  and  arrangement  of  the  stomates  (Fig.  153).  In 
mature  vegetative  organs  such  as  leaves  the  cells  may  be  larger  and  fewer 
than  in   the   diploids;   sometimes   they   are   not.     In   the   meristematic 


W^,e#-* 


*     .' 

Fig.  154. — Synaptic  configurations  in  heteroploid  plants,  a,  trivalent  at  zygotene  in 
triploid  tulip,  b,  postdiplotene  trivalent  in  triploid  tulip,  c,  quadrivalent  at  zygotene  in 
tetraploid  hyacinth,  d,  postdiplotene  quadrivalent  in  pentaploid  tulip.  {After  TV.  C.  F. 
Newton  and  C.  D.  Darlington.) 

tissues  of  maize  the  number  of  cells  is  the  same  as  in  the  diploids,  but 
with  the  greater  cell  size  is  associated  the  development  of  larger  organs. 
There  are  also  physiological  differences  between  diploids  and  tetraploids. 
Some  tetraploid  plants  are  able  to  grow  well  in  a  wider  range  of  ecological 
habitats  than  the  related  diploids.  Fruits  borne  on  tetraploid  tomato 
plants  derived  from  dii:)loids  have  a  higher  vitamin  C  content  than  those 


208 


FUNDAMENTALS  OF  CYTOLOGY 


borne  on  the  diploids.     Similarlj^  yellow  kernels  produced  by  tetraploid 
maize  plants  have  more  vitamin  A  than  those  from  diploids. 

Chromosome  behavior  in  autotetraploid  plants  is  normal  throughout 
somatic  development.  At  meiosis,  however,  certain  irregularities  arise 
when  all  or  some  of  the  chromosomes  form  quadrivalent  groups  at 
synapsis  (Fig.  154).  This  may  lead  to  some  irregularity  in  distribution 
at  anaphase  /  and  add  thus  to  the  sterility  attributable  to  pther  genetical 
causes.  In  colchicine-induced  autotetraploids,  fertility  ranges  from  a 
fairly  high  value  comparable  to  that  in  many  natural  tetraploids  down  to 
complete  sterility.     Later  it  will  be  pointed  out  that  in  allotetraploid 


Fig.  155. — Selted  ears  from  diploid  (above)  and  tetraploid  (below)  plants  heterozygous 
for  the  color-factor  pair  Rr.  Segregation  for  color  is  about  3:1  in  the  diploid  and  35: 1  in 
the  tetraploid.      Note  difference  in  size  of  kernels.      {After  L.  F.  Randolph.) 

plants  the  fertility  may  be  much  higher  than  in  the  diploids  from  which 
they  are  derived  (page  221).  In  tetraploid  Unes,  diploid  individuals 
appear  on  rare  occasions  as  a  result  of  parthenogenesis,  just  as  haploids 
sometimes  appear  among  diploid  organisms. 

Genetical  ratios  for  characters  of  autotetraploid  plants  tend  to  be 
unlike  those  of  diploids,  for  each  chromosome  is  present  in  quadruplicate. 
Assuming  a  random  distribution  of  the  four  chromosomes  bearing  a 
given  gene,  the  expected  phenotypic  ratios  after  selfing  are  1:0  for  a 
plant  with  A  AAA  or  AAAa,  35: 1  for  a  plant  with  AAaa,^:l  for  a  plant 
with  Aaaa,  and  0:1  for  one  with  aaaa.  The  corresponding  tcstcross 
ratios  are  1:0,  5:1,  1:1,  and  0:1.  These  expectations  are  for  chaiacters 
controlled  by  genes  in  regions  near  tlie  kinetochore,  where  the  four 
chromosomes  are  distributed  at  random  but  the  eight  chromatids  are 
not,  owing  to  the  fact  that  sister  clu-omnlids  tend  to  pass  regularl>'  to 


CHROMOSOME  NUMBERS  AND   THEIR  ALTERATION  209 

the  same  pole,  (see  i)age  106).  ('hiasinata  liberate  the  chromatids  from 
this  restriction  in  regions  far  from  the  kinetochores;  hence  in  such  regions 
they  are  (hstributerl  at  random.  On  the  basis  of  such  random  distribu- 
tion of  eight  chromatids  in  these  regions,  the  ratios  for  genes  located 
there  would  be  the  following.  After  selfing:  AAA  A,  1:0;  AAAa,  783 : 1 ; 
AAaa,  21:1;  Aaaa,  2.48 : 1 ;  aaaa,  0:1.  After  a  testcross  the  correspond- 
ing ratios  would  be  1:0,  27:1,  3.7:1,  13:15,  and  0:1.  In  a  number  of 
researches  expected  ratios  have  been  found  to  be  rather  closely  approxi- 
mated (Fig.  155). 

The  breeding  behavior  of  autopolyploids  differs  markedly  from  that  of 
diploids  because  of  the  relative  infrequency  with  which  recessive  char- 
acters reappear  in  succeeding  generations.  Thus  the  F^  generation  is 
more  uniform  than  in  diploids.*  Another  fact  of  genetical  and  practical 
interest  is  that  tetraploid  lines  in  many  cases  are  kept  distinct  from  the 
diploids  by  cross  incompatibility  and,  in  case  fertilization  is  accomplished, 
by  the  failure  of  the  resulting  triploid  lines  to  compete  successfully  with 
the  diploids  and  tetraploids. 

Triploidy.^ — Triploid  plants  almost  always  arise  from  the  union  of  a 
haploid  gamete  with  a  diploid  one,  the  latter  having  been  produced  regu- 
larly by  a  tetraploid  plant  or  after  a  failure  of  haplosis  in  a  diploid  plant. 
Such  plants,  like  tetraploids,  show  good  vegetative  growth  and  are  fre- 
quently somewhat  larger  than  the  diploids.  Although  the  sexual  fertility 
of  some  triploids  is  low  because  of  the  irregular  meiosis  where  three 
genomes  are  present,  others,  e.g.,  maize  and  iris,  show  good  fertility'. 
Progenies  derived  from  triploid  plants  tend  to  have  a  low  sur^'ival  "\-alue 
because  of  their  aneuploid  chromosome  numbers,  and  this  greatly  restricts 
the  value  of  triploidy  in  the  development  of  new  types.  As  a  general  rule 
triploid  plants  are  unsuccessful  in  nature  unless  they  have  some  form  of 
asexual  reproduction  upon  which  they  can  rely;  hence  they  are  rarely 
found  established  among  sexually  reproducing  species  in  the  field. 
Numerous  highly  valued  plants  of  the  orchard  and  garden,  e.g.,  certain 
varieties  of  apples,  tulips,  iris,  and  hyacinths,  are  triploid,  but  they  are 
normally  propagated  ])y  vegetative  methods. 

Higher  Degrees  of  Polyploidy. — Species  with  6,  8,  and  10  or  more 
genomes  are  found  in  nature  and  show  a  high  degree  of  fertility.  That 
their  establishment  probal)ly  was  accomplished  gradually  and  may  ha\e 
involved  some  hybridization  is  suggested  by  experimentally  produced 
autopolyploids  with  such  numbers  of  genomes.  In  maize,  to  select  an 
example  from  a  considerable  number  of  known  cases,  induced  octoploid 
plants  are  far  less  vigorous  than  the  tetraploids  and  are  completely  sterile. 
Again,  when  doubling  is  induced  in  commercial  varieties  of  potatoes, 
which  are  tetraploids,  the  resulting  octoploid  plants  and  tubers  are  inferior 
to  the  tetraploids;  whereas,  when  related  diploid  species  are  doubled,  the 


210  FUNDAMENTALS  OF  CYTOLOGY 

resulting  tetraploids  tend  to  be  more  vigorous  and  highly  fertile.  The 
sterility  of  higher  autopolyploids  is  due  in  part  to  a  tendency  to  form 
multivalents  (Fig.  156)  which  are  distributed  with  some  degree  of  irregu- 
larity in  meiosis. 

In  general  the  results  obtained  so  far  with  agricultural  and  ornamental 
plants  indicate  that  a  limit  of  improvement  through  induced  chromosomal 
doubling  is  ordinarily  reached  at  or  near  the  tetraploid  level.  Further- 
more, a  deleterious  effect  of  the  chromosomal  change  may  offset  the 
improvement.  Thus  doubling  in  tobacco  plants  results  in  an  increase  of 
as  much  as  one-third  in  the  percentage  of  nicotine  in  the  leaves,  but  this 
is  more  than  offset  by  a  reduction  of  50  per  cent  in  the  dry  weight  of  the 
leaves.  In  several  cereals  it  has  been  found  that  although  the  doubled 
varieties  bear  larger  kernels  the  total  yield  is  not  larger,  for  an  accompany- 
ing reduction  in  fertility  decreases  the  number  of  kernels  obtained. 

Fig.  156. — Chromosomes  from  normal  and  colchicine-induced  polyploid  Petunia  plants 
at  first  meiotic  metaphase:  two  bivalents,  two  trivalents,  two  quadrivalents,  two  quinque- 
valents,  one  heptavalent,  and  one  octovalent.      {After  A.  Levan.) 

The  problem  of  plant  improvement  by  chromosomal  doubling  is  there- 
fore far  from  being  a  simple  one.  It  is  found  that  different  kinds  of 
plants  (species,  varieties,  inbred  lines)  often  show  widely  different 
responses  to  the  doubling,  for  the  type  and  degree  of  change  exhibited 
evidently  depend  in  part  upon  the  genie  composition  and  physiological 
state  of  the  material  treated.  At  present  the  results  of  a  given  experi- 
ment cannot  be  predicted  with  any  degree  of  certainty,  nor  can  the  limits 
of  the  method's  usefulness  be  stated.  Nevertheless,  investigators  are 
confident  that  with  proper  attention  to  the  genotype  and  a  judicious  use 
of  selection  and  crossing  much  of  value  will  be  accomplished,  even  though 
the  ratio  of  error  to  trial  may  still  remain  high.  We  shall  revert  to  this 
topic  in  a  discussion  of  hybridity  in  the  next  chapter. 

Although  clear  cases  of  polyploidy  are  relatively  rare  among  animals, 
the  high  numbers  in  some  genera  strongly  suggest  changes  in  number 
in  the  evolution  of  certain  natural  groups.  Known  somatic  numbers  in 
carnivores  range  up  to  78  in  the  dog,  and  a  comparable  range  is  found  in 
rodents.  Most  of  the  investigated  primates  including  man  have  48. 
Single  individuals  or  strains  with  increased  chromosome  numbers  are 
occasionally  encountered  in  the  field  and  in  the  laboratory.  Diploid, 
tetraploid,  and  octoploid  races  of  the  brine  shrimp  (Artemia)  exist  in 


CHROMOSOME  NUMBERS  AND  THEIR  ALTERATION 


211 


nature,  and  it  has  been  found  that  the  development  of  tetraploid  indi- 
\iduals  can  be  induced  in  the  laboratory  by  refrigerating  eggs  that  would 
normally  have  developed  parthenogenetically  into  diploids.  Another 
notable  case  is  a  series  of  polyploid  salamanders  that  appeared  spon- 
taneously in  laboratory  cultures  (Fig.  157).  A  beginning  has  also  been 
made  on  the  investigation  of  colchicine-induced  chromosome  doubling  in 
animal  cells. 


izm 


Fig.  157. — Larvae  of  the  common  newt  {Triturus  viridcscens)  with  different  numbers  of 
genomes.  From  left  to  right:  pentaploid,  tetraploid,  triploid,  diploid,  and  monoploid 
specimens.  Below  them  are  mitotic  metaphases  observed  in  bits  of  tail  fins  stained  and 
mounted  without  sectioning.  The  normal  diploid  larva  has  22  chromosomes.  The  size 
of  nuclei  and  cells  increases  roughly  in  proportion  to  the  chromosome  number,  but  the 
body  size  does  not;  this  indicates  a  lower  cell  number  in  the  polyploids.  (Courtesy  of  G. 
Fankhausrr.) 

Other  Types  of  Heteroploidy. — In  the  sporophytes  of  plants  true 
monoploidy  occurs  very  rarely.  Such  plants  have  appeared  in  cultures  in 
the  case  of  several  angiosperm  genera  (Datura,  Zea,  Crepis,  Nicotiana, 
Oenothera,  Triticum,  and  others),  but  nowhere  have  such  monoploids 
become  estabUshed  in  nature,  so  far  as  we  know.  The  main  reason  for 
this  appears  to  be  their  high  degree  of  sexual  sterility.  Although  they  are 
usually  somewhat  smaller  and  less  vigorous  than  diploids,  the}^  often 
grow  well,  but  at  the  time  of  meiosis  the  chromosomes  of  the  single 
genome,  having  no  synaptic  partners,  behave  so  irregularly  that  practi- 
cally no  functional  spores  or  gametes  are  produced.  Rarely  all  the 
chromosomes  are  included  in  a  single  spore,  so  that  after  many  trials  a 
diploid  plant  is  sometimes  obtained  by  selfing  a  monoploid  one.     Such  a 


212 


FUNDAMENTALS  OF  CYTOLOGY 


plant  is  of  interest  in  genetical  studies,  for  it  is  completely  homozygous, 
except  for  possible  new  mutations.  It  seems  possible  that  a  monoploid 
race  might  be  established  in  nature  if  the  plant  had  efficient  means  of 
vegetative  reproduction,  but  no  such  case  has  been  discovered. 

Among  the  many  types  of  aneuploidy,  in  which  one  or  more  full 
genomes  are  accompanied  by  one  or  more  additional  chromosomes  not 
constituting  a  full  genome,  the  commonest  and  probably  the  most  impor- 
tant is  the  simple  trisomic  condition  (2/i  +1).  Here  the  plant  has  all  its 
chromosomes  in  duplicate  except  one,  which  is  present  in  triphcate.  If 
two  members  of  the  genome  are  in  triplicate,  the  plant  is  said  to  be 
doubly  trisomic  (2n  +1  +  1),  etc.  Simple  trisomic  plants  are  of  special 
value,  for  they  have  normal  fertility,  transmit  the  extra  chromosome  to 


C 


)^ 


y 


to 


/\  > 


Fig.  158. — -Flower  heads  borne  on  monoploid 
individuals  of  Crepis  capillaris.  Left:  monoploid 
head.  Middle:  diploid  head,  after  chromosomal 
doubling.  Right:  head  with  monoploid  and  diploid 
sectors  arising  after  local  doubling.  {After  L. 
H  oiling  shead.) 


Fig.  159. — Chromosomes  in 
pollen  grain  from  trisomic  Datura 
stramonium.  This  grain  carries 
an  extra  3.4  chromosome.  (After 
S.  Satina,  D.  Bergner,  and  A.  F. 
Blakeslee.) 


some  of  their  progeny,  though  onlj^  rarely  from  the  pollen  parent.  The>' 
thus  serve  to  reveal  the  special  functions  of  each  chromosome  of  the 
genome.  This  point  has  been  explained  on  page  174,  where  the  effect 
upon  genetical  ratios  was  described. 

Since  each  chromosome  of  the  genome  differs  from  the  others  with 
respect  to  the  group  of  genes  it  carries,  plants  trisomic  for  different 
chromosomes  are  expected  to  differ  in  visible  characters.  This  expecta- 
tion is  met  in  some  degree  in  the  species  investigated.  In  Datura  stramo- 
nium each  of  the  12  possible  trisomic  types  can  be  distinguished  from  the 
others  (Figs.  159,  160).  The  same  is  true  of  the  trisomic  types,  also  12 
in  number,  in  Nicotiana  sylvestris.  In  Zea  mays  9  of  the  10  possible 
trisomic  types  have  been  obtained,  and  most  of  these  are  distinguishable, 
although  the  differences  are  less  than  might  be  expected.  Moreover, 
there  are  certain  features,  notably  the  reduced  size  of  the  plants  and 
seeds,  that  characterize  all  the  maize  trisomies  in  common.  In  addition  to 
these  primary  trisomic  plants,  in  which  the  three  chromosomes  composing 


niUOMOSOME  NUMBERS  AND  THEIR  ALTERATION  213 


NORMAL 


ROLLED 


GLOSSY 


BUCKLING  ELONGATE 


ECHINUS 


COCKLEBUR  MICROCARPIC  REDUCED 


POINSETTIA 


SPINACH 


GLOBE 


ILEX 


Fig.  IGO. — Seed  capsules  of  iioriaal  and  primary  tiisoniic  types  of  the  Jinisoii  weed 
(Datura  stramonium).  Each  of  the  12  members  of  the  genome  (see  Fig.  72)  produces  a 
cliaracteristic  visible  effect  when  present  in  triplicate.  In  "rolled"  the  extra  member  is 
cnromosome  1.^,  in  "glossy"  it  is  3.4,  and  so  on  to  "ilex"  with  23.24.  The  plants  show 
other  differences  also.     {Courtesy  of  A.  F.  Blakeslee.) 


214 


FUNDAMENTALS  OF  CYTOLOGY 


the  trisome  are  all  alike,  Datura  has  been  found  to  have  a  number  of 
secondary  trisomic  types,  in  which  the  extra  chromosome  consists  of  two 
similar  arms  as  the  result  of  some  form  of  aberration  (Fig.  161).  Since 
there  are  12  chromosomes  in  the  genome,  24  secondaries  should  be  pos- 
sible if  each  of  the  two  arms  of  every  chromosome  were  to  give  rise  to  such 
a  chromosome  with  similar  arms.  More  than  half  of  these  have  actually 
been  found  in  Datura.  The  same  phenomenon  is  also  known  to  occur  in 
maize,  although  not  so  many  of  the  secondaries  have  yet  been  discovered. 
The  special  characters  of  these  plants  show  that  each  half  of  each  chromo- 
some has  its  own  distinctive  effect  upon  the  many  reactions  involved  in 
development,  which  is  what  one  should  infer  from  the  fact  that  they  carry 
different  groups  of  genes. 

Monosomic  plants,  with  one  less  than  the  normal  diploid  chromosome 
number  {2n  —  1),  are  rarely  encountered,  evidently  because  of  the  serious 


Fig.  161. — Diagram  of  the  production  of  chi'omosomes  with  two  similar  arms  ("secondary" 
chromosomes)  by  the  misdivision  of  the  kinetochore. 

unbalance  in  the  chromosome  complement  caused  by  the  loss  of  one  mem- 
ber. Autopolyploids,  however,  are  frequently  found  with  a  chromosome 
missing  as  a  result  of  irregular  disjunction  of  multivalents;  here  the  loss  of 
a  single  member  causes  less  unbalance.  In  tobacco  plants,  which  are 
allotetraploid  (page  222),  nearly  all  the  24  possible  types  lacking  one 
chromosome  have  been  found. 

Other  aneuploid  types  \\ith  varying  numbers  of  additional  chromo- 
somes are  found  in  considerable  variety,  especially  in  hybrids  between 
members  of  a  polyploid  series.  The  number  of  extra  chromosomes  in 
some  of  these  is  rather  high.  Ordinarily  fertility  and  vigor  among  aneu- 
ploids  approach  the  normal  as  the  composition  of  the  complement 
approaches  that  in  a  plant  with  complete  genomes. 

Significance  of  Autoheteroploidy  in  Nature.^ — After  observing  the 
alterations  in  characters  following  the  spontaneous  or  induced  doubling  of 
the  chromosome  number,  and  after  considering  the  prevalence  of  poly- 
ploidy among  angiosperms  in  nature  as  revealed  by  lists  of  reported 
chromosome  numbers,  the  conclusion  that  doubling  and  character 
differentiation  have  been  causally  related  in  the  natural  evolution  of  these 


CHROMOSOME  NUMBERS  AND   THEIR  ALTERATION  215 

plants  is  one  that  can  hardly  be  escaped.  To  what  extent  the  poly- 
ploidy in  nature  has  arisen  by  doubling  in  relatively  pure  strains  (auto- 
polyploidy),  or  b}^  the  doubling  in  hybrids  (allopolyploidy)  to  be  discussed 
in  the  next  chapter,  is  a  difficult  problem  to  solve.  At  present  the  relative 
importance  of  the  two  processes  is  a  subject  of  debate,  and  it  is  well 
realized  that  much  observational  and  experimental  work  must  be  done 
before  the  tangled  situation  in  nature  can  be  very  thoroughly  understood. 
In  the  meantime  it  is  to  be  borne  in  mind  that  the  two  forms  of  natural 
polyploidy  differ  in  degree  rather  than  in  kind:  to  form  a  hybrid  at  all, 
two  species  must  have  a  considerable  degree  of  similarity  in  their  genomes. 

The  significance  of  autopolyploidy  in  the  origin  of  polyploid  species 
in  nature  is  strongly  suggested  by  the  fact  that  experimentally  produced 
polyploids  and  those  in  the  field  show  many  resemblances.  They  tend  to 
differ  morphologically  from  related  diploids  in  the  same  way;  they  often 
show  similar  phj^siological  peculiarities  and  ecological  adaptability;  both 
show  the  same  type  of  meiotic  irregularity,  including  multivalent  associa- 
tions and  sterility  among  the  spores  or  gametophytes.  The  natural 
polyploids  are  irregular  in  a  less  degree,  presumably  because  of  the  past 
action  of  natural  selection. 

Whether  induced  autopolyploids  gradually  improve  in  fertility  and 
regularity  of  meiotic  behavior  after  many  generations  is  a  significant 
question  now  being  studied.  Should  they  do  so  and  become  distinct  new 
types  with  regular  bivalent  formation,  it  is  possible  that  they  might  have 
greater  genetic  stability  than  their  ancestral  diploid  types,  for  the  reason 
that  a  new  recessive  mutation  would  remain  hidden  longer  and  affect  the 
phenotype  only  in  those  rare  individuals  having  all  the  increased  number 
of  controlling  factors  in  the  recessive  state. 

The  role  of  allopolyploidy  is  suggested  by  the  absence  of  multivalents 
in  many  natural  polyploids,  for  when  the  high  chromosome  number 
results  from  a  combination  of  genomes  each  of  which  is  present  onl}^ 
twice,  bivalents  only  are  expected  at  meiosis.  Furthermore,  as  will  be 
pointed  out  in  the  next  chapter,  chromosomal  doubling  in  diploid  hybrids 
is  often,  though  not  always,  accompanied  by  increased  fertility,  and  this, 
together  with  the  increased  vigor  and  new  character  combinations  due  to 
the  hybridity,  should  contribute  much  to  the  success  of  such  newly  formed 
types  in  nature. 

Answ^ers  to  many  questions  like  those  suggested  above  will  be  required 
before  the  evolutionary  role  of  heteroploidy  can  be  described  \\dth  any 
degree  of  precision.  A  role  it  surely  has,  but  just  how  it  should  ho  ranked 
among  other  factors  of  speciation  in  different  families  of  organisms  we  do 
not  know. 


CHAPTER  XV 
CYTOLOGICAL  ASPECTS  OF  HYBRIDITY 

The  traditional  conception  of  a  hybrid  was  that  it  was  the  offspring  of 
parents  belonging  to  different  species.  From  a  practical  point  of  view,  a 
hybrid  was  an  individual  manifesting  a  combination  or  blend  of  characters 
from  those  species.  The  advances  in  modern  genetics  and  cytology  have 
led  to  an  interpretation  that  is  at  once  broader  in  its  basis  and  more 
specific  in  its  designation  of  what  constitutes  hybridity.  It  is  now  usually- 
stated  that  a  hybrid  is  the  product  of  the  union  of  two  genetically  unlike 
gametes,  whatever  their  source.  They  may  come  from  two  species, 
varieties,  or  inbred  lines,  or  even  from  the  same  heterozygous  bisexual 
individual.  They  may  or  may  not  manifest  combinations  or  blends  of 
characters,  for  dominance  often  renders  them  indistinguishable  from  one 
parent.  Hence,  in  the  modern  view,  the  essence  of  hybridity  lies  in  the 
genetical  constitution  of  the  individual  itself  rather  than  in  the  taxonomic 
relationship  of  the  parents  which  contributed  to  this  constitution, 
although  it  is  of  course  realized  as  fully  as  ever  that  the  crossing  of  indi- 
viduals with  greater  than  vaz'ietal  differences  is  of  special  importance  in 
the  evolution  of  natural  types. 

The  essential  constitutional  state  of  an  ordinary  diploid  hybrid  lies  in 
the  unlikeness  of  its  two  genomes.  The  smallest  degree  of  difference 
between  them  is  seen  in  an  individual  heterozygous  for  only  one  gene. 
(It  may  be  pointed  out  in  passing  that  this  condition  may  also  arise  by 
mutation  in  the  individual,  rather  than  by  gametic  union.)  Heterozj^- 
gosity  in  its  many  possible  degrees  thus  constitutes  hybridity  in  modern 
genetics.  The  two  genomes  may  also  differ  in  the  arrangement  of  the 
genes  in  regions  of  certain  chromosomes,  as  in  the  plants  heterozygous 
for  translocations  and  inversions  described  in  Chap.  XIII.  Such  plants 
are  referred  to  as  structural  hybrids,  even  though  the}"  may  conceivably' 
contain  no  heterozygous  pairs  of  genes.  Furthermore,  one  genome  may 
have  genes  with  no  counterpart  in  the  other,  as  in  plants  with  a  deletion 
in  one  genome,  or  in  a  hybrid  between  two  rather  distantly  related  types. 
The  number  of  chromosomes  may  even  differ  in  the  two  genomes.  The 
present  tendency  is,  therefore,  to  include  a  wider  variety  of  cases  under 
the  heading  of  hybridity,  but  to  state  more  specifically  what  is  common  to 
all  of  them:  a  dissimilarity  in  the  genomes  responsible  for  their  cytogenet- 
ical  behavior. 

216 


CYTOLOGIGAL  ASPECTS  OF  HYBRIDITY  217 

The  genomic  composition  of  a  hybrid  usually  has  well-known  conse- 
quences. Among  these  are  the  appearance  of  certain  parental  characters 
to  the  exclusion  of  others,  the  appearance  of  a  condition  intermediate 
between  the  parents  as  in  "blending"  or  quantitative  inheritance,  the 
production  of  gametes  unlike  in  genetical  constitution,  and  often  a  reduc- 
tion in  the  degree  of  fertility.  Hybrids  resulting  from  narrow  crosses, 
such  as  those  cited  as  illustrations  of  Mendelian  heredity  in  Chap.  XII, 
commonly  show  regular  chromosome  behavior  and  good  fertility.  It  is 
mainly  in  hybrids  resulting  from  wider  crosses,  i.e.,  in  hybrids  in  the 
traditional  sense,  that  more  extensive  genie  and  structural  differences  in 
the  chromosomes  lead  to  the  cytological  and  genetical  abnormalities  to 
be  reviewed  in  this  chapter. 

Chromosome  Behavior  in  Diploid  Hybrids. — The  fundamental  reason 
why  it  is  often  difficult  or  impossible  to  obtain  hybrids  between  members 
of  different  species  or  genera  lies  in  the  genie  dissimilarity  of  the  parents. 
The  effects  of  this  dissimilarity  are  various.  The  pollen  of  one  species 
may  not  grow  successfully  in  the  style  of  the  other;  the  gametes,  if  they 
meet,  may  not  actually  fuse;  if  fusion  does  occur,  the  zygote  may  fail  to 
develop  because  of  disharmony  within  its  chromosome  complement  or 
between  it  and  the  surrounding  tissues.  In  certain  cases,  on  the  other 
hand,  the  cross  results  in  a  hybrid  that  develops  well,  even  with  greater 
vigor  than  was  shown  by  the  parents.  The  two  parental  genomes,  in 
spite  of  their  differences,  may  thus  constitute  a  single  harmonious  system 
during  ontogenetic  development. 

In  most  such  hybrids,  abnormalities  appear  at  some  stage  in  the 
development  of  reproductive  cells  that  render  them  partially  or  com- 
pletely sterile.  Although  degenerative  changes  may  set  in  at  an  early 
stage  of  flower  development,  the  most  characteristic  cytological  aberra- 
tions appear  during  the  meiotic  prophase  and  affect  especially  the  course 
of  synapsis.  Synapsis  may  be  normal,  indicating  a  close  genie  similarity 
in  the  genomes.  More  often  synapsis  fails  to  occur  between  some  or  all 
of  the  chromosomes  (asynapsis),  or  after  synapsing  the  chromosomes 
may  separate  prematurelj''  (desynapsis) .  Although  asynapsis  usualh'^ 
indicates  a  considerable  degree  of  genetical  dissimilarity  in  the  genomes, 
it  does  not  always  do  so,  for  even  in  pure  lines  and  narrow  crosses  synapsis 
sometimes  fails  because  of  certain  mutant  genes  influencing  the  course  of 
meiosis  or  because  of  temporary  physiological  conditions  induced  by  the 
environment.  Obviously,  caution  must  be  used  in  depending  upon 
synapsis  as  a  criterion  of  relationship. 

The  usual  consequence  of  normal  synapsis  is  regularity  in  the  ana- 
phasic  distribution  of  the  chromosomes,  each  spore  and  gamete  bearing  a 
complete  genome.  This  is  distinctly  favorable  to  fertility,  yet  it  does  not 
guarantee  it,  for  some  of  the  genomes  produced  may  haA'e  chromosomes 


218  FUNDAMENTALS  OF  CYTOLOGY 

that  do  not  constitute  a  harmonious  system.     Some  of  the  foregoing 
points  are  iUust rated  in  the  following  cases. 

Maize  (Zea  mays)  and  Alexican  teosinte  {Euchlaena  mexicana)  each 
have  10  chromosomes  in  the  gamete.  AVhen  the  two  are  crossed,  the 
resulting  hybrids  vary  in  fertility  depending  in  part  upon  what  race  of 
teosinte  is  used.  Synapsis  tends  to  be  regular  (Fig.  162),  and  crossing 
over  takes  place.  In  subsequent  generations  some  of  the  plants  show 
various  parental  character  combinations,  while  others  are  precisel}^  like 
maize  or  teosinte.  This  indicates  a  degree  of  cytological  and  genetical 
similarity  unusually  high  for  plants  assigned  to  different  genera.  On  the 
basis  of  a  vaiiety  of  e^ddences  it  has  recently  been  proposed  that  the  two 

species  of  Euchlaena  be  transferred  to 
the  genus  Zea. 

Chromosomal  compatibility  and 
fertility  are  also  shown  in  various 
degrees  in  crosses  of  European  and 
American  species  of  grape,  sycamore, 
and  larch,  as  well  as  in  certain  inter- 
generic  moth  hybrids.  After  crossing 
the  garden  pea  {Pisum  sativum)  with. 
P.  humile,  each  with  seven  chromo- 
FiG.  162 —Pachytene  stage  ill  micro-     ^^^^^  ^^  ^^le  gamete,  the  hybrids  in 

sporocyte  of  a  teosmte-maize  /<  i  hybrid,  . 

showing   the    nearly    perfect    synapsis     Subsequent  generations  show  regular 
of    the    chromosomes.    {After    A.    E.     nieiosis,  but  the  Occurrence  of  abnor- 

Longley.) 

mal  types  and  much  sterility  indicates 
disharmonies  in  many  of  the  complements  produced  by  the  random 
assortment  of  the  parental  chromosomes. 

When  the  foxglove  (Digitalis  purpurea)  is  crossed  with  D.  ambigua, 
each  having  14  chromosomes  in  the  gamete,  the  number  of  bivalents 
formed  at  meiosis  in  the  hybrid  varies  from  5  to  12,  the  rest  of  the  chromo- 
somes remaining  univalent.  Such  behavior  is  exhibited  by  many  other 
interspecific  hybrids,  and  the  irregularity  may  vary  in  amount  with  the 
cultural  conditions.  One  result  of  this  partial  asynapsis  is  irregular  ana- 
phasic  distribution:  the  bivalents  disjoin  normally,  but  the  univalents 
either  pass  in  various  numbers  to  the  poles  or  undergo  equational  division. 
Further  irregularity  follows  in  the  second  division,  so  that  numerous 
abnormal  complements  and  much  sterility  result.  Irregularity  in  chro- 
mosome distribution  often  leads  to  the  formation  of  microspore  groups 
comprising  spores  varying  in  size  and  number  (polyspory)  instead  of 
normal  quartets.  Such  a  condition  can,  however,  arise  from  temporary 
environmental  causes  such  as  extreme  fluctuations  in  temperature; 
consequently,  nonuniform  pollen  is  not  a  sure  sign  of  hybridity.  Along 
mth  the  in  viable  chromosomal  combinations  are  some  that  are  successsful. 


CYTOLOGICAL  ASPECTS  OF  HYBRIDITY  219 

This  may  be  the  case  even  when  the  genomes  of  the  parents  differ  in  the 
number  of  members.  Thus  in  Fx  pansy  hybrids  between  Viola  arvensis 
(n  =  17)  and  V.  tricolor  (n  =  13)  the  number  of  bivalents  varies  and  there 
is  some  steriUty,  but  later  generations  are  made  up  of  both  the  parental 
types  and  a  number  of  new  ones  that  are  fertile  and  breed  true. 

When  asynapsis  is  complete,  total  sterilit}^  may  be  expected.  Occa- 
sionally^ there  is  a  different  result:  all  the  chromosomes  may  undergo  a 
single  equational  division,  two  large  spores  then  being  formed  each  with 
the  hybrid's  diploid  complement.  Rarely  such  a  spore  may  function  in 
the  production  of  polyploid  offspring. 

Hybridity  Involving  Pol5^1oidy. — Hybrids  containing  different  num- 
bers of  genomes  may  be  obtained  by  intercrossing  members  of  a  polyploid 
series.  For  example,  crosses  of  diploid  and  tetraploid  species  may  yield 
triploid  hybrids,  those  between  tetraploids  and  octoploids  may  produce 
hexaploid  hybrids,  etc.  Polyploid  hybrids  may  also  be  obtained  by 
inducing  chromosome  doubling  in  plants  already  hybrid  in  constitution. 
The  characters  exhibited  by  such  plants,  provided  they  develop  success- 
fully, depend  not  only  upon  the  kinds  of  parental  genes  and  their  inter- 
action, but  often  upon  the  relative  number  of  parental  genomes  as  well. 
Thus  in  maize-teosinte  hyl)rids  the  tetraploid  type  with  two  genomes  from 
each  parent  resembles  the  diploid  hybrid,  whereas  the  triploid  hj^brid  with 
one  maize  and  two  teosinte  genomes  looks  more  like  teosinte.  The  same 
tendenc}^  is  strikingly  shown  in  more  extensive  series  of  radish-cabbage 
hybrids  and  moss  hybrids. 

The  breeding  behavior  of  these  allopolyploid  plants  depends  of  course 
upon  the  number  of  genomes,  the  type  and  regularity  of  synapsis,  the 
viability  of  spores,  gametes,  and  zygotes,  the  ratio  of  genome  numbers 
in  embryo  and  endosperm,  and  other  factors.  The  calculation  of  expected 
genetical  ratios  becomes  a  complex  matter,  yet  for  certain  types,  notably 
the  one  in  which  there  are  two  genomes  from  each  parent  (see  next 
section),  the  expectations  have  in  several  instances  been  approximated  by 
latios  observed  in  the  breeding  plot. 

Chromosome  behavior  at  meiosis  in  polyploid  hybrids  is  illustrated 
in  the  following  cases.  In  a  fertile  hexaploid  hybrid  poppy  formed  by 
crossing  the  diploid  Papaver  nudicaule  (7  chromosomes  in  gamete)  with 
the  decaploid  P.  striatocarpum  (35  chromosomes  in  gamete)  the  micro- 
sporocytes  showed  21  bivalents:  all  the  chromosomes  found  mates.  In 
contrast  to  this,  a  hexaploid  hybrid  rose  formed  by  crossing  a  diploid 
form  (7  in  gamete)  with  a  decaploid  form  (35  in  gamete)  showed  onl}^  7 
bivalents,  the  remaming  28  chromosomes  appearing  as  univalents.  In 
hybrids  like  the  latter  the  chromosomes  often  show  a  very  characteristic 
type  of  subsequent  behavior:  the  bivalents  disjoin  and  pass  poleward, 
after  which  the  univalents,  now  longitudinally  double,  occupy  the  equator 


220 


FUNDAMENTALS  OF  CYTOLOGY 


and  separate  equationally.  All  may  succeed  in  reaching  the  poles  iti 
time  to  be  included  in  the  telophase  nuclei,  or  some  may  fail  and  become 
the  nuclei  of  small  extra  microspores  (Fig.  163).  Such  irregularity- 
results  in  various  degrees  of  sexual  sterility. 

The  difference  in  behavior  shown  by  the  poppy  and  rose  hybrids  is 
interpreted  on  the  basis  of  chromosomal  homology  as  follows.  The 
genomes  involved  in  the  poppy  cross  are  of  five  kinds:  A  B  Bi  C 
Ci.  The  diploid  species  has  in  the  soma  two  A  genomes  only,  one  being 
transmitted  by  each  gamete.  The  decaploid  species  has  all  five  in  the 
gamete.  Hence  after  a  cross  the  pairing  in  the  hybrid  is  AA  BBi  CC\, 
making  21  bivalents  in  all.  In  the  roses  there  are  also  five  genomes, 
ABODE,  but  their  degree  of  difference  is  great  enough  to  preclude 
synapsis  between  them.     The  diploid  type  furnishes  a  gamete  with  A 


Fig.  163. — 1,  2,  meiosis  in  miciosporocytes  of  an  interspecific  wheat  hybrid.  After  the 
bivalents  disjoin  and  pass  poleward  in  division  /,  the  univalents  occupy  the  equator  and 
separate  equationally.  3,  supernumerary  microspores  in  a  wheat-rye  hybrid.  {After 
K.  H.  von  Berg.) 

and  the  decaploid  type  one  with  A  B  C  D  E,  so  that  in  the  hybrid  only 
the  two  A  genomes  form  bivalents.  The  hypothesis  that  a  differentiation 
of  the  fundamental  genome  of  a  genus  into  several  kinds  has  occurred  in 
different  strains  with  the  passage  of  time  has  the  support  of  other  similar 
cases.  From  such  synaptic  behavior,  conclusions  are  drawn  regarding 
the  degree  of  residual  homology  in  these  genomes  and  the  degree  of  rela- 
tionship of  the  plants  containing  them.  This  method  of  analysis  has  been 
carried  out  on  a  very  extensive  scale  with  different  types  of  wheat, 
goat  grass  (Aegilops) ,  rye,  and  their  various  interspecific  and  intergeneric 
hybrids.  Here,  as  in  the  roses,  there  are  genomes  of  several  types  each 
consisting  of  seven  chromosomes,  and  these  are  combined  in  different 
ways  in  the  genera,  polyploid  species,  and  hybrids. 

Amphidiploidy. — Special  consideration  should  be  given  to  the  type  of 
polyploid  hybrid  having  two  genomes  from  each  of  two  species.  Such  a 
plant  is  said  to  be  aniphidiploid,  since  it  is  diploid  for  both  parental 
genomes.     (It  is  also  allotetraploid,  but  a  plant  combining  three  genomes 


CYTOLOGICAL  ASPECTS  OF  HYBRIDITY 


221 


from  one  parent  with  one  of  the  other  is  hkewise  allotetraploid.)  Amplii- 
diploid  hybrids  are  of  special  importance  because  they  are  usually  fertile, 
occur  rather  widely  among  angiosperms  in  nature,  afford  clues  to  the 
relationship  of  certain  species,  and  open  a  new  path  to  the  improvement 
of  cultivated  plants. 

Amphidiploidy  commonly  arises  through  a  doubling  of  the  chromo- 
some number  in  the  somatic  cells  of  a  diploid  hybrid.  Such  doubling  may 
occur  spontaneously,  and  in  numerous  cases  it  has  been  induced  with 


Fig.  164. — Hybridity  and  polyploidy  in  Calendula.  From  right  to  left:  flower  heads 
of  C.  suffruticosa,  C.  officinalis,  a  diploid  officinalis  X  suffruticosa  hybrid,  a  tetraploid 
officinalis  X  suffruticosa  hybrid  resulting  from  colchicine  treatment.  {Courtesy  of  C. 
Weddle.) 


colchicine.  If  it  takes  place  in  the  zygote  very  soon  after  syngamy,  the 
whole  plant  is  amphidiploid;  if  later,  e.g.,  in  a  bud,  only  a  branch  or  other 
portion  shows  this  condition.  The  most  valuable  feature  of  these  plants 
is  the  increased  fertility  that  many  of  them  show  over  the  diploids  from 
which  they  arose.  It  is  largely  in  the  hope  of  conferring  some  degree  of 
fertihty  upon  sterile  hybrids  with  desirable  combinations  of  characters 
that  the  doubling  technique  is  applied  in  plant-improvement  programs, 
and  in  many  cases  the  results  have  been  successful.  In  addition,  the 
plants  obtained  may  show  certain  characters  associated  with  tetraploidy 
itself,  such  as  sturdier  habit  or  greater  flower  size  (Fig.  164).  They  also 
frequently  exhibit  pronounced  hybrid  vigor. 


222 


FUNDAMENTALS  OF  CYTOLOGY 


One  of  the  earliest  known  amphidiploid  hybrids  appearing  in  culture 
was  the  fertile  Primula  kewensis,  with  36  somatic  chromosomes.  A 
cross  between  P.  florihunda  (2n  =18)  and  P.  verticillata  (2n  =  18)  had 
yielded  the  sterile  diploid  P.  kewensis  (2n  =  18)  with  one  genome  from 
each  parent  species.  From  a  lateral  bud  on  this  plant  there  arose  spon- 
taneously a  tetraploid  shoot  with  two  genomes  from  each  pai'ent,  and  this 
proved  to  be  fertile.  The  numerical  changes  may  be  represented  as 
follows:  (9  +  9)  X  2  =  36. 

An  example  of  doubling  which  evidently  occurred  after  sj^ngamy  is 
afforded  by  a  fairly  fertile  amphidiploid  columbine  that  appeared  after  a 
cross  of  the  two  diploid  species  Aquilegia  chrysantha  and  A.  flahellafa  nana: 
(7  +  7)  X  2  =  28.  A  general  formula  for  such 
cases  would  be  (n  +  7i)2  =  4n.  Among  com- 
mercially important  plants  evidently  ha\'ing  such 
a  constitution  are  the  pink-flowered  ornamental 
tree,  Aesculus  carnea,  and  tobacco,  Nicotiana 
fahacum.  The  former  arose  in  cultivation  as  a 
hybrid  between  the  horse  chestnut,  .4.  hippo- 
castanum,  and  A.  pavia:  (20  +  20)  X  2  =  80. 
The  latter  has  been  shown  by  a  long  series  of 
studies  to  represent  in  all  probability  an  am- 
phidiploid hybrid  derived  from  a  cross  of  N. 
sylvcstris  and  another  species  which  now  appears 
to  have  been  A^.  otophora:  (12  -^  12)  X  2  =  48. 

Some  plants  spoken  of  as  amphidiploids  have 
arisen  from  crosses  of  species  differing  in  chromo- 
some number.  The  chromosome  complement  of 
such  an  amphidiploid  Crepis  plant  is  shown  in 
Fig.  165.  Nicotiana  "  digluta''  arose  ivom  a,  cross 
of  N.  gliitinosa  (24  somatic  chromosomes)  and  N.  tahacum  (48  somatic 
chromosomes):  (12  +  24)  X  2  =  72.  If,  as  indicated  in  the  preceding 
paragraph,  A^.  tahacum  is  tetraploid  with  2  genomes  from  each  of 
two  species,  N.  ^'digluta"  in  terms  of  the  basic  number  for  the 
genus,  12,  would  be  allohexaploid  with  4  genomes  from  one  species 
and  2  from  the  other.  The  general  formula  for  such  cases  would 
be  (n  +  2n)2  =  6n.  Whether  such  plants  are  regarded  as  amphi- 
diploid because  they  carry  the  combined  somatic  complements  of  the 
parents  or  as  allohexaploid  because  of  their  number  of  basic  genomes, 
they  have  the  degree  of  fertility  exhibited  by  other  hybrids  in  which 
each  genome  has  a  duplicate  with  which  to  pair  at  meiosis.  Further 
examples  of  this  type  of  hybrid  are  the  domestic  plum,  (8  -f  16)  X  2 
=  48;  certain  hyln-id  mints,  (48  -|-  12)  X  2  =  120;  and  certain  hybrid 


Fig.  165. — Somatic 
chromosome  complement 
in  an  amphidiploid  hybrid 
between  Crepis  capillaris 
and  C  tectorum.  It  com- 
prises two  genomes  of  four 
members  each  from  tec- 
torum (7')  and  two  gen- 
omes of  three  members 
each  from  capillaris  (C). 
Satellites  on  tectorum  D- 
chromosomes  do  not  show 
at  metaphase  in  this  hy- 
brid. {After  L.  Hollings- 
head.) 


CYTOLOGICAL  ASPECTS  OF  HYBRIDITY  223 

cottons,  (26  +  13)  X  2  =  78.     In  the  mints  and  cottons  the  new  types 
were  developed  with  the  aid  of  colchicine. 

Ampliidiploids  sometimes  arise  in  ways  other  than  by  somatic  chromo- 
some doubling.  Diploid  spores  and  therefore  gametes  may  appear  after 
haplosis  has  failed  in  sporogenesis  in  a  diploid  hybrid,  two  diploid  gametes 
then  uniting.  Although  the  chance  of  obtaining  such  plants  in  this 
manner  seems  to  be  relativelj'  small,  they  have  evidently  arisen  thus  from 
interspecific  diploid  hybrids  in  Nicotiana,  Triticum,  and  Digitalis,  and  in  a 
Raphanus-Brassica  hybrid.  A  recentl}^  observed  case  is  that  in  Madia, 
a  genus  of  western  composites.  Two  rare  and  self-sterile  species,  M. 
nutans  {n  =  9)  and  M.  Rammii  (n  =  8)  produced  a  nearly  sterile  diploid 
hybrid  (2/(  =  17).  Among  several  types  in  Fo  there  were  two  plants  with 
34  chromosomes  which  showed  almost  perfect  synapsis  into  17  bivalents. 
After  four  generations  the  plants  of  this  line  were  vigorous,  fertile,  true 
lireeding,  and  different  in  several  morphological  characters  from  their 
parents  and  all  other  species  of  the  genus. 

A  third  important  method  is  illustrated  by  amphidiploid  snapdragons 
(Antirrhinum)  obtained  by  crossing  two  different  autotetraploid  strains. 

It  should  now  be  evident  why  polyploid  hybrids  confront  the  geneticist 
with  difficulties,  as  suggested  earlier  in  the  chapter.  Even  in  the  typical 
amphidiploids  with  two  genomes  from  each  parent,  much  depends  upon 
the  manner  and  degree  in  which  synapsis  is  carried  out.  In  the  most 
nearly  true  breeding  of  them  the  homologous  chromosomes  from  the  same 
parent  form  bivalents,  while  those  from  the  other  parent  do  likewise 
(autosynapsis).  The  result  is  that  the  genomes  of  the  parent  species 
remain  distinct  in  successive  generations,  the  hybrid  therefore  breeding 
true  for  characters  due  to  interspecific  hybridity.  The  genetical  results  in 
such  a  case,  where  the  two  parents  seem  to  have  had  relatively  few  genes 
in  common,  are  as  though  the  plant  were  diploid  with  a  large  chromosome 
number:  the  Mendelian  characters  of  each  original  parent  continue  to 
show  disomic  ratios.  If  the  particular  genes  concerned  are  present  in  the 
chromosomes  from  both  parents,  there  appear  certain  tetrasomic  ratios 
like  those  in  experimentally  induced  autotetraploids  (page  208).  When 
some  or  all  of  the  chromosomes  from  one  parent  synapse  with  members 
from  the  other  parent  (allosynapsis) ,  the  genetical  data  become  even  more 
difficult  to  analyze.  Since  two  crossable  species  may  have  genes  in 
common  as  well  as  unlike  genes,  the  type  of  synapsis,  degree  of  fertihty, 
and  genetical  ratios  tend  to  be  variable  in  polyploid  hybrids  generally, 
even  in  the  most  regular  amphidiploids. 

Cytological  Types  of  Hybridity. — The  foregoing  descriptions  have 
been  based  upon  the  essential  feature  of  hybridity  as  it  appears  in  plants 
or  animals  with  two  or  more  genomes:  the  presence  in  the  nucleus  of 


224 


FUNDAMENTALS  OF  CYTOLOGY 


genomes  differing  in  genie  constitution  and  therefore  in  influence  upon 
characters.  This  difference  arises  in  practically  all  cases  from  the  union 
of  nuclei  from  two  sources,  each  nucleus  in  the  individual  having  a 
"hybrid"  chromosome  complement  when  the  two  sources  contribute 
unlike  sets  of  genes.  This  is  evident  enough  in  the  ordinary  diploid 
hybrid  employed  in  most  cytogenetical  researches,  but  because  there 
are  other  ways  in  which  unlike  outfits  of  genes  may  be  associated  in  an 
individual,  it  will  be  well  at  this  point  to  pass  them  in  brief  review, 
beginning  with  the  ordinary  intraspecific  hybrid  (see  Fig.  166). 


5                                              6  7                                             3 

Fig.    166. — Diagram    illustrating  various    ways    in    which    unlike    genetical    elements 

may  be  combined  in  an  organism.  Rectangles  and  circles  represent  cells  and  nuclei, 
respectively.     Explanation  in  text. 

(1)  Two  similar  genomes  with  one  or  more  genes  in  the  heterozygous 
state.  (2)  Two  genomes  similar  in  their  genes  but  differing  in  the 
arrangement  of  these  in  the  chromosomes:  "structural  hybrids"  with 
chromosomal  regions  translocated  or  inverted  in  one  of  the  genomes. 

(3)  Two  genomes  with  \vider  genie  differences  and  often  differing  in 
chromosome  number:  diploid  interspecific  hybrids  with  synapsis  and 
reassortment   of  specific   characters,    or   with   asynapsis   and   sterility. 

(4)  More  than  two  genomes  from  two  species  ^^dth  regular  or  irregular 
cytogenetical  behavior:  polyploid  hybrids,  most  of  whose  gametes  are 
themselves  "hybrid"  in  constitution.  (5)  Two  unfused  monoploid 
nuclei  carrying  different  genomes:  dikaryotic  hybrids  in  certain  fungi 
(page  163).  (6)  One  genome  only,  this  being  composed  of  chromosomes 
and  genes  from  different  sources:  "monoi^loid  hybrids,"  or  "ha])l()micts," 


CYTOLOa/CAL  ASPECTS  OF  IIYBRIDITY  225 

ill  nionoploid  algae  and  in  bryophytc  gametophytes  which  exhibit  com- 
binations of  characters  from  two  unlike  parents.  (7)  A  nucleus  from 
one  species  in  cytoplasm  of  another  species,  the  cytoplasm  carrying  a 
nongenic  element  having  a  distinct  and  persistent  effect  upon  characters: 
cases  of  "cytoplasmic  inheritance"  described  in  the  next  chapter.  (8) 
There  is  no  hybridity  within  the  protoplast,  yet  two  geneticall}^  vmlike 
kinds  of  protoplast  are  so  intimately  associated  in  a  t>ody  that  both  share 
in  determining  its  characters. 

Examples  of  the  last  condition  in  the  foregoing  list  may  be  given  here. 
In  a  chimera  two  genetically  unlike  tissues  together  constitute  an  indi- 
vidual plant  as  a  result  of  local  somatic  mutation  or  of  a  graft  involving 
two  species.  In  periclinal  chimeras,  which  have  one  type  of  tissue  over- 
lying the  other  like  a  glove  over  a  hand,  the  plant  may  combine  characters 
of  the  two  species  involved.  The  form  of  the  leaf,  for  example,  may  be 
determined  by  the  inner  component,  while  the  character  of  its  surface 
is  that  of  the  outer  component.  Sometimes  the  characters  of  the  oviter 
component  are  apparently  affected  by  the  genotype  of  the  inner  com- 
ponent. The  chimeral  condition  may  be  reproduced  vegetatively  but  not 
sexually,  since  the  spores  are  developed  solely  by  one  component  or  the 
other — normally  the  one  constituting  the  subepidermal  cell  layer. 
Another  example  of  such  cellular  association  is  afforded  by  certain  slime 
molds  in  which  the  numerous  ameboid  cells  do  not  lose  their  boundaries 
when  they  unite  to  form  a  pseudoplasmodium  from  which  fruiting  bodies 
(sorocarps)  develop.  When  pseudoplasmodia  of  two  species  are  thor- 
oughly mixed  and  grown  under  certain  cultural  conditions,  the  mixture 
produces  not  only  sorocarps  of  the  two  specific  types  but  also  some  com- 
bining in  various  ways  the  characters  of  the  two  species.  An  extreme 
example  of  the  association  of  unlike  protoplasts  is  seen  in  lichens,  whose 
l)odies  are  made  up  of  a  fungus  and  an  alga  living  in  symbiotic  union. 

This  section  is  included  in  the  chapter  not  to  confuse  our  conceptions 
of  hybridity,  but  rather  in  the  hope  of  supplying  them  with  a  broader 
basis.  The  physical  basis  of  heredity  shows  a  striking  fundamental 
similarity  throughout  practically  the  entire  organic  world,  yet  it  has 
several  variants  in  different  groups  of  organisms.  Cytogenetical 
researches  are  being  extended  into  more  groups  as  time  goes  on.  Hj^brid- 
ity,  the  presence  of  unlike  genetical  protoplasmic  elements  from  different 
sources,  exists  in  some  degree  nearly  everywhere,  but  if  the  investigator 
expects  the  physical  mechanism  of  inheritance  to  be  of  exactly  the  same 
standard  type  in  every  organism  he  will  meet  many  puzzles.  It  has 
sometimes  happened  that  an  elaborate  and  ingenious  hypothesis  has  been 
formulated  to  account  for  aberrant  genetical  data  when  an  awareness  of  a 
peculiar  cytological  or  histological  condition  in  the  organism  would  have 
suggested  a  much  simpler  explanation. 


226  FUNDAMENTALS  OF  CYTOLOGY 

Conclusions. — We  may  summarize  here  the  contributions  that 
cytology  has  made  to  our  understanding  of  hybridity. 

It  has  revealed  much  of  the  physical  basis  of  peculiar  modes  of  geneti- 
cal  behavior  and  grades  of  sexual  sterility  in  known  hybrids. 

It  has  furnished  a  means  of  detecting  hybridity  and  probable  origin 
in  some  organisms  not  giving  other  clear  evidence.  For  example,  it  has 
shown  why  some  plants  that  breed  true  must  be  regarded  as  hybrids. 

It  has  shown  that  a  significant  association  often  exists  between 
hybridity,  heteroploidy,  apomixis,  and  certain  types  of  mutation. 

It  has  afforded  a  partial  explanation  of  how  unstable  hybrids  may, 
after  some  generations,  yield  new  stable  and  fertile  types. 

It  has  revealed  in  chromosomal  alterations  an  inner  evolution  which 
plays  some  role  in  the  evolution  of  external  diversity  among  organisms. 

It  has  offered  suggestions  as  to  modes  of  procedure  in  attempts  to 
produce  new  fertile  types  through  hybridization. 

It  has  devised  artificial  methods  of  conferring  fertility,  with  tetra- 
ploidy,  upon  desirable  but  sterile  hybrids. 

It  has  revealed  a  number  of  variants  of  the  fundamental  physical 
basis  of  hybridit}^  among  organisms. 

Finally,  it  has  given  us  a  far  better  conception  of  the  cytogenetical 
history  of  present  organisms,  enabling  us  better  to  predict  what  their 
future  may  possibly  be.  Change  is  the  rule  in  natvire,  and  cytogenetics 
has  shown  us  some  of  its  inner  causes. 


CHAPTER  XVI 

THE  ROLE  OF  THE  CYTOPLASM  IN  DEVELOPMENT 
AND  HEREDITY 

In  the  preceding  chapters  it  has  been  shown  why  and  how  the  chromo- 
somes have  been  assigned  a  special  role  in  the  development  and  inherit- 
ance of  the  organism's  characters.  When  two  individuals  develop  similar 
characters  under  the  same  environinental  conditions,  it  is  concluded  that 
their  protoplasmic  constitutions  are  alike,  for  development  is  the  result 
of  interactions  of  the  protoplasmic  sj'stem  ^^ith  its  environment  and 
among  its  own  components.  When  the  interactions  and  characters  are 
the  same  in  successive  generations,  the  characters  are  said  to  be  inherited, 
although  this  actually  means  that  they  have  been  redeveloped  in  the 
offspring  because  its  protoplasm  is  like  that  of  the  parent.  When  two 
individuals,  whether  they  are  brothers  or  parent  and  offspring,  develop 
different  characters  in  the  same  kind  of  environment,  the  differences  are 
attributed  to  dissimilarities  in  the  constitution  of  their  protoplasms,  in 
particular  to  differential  factors  in  their  chromosomes.  It  is  largely  the 
Mendelian  phenomenon  and  the  numerous  refinements  in  experimental 
procedure  that  have  made  this  correlation  possible.  We  now  face  the 
question,  "Are  there  differential  factors  elsewhere  in  the  protoplast?" 

Reciprocal  Crosses. — A  method  for  ascertaining  whether  cytoplasmic 
elements  participating  in  character  formation  ever  act  differentially  is  that 
of  comparing  the  results  of  reciprocal  crosses.  In  angiosperms  the  cyto- 
plasm is  derived  mainh-  or  entirely  from  the  maternal  parent.  When, 
therefore,  two  inbred  types  of  plant  are  crossed  reciprocally,  the  offspring 
of  the  two  crosses  have  nuclei  that  are  alike  but  cytoplasms  that  are 
different.  Differences  between  the  two  classes  of  offspring  might  then 
be  attributed  to  the  differential  action  of  the  parental  cytoplasms.  This 
is  the  converse  of  the  situation  in  crosses  employed  in  most  cytogenetic 
investigation,  where  the  nuclear  constitution  is  made  to  vary  in  a  uniform 
cytoplasm.  The  usual  absence  of  differences  between  such  reciprocal 
intraspecific  hybrids  indicates  the  absence  of  such  cytoplasmic  differences 
between  the  two  types  crossed  as  would  lead  to  differences  in  character 
development.  In  some  interspecific  and  intergeneric  hybrids,  however, 
such  differences  are  frequently  observable.  Often  the  difference  dis- 
appears in  the  course  of  one  generation  or  more.  This  is  interpreted  to 
mean  that  the  egg  carries  in  its  cytoplasm  a  lingering  effect  impressed 

227 


228 


FUNDAMENTALS  OF  CYTOLOGY 


upon  it  by  the  plant  which  bore  it  and  that  this  effect  is  soon  nullified  as 
new  cytoplasm  is  produced  under  the  influence  of  the  hybrid  nuclei  in  the 
plant  into  which  the  fertilized  egg  grows. 

An  example  of  the  brief  temporary  effect  of  the  maternal  cytoplasm 
upon  the  offspring  is  seen  in  seeds  of  stocks  (Matthiola)  (Fig.  167).  The 
epidermis  of  the  embryo  is  dark  blue  inM.  incanasind  yellow  inilf .  glabra. 
When  the  two  are  crossed,  vnt\\  incana  as  the  maternal  parent,  the 
hybrid's  seeds  are  dark  blue.  When  the  reciprocal  cross  is  made,  the 
seeds  vary  from  clear  yellow  to  light  blue.  Thus  the  cytoplasms  of 
the  two  species  react  differently  to  the  same  heterozygous  genes  in  the 
hybrids'  nuclei:  incana  cytoplasm  develops  a  blue  color  strongly  and  at 
once,  whereas  glabra  cytoplasm  does  so  weakly  or  not  at  all.  When  the 
two  kmds  of  hybrid  are  selfed,  they  behave  alike  in  yielding  blue  seeds 
with  varying  depth  of  color  and  yellow  seeds  in  the  ratio  of  3:1. 


Fi  dark   blue 


h,  light  blue 
to  yel  low 


3  blue 

(dark  to  light) 
I    yellow 


3  blue 

(dark  to  light) 
I    yellow 


M. INCANA 
epidermis 
of  embryo 
dark  blue 

M. GLABRA 

epidermis 

of  embryo 

ye  How 

Fig.    167. — Effect    of    reciprocal    crossing    in    Matthiola    (stocks).     Explanation    in    text. 

{Based  on  data  of  C.  Correns.) 

The  same  phenomenon  is  observed  among  animals  in  the  case  of  cer- 
tain larval  characters.  For  example,  in  cross-fertilized  sea  urchin  or 
fish  eggs  the  rate  and  the  type  of  cleavage  are  the  same  as  in  the  mother, 
no  matter  what  the  direction  of  the  cross.  They  are  characters  impressed 
upon  the  egg  during  its  ovarian  history,  and  the  male  nucleus  fails  to 
change  the  condition  already  induced  in  the  egg  by  maternal  nuclear 
factors.  When  the  hybrid  matures  and  produces  eggs,  however,  it  is 
found  that  these  all  show  the  dominant  rate  and  type  of  cleavage,  no 
matter  which  parent  contributed  the  dominant  factor.  This  shows  that 
the  male  does  affect  the  character  in  the  second  generation.  Breeding 
experiments  with  moths  and  butterflies  have  given  similar  results  with 
respect  to  some  embryonic  characters.  The  conclusion  is  that  certain 
characters  whose  differentiation  is  initiated  in  the  egg  cytoplasm  before 
syngamy,  although  fundamentally  Mendelian  in  their  inheritance,  may 
be  peculiar  in  that  the  visible  effect  of  the  male  gamete  is  delayed  for  one 
generation. 


rilE  ROLE  OF  THE  CVTOFLASM 


229 


An  instance  of  cytoplasmic  influence  enduring  for  a  longer  })eri()(l  is 
afforded  by  the  protozoan,  Paramecium.  When  individuals  of  two 
races  unlike  in  size  are  allowed  to  conjugate  and  then  to  multiply  by 
fission,  the  individuals  of  both  exconjugant  lines  graduall}^  come  to  be  of 
the  same  size  after  about  22  fissions.  The  inference  is  that  there  is  here  a 
Ungering  cytoplasmic  influence  slowly  being  overcome  by  genes  affecting 
size.  This  inference  is  supported  by  the  further  observation  that  the  size 
eventually  attained  is  not  the  same  after  different  pairs  of  individuals 
of  the  same  clones  have  conjugated,  for  this  indicates  the  formation  of 
various  genie  combinations  in  the  meiosis  and  syngamy  occurring  at  the 
time  of  conjugation. 


o 

? — ► 

G) 

?x 

0 

► 

« 

L 

L 

14 
generations 

of                     ~« 
back  -crosses 

to 

L 

L 

E.  luteum 

E.  luteum 

'^  alike 

/^~N 

/"^X 

/-—X 

X — X 

^ 

/ 

/ 

(^ 

E.  hirsutum 

^ 

w 

H 

/^ 

H 

^,  — —  » 

L 

d"/ 

L 

r    w:....i:. 

^*- 

_-  -  "~ 

Fui.  168. — Diagram  illustrating  cytoplasmic  inheritance  in  species  of  Epilohium 
(willow  herbs).  Rectangles  represent  plant.s;  circles  represent  their  nuclei.  L,  luteum 
cytoplasm;  H,  hirsutum  cytoplasm;  I,  luteum  genome;  h,  hirsutum  genome.  The  third 
plant  in  the  second  row  is  a  "nucleocytoplasmic  hybrid"  (compare  Fig.  166,  7).  Further 
explanation  in  text.      {Based  on  data  of  P.  Michaelis.) 

In  another  class  of  cases  the  story  is  a  different  one:  the  cytoplasmic 
effect  does  not  disappear,  but  persists  indefinitely  even  in  spite  of  attempts 
to  increase  the  influence  of  the  genes.  In  Epilohium  (Fig.  168)  the 
hybrids  derived  from  reciprocal  crosses  of  E.  luteum  and  E.  hirsutum  are 
unlike  in  various  vegetative  characters  and  fertility.  The  hybrid  type 
containing  luteum  cytoplasm  retains  its  distinctive  characters  even  after 
back  crossing  to  hirsutum  for  14  generations.  After  such  a  number  of 
backcrosses  the  nuclei  in  all  probability  have  only  hirsutum  chromosomes, 
yet  the  effect  of  the  original  maternal  cytoplasm  persists.  When  such 
plants  containing  hirsutum  chromosomes  and  luteum  cytoplasm  are 
crossed  reciprocally  with  E.  luteum,  the  hybrids  obtained  are  alike  in 
spite  of  the  fact  that  the  nuclear  relations  are  the  same  as  in  the  original 
cross  between  the  two  species,  where  the  reciprocal  hybrids  w;ere  not  alike. 


230 


FUNDAMENTALS  Of  CYTOLOGY 


This  supports  the  conckision  that  the  characters  involved  have  a  differen- 
tial basis  in  the  cytoplasm. 

A  comparable  example  is  furnished  by  certain  moss  hybrids,  in  which 
gametophytic  characters  are  involved.  When  Funaria  hygrometrica  and 
Physcomitrium  pyriforme  are  crossed  reciprocally,  the  resulting  hybrid 
sporophytes  show  characteristic  structural  differences,  as  do  the  diploid 
hybrid  gametophytes  produced  from  them  by  regeneration.  Some  of 
these  gametophytic  characters,  notably  the  length  of  the  leaf  midrib,  per- 
sist throughout  subsequent  backcross  generations  of  monoploid  offspring 
grown  from  spores,  indicating  a  strong  and  persistent  cytoplasmic  effect. 

The  shape  of  the  leaf  shows  the  effect  some- 
what less  strongly,  and  the  form  of  the 
paraphyses  shows  it  still  less.  The  conclusion 
is  that  in  these  mosses,  and  presumably  in 
other  plants  showing  strongly  persistent 
cj^toplasmic  effects,  there  is  a  stable  element 
in  the  cytoplasm  that  acts  differentially  upon 
characters.  This  element  in  mosses  is  called 
the  plasmone.  In  the  case  of  some  characters, 
such  as  midrib  length  in  the  above-mentioned 
hybrids,  it  is  the  plasmone  that  is  responsible 
for  differences  in  the  character,  while  other 
characters  may  be  acted  upon  differentially 
by  both  plasmone  and  genome  or  by  the 
genome  alone. 

Peculiar  interest  attaches  to  a  case  of 
cytoplasmic  inheritance  in  Zea  because  of  a 
visible  difference  in  the  cytoplasm  correlated 
with  the  character  involved.  In  a  certain 
race  of  maize  the  pollen  degenerates  partially 
or  completely,  usually  after  the  formation  of  the  generative  cell.  This 
character,  male  sterile,  is  transmitted  through  the  eggs  to  the  next  genera- 
tion, but  not  by  the  few  good  pollen  grains  from  partially  sterile  plants. 
That  the  cause  of  the  defect  is  actually  in  the  cytoplasm  and  not  in  the 
genes  has  been  shown  by  a  series  of  crosses  involving  each  of  the  10  chro- 
mosomes. In  cells  that  are  to  ^deld  normal  pollen,  certain  bodies,  pre- 
sumably the  proplastids,  are  rod-shaped,  whereas  in  cells  about  to 
produce  degenerating  pollen  they  are  spherical  (Fig.  169).  These  bodies, 
if  not  the  cause  of  the  defect,  are  at  least  indicators  of  a  determining 
influence  in  the  cytoplasm. 

Chlorophyll  Characters. — Two  successive  generations  of  cells  repro- 
ducing by  division  resemble  each  other  partly  because  the  organs  of  a 
given  cell  may  actually  continue  as  the  corresponding  organs  of  its  daugh- 


h'lo.  169. — Two  sporocytes 
in  a  partially  male-sterile  indi- 
vidual of  maize.  Normal  cell 
above,  affected  one  below. 
See  text.  {Courtesy  of  M.  M. 
Rhoades.) 


THE  ROLE  OF  THE  CYTOPLASM  231 

ter  cells.  In  a  unicellular  green  alga  the  daughter  individuals  ai-e  like  tlu^ 
mother  in  being  green  because  the  chloroplast  of  the  mother  ('(>11  is  divided 
and  passed  on  directly  to  them.  In  those  algae  in  which  a  swarm  spore 
germinates  to  produce  a  multicellular  individual  or  associates  with  others 
of  its  kind  to  form  a  colony,  the  color  of  the  successive  colonies  or  indi- 
A'iduals  is  a  character  transmitted  directly  by  the  repeated  division  of 
chloroplasts. 

A  somewhat  similar  interpretation  has  been  placed  upon  the  inherit- 
ance of  chlorophyll  characters  in  the  higher  plants,  the  supposition  being 
that  plastids,  multiplying  only  by  division,  are  responsible  for  the  dis- 
tribution, in  the  individual  plant  and  through  successive  generations,  of 
those  characters  which  manifest  themselves  in  these  organs.  Abnormali- 
ties in  chlorophyll  coloring,  such  as  pale  greenness,  whiteness,  and 
variegation,  are  accordingly  attributed  to  an  abnormal  condition  in  the 
chloroplast  or  the  surrounding  cytoplasm.  Since  the  color  itself  is  not 
present  in  the  plastids  of  angiosperm  gametes,  this  character  may  resem- 
ble ordinary  Mendelian  characters  in  being  developed  anew  in  each 
generation,  but  it  differs  from  them  in  depending  upon  the  reproduction 
and  distribution  of  differentiated  cytoplasmic  organs,  the  plastids. 
Indeed,  it  has  been  shown  that  the  various  known  chlorophyll  characters, 
even  those  appearing  much  alike,  fall  into  two  categories:  (1)  those 
inherited  according  to  ordinary  Mendelian  rules,  which  is  taken  to  mean 
that  the  processes  concerned  in  their  color  development  are  under  the 
influence  of  differential  nuclear  factors;  and  (2)  those  not  so  inherited 
and  therefore  having  their  differential  in  the  cytoplasm.  Both  types  may 
appear  in  the  same  genus  or  species,  as  in  maize.  It  is  to  be  emphasized 
that  the  characters  in  both  categories  are  developed  under  the  influence 
of  both  nucleus  and  cytoplasm  but  that  they  differ  with  regard  to  the 
location  and  nature  of  the  factors  acting  differentially. 

A  classic  example  of  the  non-Mendelian  type  is  a  variegated  four- 
o'clock,  Mirahilis  jalapa  albomaculata.  Plants  of  this  kind  have  some 
branches  with  normal  green  leaves,  some  with  white  leaves,  and  some 
A\T.th  variegated  leaves.  Flowers  are  borne  on  branches  of  all  three  types. 
Crosses  between  unlikes  result  in  seedlings  with  the  color  of  the  maternal 
l^arent  or  branch.  For  instance,  when  a  flower  on  a  green  branch  is 
l^ollinated  with  pollen  from  a  flower  on  a  white  branch,  the  oft'spring  are 
all  green.  In  the  reciprocal  cross  the  offspring  are  all  white  and  soon  die 
])ecause  of  the  lack  of  chorophyll.  If  flowers  on  variegated  branches  are 
l)ollinated,  offspring  of  all  types  may  result.  In  no  case  does  the  pollen 
affect  the  color  of  the  progeny. 

The  hypothesis  proposed  to  account  for  these  facts  is  that  there  is 
present  in  the  plant  an  abnormal  cytoplasmic  condition  that  prevents 
the  normal  development  of  the  chloroplasts.     It  is  delivered  directly 


232  FUNDAMENTALS  OF  CYTOLOGY 

to  the  next  generation  through  the  egg  cytoplasm  but  is  not  transmitted 
by  the  male  parent  because  the  male  gamete  brings  no  functional  cyto- 
plasm into  the  egg  at  syngamy.  If  the  condition  were  under  the  differen- 
tial control  of  nuclear  factors,  it  would  be  transmitted  equally  well  by 
male  and  female  gametes,  since  the  nuclear  contributions  of  the  two  are 
equivalent.  There  are  other  plants  in  which  chlorophyll  characters  of 
this  non-Mendelian  type  are  inherited  from  the  male  parent  as  well  as 
from  the  female,  indicating  a  participation  of  the  male  gamete's  cyto- 
plasm in  the  formation  of  the  zygote. 

There  is  much  concerning  the  inheritance  and  development  of  chloro- 
phyll characters  that  is  not  well  understood.  The  cytological  mechanism 
of  variegation  is  particularly  obscure  in  cases  like  the  above,  especially 
where  the  color  pattern  does  not  coincide  with  the  pattern  of  tissue  devel- 
opment. There  is  much  more  to  be  learned  about  plastids,  the  nature  of 
the  cytoplasmic  differential  factor,  and  the  causes  of  differentiation  in 
general  before  such  problems  can  be  solved. 

Conclusions. — The  subject  of  this  chapter  leads  us  back  to  a  concept 
stressed  in  early  pages  of  the  book,  viz.,  that  of  the  living  individual  as  an 
organized  protoplasmic  system  with  many  specialized  regions  contributing 
to  the  orderly  activity  of  the  whole.  Accounts  of  the  remarkable  role 
of  the  nucleus  in  heredity  like  that  in  the  more  recent  chapters  sometimes 
suggest  that  the  nucleus  is  the  sole  arbitrary  determiner  of  the  proto- 
plast's activities  and  their  consequences,  the  cytoplasm  being  merely  a 
complex  organic  culture  medium  in  which  it  performs  its  functions. 
Whatever  may  have  been  the  historical  origin  of  the  nucleus-cytoplasm 
type  of  organization — and  what  it  was  w^e  should  like  very  much  to  know 
— the  fact  that  the  cytoplasm  participates  in  at  least  the  development  of 
characters  is  now  obvious. 

That  the  cytoplasm  also  shares  in  determining  what  kind  of  characters 
shall  develop  is  evident  in  the  phenomena  reviewed  in  this  chapter. 
Nuclei  never  develop  alone :  it  is  always  a  nucleocytoplasmic  S3^stem  that 
undergoes  development.  When  the  type  of  cytoplasm  associated  with 
the  nucleus  is  sufficiently  altered,  as  in  certain  wide  crosses,  the  charac- 
ters are  also  altered,  showing  the  importance  of  nucleocytoplasmic  inter- 
action in  character  development.  Hence  in  pure  lines  the  cytoplasm  at 
least  contributes  to  the  similarity  of  individuals,  whether  these  are 
brothers  or  parent  and  oft'spring,  and  similarity  is  a  principal  feature  in 
heredity.  The  visible  results  of  a  strongly  differential  action  of  the  cyto- 
plasm sometimes  observed  in  reciprocal  interspecific  crosses,  and  the  fact 
that  in  attempted  crosses  between  very  distantly  related  organisms  the 
nucleus  and  cytoplasm  will  not  interact  at  all,  indicate  that  the  cytoplasm 
is  in  some  measure  responsible  for  the  differences  between  those  organisms. 
They  also  suggest  that  if  such  very  wide  crosses  were  successful  and  the 


\ 


THE  ROLE  OF  THE  CYTOPEASM  233 

hybrids  obtained  were  fertile,  the  cytoplasm  would  be  found  to  play  a 
more  prominent  differential  role  in  character  determination  than  it  does  in 
l^ure  lines  and  intraspecific  hybrids. 

Emphasis  upon  the  nucleus  as  a  system  of  elements  necessary  to 
development  and  upon  its  chromosomal  organization  as  the  key  to  the 
Mendelian  phenomenon  is  surely  warranted,  but  it  should  not  obscure  the 
fact  that  "the  physical  basis  of  heredity"  in  a  broad  sense  includes  all 
relatively  stable  protoplasmic  elements  affecting  the  characters  developed, 
wherever  these  elements  are  located  and  whether  their  activitj'  results 
in  likeness  or  unlikeness  in  the  characters.  Even  if  one  inclines  to  view 
the  cytoplasm  as  a  "culture  medium"  in  which  the  nucleus  somehow 
works  out  the  characters,  it  must  be  remembered  that  this  medium,  even 
more  than  the  characters  themselves,  is  a  direct  inheritance  from  previous 
generations. 


CHAPTER  XVII 
CYTOLOGY  AND  TAXONOMY 

In  recent  years,  cytology  has  found  a  new  field  of  usefulness  as  an  ally 
of  taxonomy.  As  every  student  of  biology  is  aware,  the  task  of  taxonomy 
is  that  of  arranging  animals  and  plants  into  a  hierarchy  of  systematic 
units — species,  genera,  families,  orders,  etc. — according  to  the  degree 
of  their  relationship.  In  earlier  days  "relationship"  meant  merely 
resemblance  in  externally  visible  characters  or  nearness  of  approach  to 
certain  ideal  standards.  Modern  taxonomy  is  much  more  than  this. 
It  strives  to  improve  sj^stems  of  groupings  made  for  the  sake  of  con- 
venience, but  it  also  seeks  the  natural  causes  of  the  likenesses  and 
differences  in  character  observed,  and  it  does  this  knowing  that  true 
relationship  has  its  basis  in  community  of  origin. 

Modern  taxonomy  differs  from  that  of  earlier  centuries  in  two  other 
important  respects:  it  makes  use  of  a  greater  diversity  of  evidence,  and 
like  other  branches  of  biology  it  has  supplemented  observation  with 
experiment.  In  its  effort  to  discover  true  relationships  and  origins,  it 
not  only  makes  use  of  the  usual  morphological  characters,  but  is  quick 
to  seize  upon  evidence  afforded  b}^  physiological  behavior,  ecological 
relations,  geographical  distribution,  serological  interactions,  cytological 
characters,  genetical  behavior  in  controlled  crosses,  and,  in  some  measure, 
the  fossil  record.  In  short,  it  is  now  more  conscious  of  its  integral  share 
in  accounting  for  the  distribution  of  organic  types  in  space  and  time. 

Tliis  chapter  affords  a  glimpse  of  cytotaxonomy ,  in  which  cytological 
characters,  chiefly  the  number,  morpholog}^,  and  behavior  of  chromo- 
somes, are  employed  in  the  task  of  determining  true  natural  relationships. 
The  immediate  aim  of  this  new  alliance  of  cytologj^  and  taxonomy  is  to 
establish  and  measure  correlations  between  such  cytological  characters 
and  the  natural  taxonomic  units  founded  on  field  observation  and  con- 
trolled experiment.  Its  further  aim  is  to  evaluate  alterations  in  chromo- 
some complements  as  factors  in  the  development  and  diversification  of 
these  units,  and  so  to  gain  a  more  broadly  based  conception  of  the  origin 
of  the  diversity  observed  in  the  living  world. 

This  type  of  study  has  been  carried  on  more  extensively  with  plants 
than  with  animals.  This  is  due  in  large  measure  to  the  fact  that  poly- 
ploidy occurs  very  widely  in  plants,  especially  among  angiosperms, 
whereas  among  animals  it  is  comparativelj^  rare.  In  both  kingdoms, 
however,  significant  results  are  being  achieved. 

234 


CYTOLOGY  AND  TAXONOMY  235 

Cytological  Evidence  of  Relationship. — Cytological  evicU^ice  of  valiu^ 
to  the  taxonomist  is  of  four  chief  kinds:  chromosome  number,  chromo- 
some morphology,  chromosome  behavior  in  crosses,  and  aben-ations  in 
reproduction. 

Chromosome  Number. — The  number  of  chromosomes  has  now  been 
ascertained  in  many  hundreds  of  species  of  animals  and  plants.  The 
reported  numbers  have  been  assembled  in  lists  which  every  worker  in 
this  field  must  have  ready  to  hand.  Unfortunately,  the  usefulness  of 
many  original  reports  is  impaired  by  a  lack  of  completeness  in  the 
taxonomic  designations  and  by  failure  to  make  permanent  records  of  the 
plants  in  the  form  of  herbarium  specimens  or  photographs.  Furthermore, 
the  reliability  of  the  numbers  varies,  for  in  many  cases  the  coun,ts  have 
been  made  upon  too  few  or  sometimes  atypical  specimens.  As  the 
science  of  cytotaxonomy  develops  further,  it  is  obvious  that  more  care 
A\dll  be  required  with  respect  to  these  points. 

One  does  not  look  far  in  a  general  list  of  chromosome  numl^ers  without 
being  struck  by  the  fact  that  the  species  of  a  genus  tend  strongly  to  show 
numbers  that  bear  some  characteristic  arithmetical  relation  to  one 
another.  This  is  most  evident  where  the  numbers  constitute  a  regular 
series  of  multiples.  In  other  genera  the  numerical  correlations  are  less 
complete,  and  in  still  others  no  significant  correlation  can  be  detected 
between  numerical  and  specific  differences.  Such  diversity  is,  of  course, 
to  be  expected  in  view  of  the  many  ways  and  degrees  in  which  chromo- 
some numbers  may  be  altered.  Such  ways  have  been  described  in 
previous  chapters.  It  is  to  be  borne  in  mind  that  it  is  primarily  the  kinds 
of  genes  present  that  determine  characters,  the  number  of  chromosomes 
in  which  they  are  carried  being  a  secondary  factor  in  producing  diversity. 

A  few  examples  of  such  numerical  relationship  will  now  be  listed.  In 
the  pond  lilies  of  the  genus  Nymphaea  the  species  stellata,  lotus,  odorata, 
Candida,  and  gigantea  have  the  following  somatic  numbers,  respective!}' : 
28,  56,  84,  112,  c.  224.  It  may  be  suspected  that  the  basic  number, 
or  original  monoploid  number,  for  this  genus  is  7,  but  no  species  with  a 
somatic  number  of  14  has  .yet  been  reported.  In  the  genus  Plantago, 
the  plantains,  the  40  species  examined  include  24  diploids  AAith  1 2  somaticr 
chromosomes,  four  diploids  with  10,  one  diploid  with  8,  one  di))loid 
with  18,  eight  tetraploids  with  24,  one  tetraploid  \Y\i\i  36,  one  octoploid 
with  48,  and  one  sixteen-ploid  with  96  (Fig.  170).  Here  it  appears  that 
there  is  more  than  one  basic  number,  and  it  is  of  interest  to  observe  that 
the  sections  of  the  genus  can  be  arranged  in  three  groups:  (1)  those 
comprising  onlj^  diploids  with  six  as  the  basic  number,  (2)  those  containing 
both  diploids  and  tetraploids  with  this  basic  number  and  types  with  a 
lower  basic  number,  and  (3)  those  containing  polyploid  species  onh'. 
The  original  basic  number  is  probably  6,  mth  5,  4,  and  9  as  derivatives. 


236  FUNDAMENTALS  OF  CYTOLOGY 

This  conclusion  is  supi)orted  by  ihv,  morphology  of  the  chromosomes 
(page  238). 

Fig.  170. — Somatic  chromosome  complements  of  several  species  of  plantains  (Plantago) . 
1,  ovata,  with  8  chromosomes;  2,  Brownii,  with  48;  3,  major,  with  12;  4,  japonica,  with  3(i; 
5,  alpina,  with  24;  6,  media,  with  24;  7,  Raoullii,  with  18;  8,  lanccolata  var.  altissima, 
with  96;  9,  serraria,  with  10.      {After  D.  McCuUagh.) 

Frequently  the  sections  of  a  genus  show  characteristic  differences  in 
chromosome  number.  In  Verbena,  the  section  Glandularia  with  a  basic 
number  of  5  includes  five  diploids  with  10  chromosomes  and  three 
triploids  with  30.  In  the  section  Verbenaca,  with  7  as  a  basic  number, 
the  Leptostachya  group  is  made  up  of  nine  diploids  with  14  chromosomes, 


CYTOLOGY  AND  TAXONOMY  237 

while  the  Pachystachya  group  comprises  one  triploid  with  21,  two 
tetraploids  with  28,  and  one  hexaploid  with  42.  In  the  genus  Triticwn 
(wheats)  the  species  of  the  einkorn  group  have  14  somatic  chromosomes, 
those  of  the  emmer  group  28,  and  those  of  the  spelta  group  42.  The 
highly  important  agricultural  wheat  species  belong  to  the  spelta  group. 
In  the  violets  {Viola)  the  morphological  characters  determining  the 
sections  are  not  well  correlated  with  chromosome  number,  other  factors 
e^•idently  having  been  more  important  in  differentiation  within  this  genus. 
In  the  sedges  (Carex)  the  species  have  a  great  variety  of  chromosome 
nimibers  that  do  not  form  any  definite  type  of  series,  although  it  is 
possible  that  they  represent  a  combination  of  several  definite  series  with 
their  modifications. 

Within  a  species  there  is  sometimes  a  small  amount  of  variation  in 
chromosome  number.  In  lists  based  on  many  collections  and  counts 
there  frequently  appears  a  "variety"  with  some  multiple  of  the  number 
characteristic  of  the  species.  Its  rarity  and  association  mth  the  preva- 
lent tvpe  may  indicate  recent  origin.  In  other  cases  its  distribution  and 
characters  suggest  its  independence  as  an  established  subspecific  unit. 
Most  often  such  plants  are  tetraploids,  though  higher  multiples  sometimes 
occur.     Aneuploids  are  very  rare. 

Within  a  family  the  related  genera  often  reveal  their  relationship  in 
their  chromosome  numbers.  Sometimes  their  numbers  are  the  same. 
When  different  numbers  occur,  these  usually  do  not  distinguish  the  genera 
as  clearh^  as  they  do  related  species  in  a  genus.  Very  often  a  polyploid 
series  runs  through  a  considerable  group  of  genera,  with  one  or  more  of 
the  multiples  of  the  basic  number  appearing  in  each  genus.  In  the 
Ericaceae  (heaths),  for  example,  at  least  ten  of  the  genera  show  one  or 
more  numbers  of  the  series  6,  12,  18,  24,  36,  and  48,  with  24  as  the  com- 
monest somatic  number.  Four  of  the  genera,  together  wdth  a  genus 
in  the  neighboring  Empetraceae,  show  26  somatic  chromosomes.  In 
the  Malvaceae  (mallow  family)  as  many  as  five  polyploid  series  are 
represented,  with  5,  6,  7,  11,  and  13  as  the  basic  numbers.  These  series, 
singty  or  in  combination,  are  more  or  less  distinctive  of  certain  groups 
of  genera  within  the  family.  In  the  Leguminosae  (pea  family)  pol^^ploidy 
appears  far  less  frequently.  Of  the  genera  studied,  42  are  completely 
diploid,  18  are  more  than  one-half  diploid,  none  is  predominantly  poly- 
ploid with  a  few  diploids,  and  10  are  completely  polyploid  with  the 
occasional  exception  of  one  species.  Polyploidy  in  this  family,  although 
occurring  in  about  23  per  cent  of  the  species  examined,  does  ser\'e  to 
differentiate  certain  related  genera  more  clearly  than  is  usual  in  the 
angios  perms. 

Chromosome  number  becomes  less  valuable  as  an  indicator  of  relation- 
ship when  groups  larger  than  genera  and  families  are  considered.     Thoie 


238  FUNDAMENTALS  OF  CYTOLOGY 

appears  to  be  little  if  any  significant  correlation  of  chromosome  number 
and  form  on  the  one  hand  with  structural  complexity  and  the  major 
divisions  of  the  plant  and  animal  kingdoms  on  the  other.  It  is  true  that 
certain  great  groups  show  definite  tendencies:  most  fungi  have  very  low 
numbers;  true  ferns  have  high  numbers;  plants  of  the  hly  family  and 
amphibians  have  large  chromosomes.  It  seems  evident,  however,  that 
the  major  evolutionary  fines  are  distinguished  by  kinds  and  assortments 
of  genes  rather  than  by  the  number,  size,  and  form  of  chromosomes. 

Chromosome  Morphology. — Species  may  differ  in  the  number  of  their 
chromosomes,  or  in  their  visible  morphology,  or  in  both.  In  plants  or 
animals  with  very  small  or  very  numerous  chromosomes  it  is  often 
impossible  to  use  chromosome  form  as  a  character  in  the  study  of  relation- 
ships. The  value  of  the  character  increases  as  the  chromosomes  become 
larger  and  fewer.  The  genus  Crepis  has  long  been  one  of  the  most 
valuable  to  the  cytologist,  for  although  the  chromosomes  are  of  only 
medium  size  they  are  well  differentiated  morphologically  and  occur  in 
genomes  composed  of  very  few  members.  In  a  majority  of  the  species 
the  gametic  number  is  4,  while  in  most  of  the  rest  it  is  only  5  or  6.  The 
characteristic  morphology  of  these  chromosomes  in  several  of  the  species 
with  the  lower  numbers  is  illustrated  in  Fig.  71.  It  is  plainly  evident 
here  that  each  species  is  characterized  by  a  chromosome  complement 
that  is  not  only  Hke  those  of  its  relatives  in  general  features  but  also 
unlike  them  in  certain  respects.  The  particular  kind  of  chromosome 
complement  characteristic  of  any  individual  or  group  of  related  organisms 
is  called  a  karyotype.  The  diagrammatic  representation  of  a  karyotype 
is  an  idiogram. 

Another  example  of  distinctive  karyotypes  in  a  genus  of  plants  is 
afforded  by  Plantago  (Fig.  170).  The  chromosome  complements  in  this 
genus  are  composed  of  members  differing  in  size  as  well  as  in  morphology 
and  number.  It  is  their  morphology  that  makes  it  possible  to  decide 
that  the  complement  of  18  somatic  chromosomes  in  one  of  the  species 
mentioned  on  page  235  consists  of  two  genomes  of  9  members  each  rather 
than  three  genomes  of  6,  and  that  each  genome  of  9  may  in  turn  represent 
a  combination  of  two  basic  genomes  with  4  and  5  members. 

In  related  genera  the  morphology  of  the  chromosomes  may,  like  their 
number,  be  strikingly  similar  or  unlike  in  various  degrees.  In  maize  and 
its  relatives  the  densely  staining  knobs  characteristic  of  the  chromo- 
somes of  these  plants  aid  in  cytological  comparisons  of  the  various  genera. 
The  genomes  of  maize  (Zea  mays),  annual  teosinte  (Euchlaena  mexicana) 
from  southern  Guatemala,  and  gamagrass  (Tripsacum  fioridanum)  are 
represented  as  idiograms  in  Fig.  171.  Inspection  of  these  idiograms 
shows  that  maize  and  gamagrass  differ  in  chromosome  number,  knob 
position,  and  arm  ratio.     Maize  and  Mexican  annual  teosinte  are  very 


CYTOLOGY  AND  TAXONOMY 


239 


closely  similar  in  the  morphology  of  their  chromosomes,  including  the 
position  of  their  knobs.  Southern  Guatemalan  teosinte  has  a  genome 
like  that  of  maize  except  for  knob  position  which  is  like  that  of  gamagrass. 
These  features,  together  with  chromosome  l)ehavior  in  the  crosses  men- 
tioned below,  have  an  important  bearing  upon  the  question  of  the  origin 
of  maize.  A  proposal  to  transfer  the  two  species  of  Euchlaena  to  Zea  has 
already  been  mentioned  (page  218). 

The  use  of  chromosome  morphology  and  number  in  the  determination 
of  generic  relationships  is  especially  well  illustrated  in  a  recent  study  of 
the  Ranunculaceae  (buttercup  family).  The  conclusions  reached  in 
this  study  are  summarized  in  the  lower  phylogenetic  chart  reproduced  in 


o  0 

I     23456     78     9   10        I     234     56789    10  123456789    10   II    12  B    14  IS  16  17    18 

Fig.  171. — Chromosome  diagrams  (idiograins)  of  maize  (left),  teosinte  from  southern 
Guatemala  (center),  and  gamagrass  (right).  Small  circles,  black  spots,  and  shaded 
regions  represent  kinetochores,  knobs,  and  nucleolus  organizers  respectively.  {After  A.  E. 
Longley.) 

Fig.  172.  The  upper  chart  is  a  similar  representation  of  the  relationships 
of  the  genera  as  conceived  under  the  Engler  system  of  classification. 
The  new  scheme  differs  conspicuously  from  the  old  in  associating  more 
closely  the  genera  with  small  chromosomes. 

Chromosome  Behavior  in  Crosses. — This  type  of  evidence  for  relation- 
ship has  a  special  value.  Chromosomes  of  two  organisms  may  be  similar 
in  form  and  visible  structure  and  j^et  be  widely  different  in  function. 
When  two  organisms  are  crossed,  however,  the  very  fact  that  a  hybrid 
results  shows  that  the  two  genomes  brought  together  are  sufficiently 
alike  to  act  iii  harmony  with  each  other  and  with  the  cytoplasm  to  permit 
ontogenetic  development.  This  is  strong  evidence  of  relationship,  for 
organisms  that  are  obviously  very  distantly  related  do  not  produce 
hybrids. 

A  fiu'tlier  critical  test  of  relationship  comes  at  the  time  of  meiosis  in 
the   hybrid.     As    already   pointed    out   in    previous    chapters,    synapsis 


240 


FUNDAMENTALS  OF  CYTOLOGY 


ktaeo  s 


Copt  is  ^  Zonrhorhiza  is 
Ntqello  i  ^         Caltha  n 

fy  AT 


Clematis  5 


I  holictrum^^I 


2S;    C^V. 

IsopfPum    f\mikaic  Anemone//o  j^^//^^^^ 

7  \      7  7  ^^  u 


(f^/)/V J  Zonfhorkzo 
1    ^---        /? 


Paeonia  5 


CYTOLOGY  AND  TAXONOMY  241 

(lept'iicLs  piimaiily  upon  the  homology,  or  genie  similarity,  of  the  chromo- 
somes concerned,  any  lack  of  synapsis  therefore  indicating  a  hick  of  close* 
relationship  between  them  (except  when  it  is  induced  b.y  environmental 
causes  or  special  mutant  genes).  In  diploid  hybrids,  synapsis  ranges  all 
the  wa}''  from  perfect  success  to  total  failure,  and  in  general,  though  not 
always,  fertility  varies  with  the  degree  of  success.  Reverting  to  Zea 
and  related  genera  mentioned  above,  it  is  found  that  when  maize  is 
crossed  with  either  annual  Mexican  teosinte  or  teosinte  from  northern 
Guatemala  the  hybrid  shows  normal  synapsis  (Fig.  162).  In  hybrids 
between  maize  and  southern  Guatemalan  teosinte,  synaptic  association 
shows  a  few  abnormalities  due  to  differences  in  the  linear  arrangement  of 
homologous  elements.  In  maize-gamagrass  hybrids  synapsis  occurs 
only  in  a  few  regions  of  the  chromosomes,  indicating  a  low  degree  of 
homology-.  Similar  beha^'ior  is  observed  in  teosinte-gamagrass  hybrids. 
In  triple  hybrids  containing  genomes  of  all  three  genera,  the  Zea  and 
Euchlaena  chromosomes  pair,  leaving  the  Tripsacum  chromosomes 
unpaired  except  for  an  occasional  trivalent.  These  cytological  findings 
are  a  valuable  supplement  to  taxonomic  and  breeding  evidence  for 
generic  relationship.  They  afford  a  visible  measure  of  the  homology 
and  arrangement  of  the  genes,  especially  since  these  plants  show  the 
process  so  clearly. 

Evidence  comparable  to  the  above  is  available  in  certain  animal 
groups  also.  It  has  been  possible  to  make  the  most  minute  comparisons 
of  chromosomes  of  related  species  in  the  Diptera  because  of  the  giant 
chromosomes  in  their  salivary  glands.  Years  ago  the  ordinary  somatic 
chromosomes  of  various  species  of  Drosophila  were  compared,  but  much 
more  can  now  be  learned  from  a  comparison  of  their  salivary-chromosome 
maps.  In  intraspecific  hybrids  the  intimate  association  of  parental 
elements  in  the  salivary-gland  chromosomes  permits  an  even  more 
precise  comparison.  In  this  way  a  point  of  special  interest  has  been 
brought  out  with  respect  to  certain  geographical  strains  of  Drosophila 
pseudoobscura.  By  studying  the  chromosomes  of  hybrids  between  these 
strains  it  was  found  that  three  of  them  have  the  genetical  elements  in 
chromosome  III  arranged  in  three  different  ways  as  the  result  of  inver- 
sions: (1)  ABCDEFGHI,  (2)  AFEDCBGHT,  (3)  AFEHGBCDI.  In 
these  arrangements  there  is  a  clue  to  the  historical  sequence  of  the  races. 
Two  successive  inversions  could  easily  give  the  sequence  (1)  -^  (2)  -^  (3) 
or  the  sequence  (3)  — ^  (2)  — >  (1).  Also,  (2)  might  have  been  the  pre- 
cursor of  both  (1)  and  (3).  On  the  other  hand,  it  is  very  highly  improb- 
able that  an  alteration  directly  from  (1)  to  (3)  or  vice  versa  would  occur 
at  one  step  through  some  more  comj^lex  aberration.  Since  the  "struc- 
tural hybrids"  between  such  inversion  strains  in  Drosophila  tend  to  be 
sterile,  it  has  been  suggested  that  such  strains,  because  of  the  consequent 


242  FUNDAMENTALS  OF  CYTOLOGY 

lack  of  effective  interbreeding,  might  represent  early  stages  in  the  differ- 
entiation of  distinct  species. 

The  use  of  synaptic  behavior  in  analyzing  the  relationships  of  poly- 
ploid plants  has  already  been  described  at  page  219. 

Aberrations  in  Reproduction. — On  several  occasions  cytology  has 
helped  to  determine  the  taxonomic  status  of  certain  plants  by  revealing 
the  presence  of  an  atypical  mode  of  reproduction.  An  instance  of  this 
is  seen  in  the  Caninac  section  of  the  genus  Rosa.  Among  these  roses  are 
several  which  had  been  regarded  as  true  species  becaiLse  of  their  constancy 
of  type  and  the  lack  of  intermediate  forms.  It  was  found,  however,  that 
they  were  polyploid  in  constitution  and  showed  the  most  characteristic 
type  of  hybrid  chromosome  behavior  at  meiosis.  The  cause  of  their 
true  breeding  in  spite  of  their  meiotic  irregularity  w^as  revealed  in  the 
discovery  that  they  are  apomictic:  their  embryos  do  not  arise  from 
sexual  cells  but  from  the  nucellus  by  adventitious  budding  (page  146). 
The  prevalence  of  such  a  mode  of  reproduction  in  a  genus  thus  tends  to 
preserve  certain  hybrids  between  the  species  and  leads  to  the  establish- 
ment of  a  group  of  many  constant  and  nearly  similar  units.  Such 
"agamic  complexes"  are  known  in  numerous  angiosperm  genera,  includ- 
ing Ruhus,  Citrus,  Potentilla,  Crepis,  Taraxacum,  Hieracium,  Festuca,  Poa, 
and  others. 

The  Role  of  Chromosomal  Changes  in  Speciation. — When  it  is 
discovered  that  the  number  and  the  morphology  of  the  chromosomes 
can  be  used  as  characters  in  classification,  one  canriot  pass  directlj^  to 
the  conclusion  that  changes  in  these  chromosomal  features  have  by 
themselves  produced  the  differentiation  of  the  taxonomic  units  in  which 
they  are  found.  Visiljle  chromosomal  changes,  like  invisible  gene 
mutations,  are  factors  in  speciation,  Ijut  there  are  many  conditions 
that  must  be  met  if  a  newly  formed  chromosomal  type  is  to  become 
established  as  a  distinct  species  or  subspecies  in  nature.  The  new  type 
must  be  physiologically  suited  to  the  habitat  in  which  it  arises.  It 
must  be  able  to  meet  competition.  It  must  either  have  sufficient  sexual 
fertility  to  maintain  itself  or  be  able  to  reproduce  vegetatively.  In 
the  latter  case  further  modification  through  gene  mutation  would,  of 
course,  be  much  slower  than  in  a  plant  capable  of  sexual  reproduction. 
It  would  be  stable  and  perhaps  distinct  in  type,  but  not  progressive. 

A  sexually  reproducing  type  should  have  some  degree  of  isolation  from 
the  parental  type  or  types,  for  as  long  as  it  crosses  freely  with  them  the 
production  of  intermediate  types  will  prevent  the  attainment  of  specific 
distinctness.  The  isolation  permitting  the  new  tj^pe  to  evolve  inde- 
pendentl}'  through  genie  variation  and  selection  may  be  of  several  kinds. 
The  type  may  be  sexually  isolated  from  the  parental  or  other  related 
types  by  difference  in  flowering  time,   poor  pollen  tube  growth  after 


CYTOLOGY  AND  TAXONOMY 


243 


cross-pollination,  or  gametic  incompatibility.  Its  independence  may 
be  almost  as  complete  even  when  hybrids  are  produced,  for  these  may  be 
sterile  or  otherwise  unable  to  compete  with  either  the  new  type  or  its 
parents.  The  genetic  mechanisms  responsible  for  hybrid  sterility  do 
not,  however,  seem  to  be  directly  correlated  with  the  genetic  changes 
producing  visible  specific  differences. 

Another  form  of  isolation  is  that  based  on  adaptability  to  ecological 
habitat.  Peculiar  physiological  characters  may  not  only  keep  the  new 
type  and  its  relatives  apart  within  a  relatively  small  region,  but  they 
may  ♦lead  to  very  wide  differences  in  geographical  range.     Examples 


Fig.  173. ^Relationship  between  polyploidy  and  geographical  distribution  in  the 
American  species  of  Tradcscantia.  Outer  heavy  line:  maximum  distribution  of  tetraploid 
species.  Inner  heavy  line:  maximum  distribution  of  diploid  species.  Heavy  cross-lined 
area:  minimum  distribution  of  diploid  species.  Centered  about  tliis  last  area  are  the 
known  areas  of  four  diploid  species  elsewhere  tetraploid.      {After  E.  Anderson.) 

of  this  are  the  following.  In  Tradescantia  virginiana  and  its  relatives 
growing  in  the  United  States  it  is  observed  that  the  diploids  grow  mostl}' 
in  the  south,  while  the  autotetraploids,  which  grow  in  a  greater  variety 
of  habitats,  have  a  much  wider  and  more  northern  distribution  (Fig.  173). 
It  is  not  thought,  however,  that  autotetraploidy  in  this  genus  ranks  with 
genie  differentiation  and  hybridization  as  a  cause  of  speciation.  In 
Biscutella,  a  genus  of  cruciferous  plants,  the  derived  tetraploid  types  are 
evidently  spreading  more  rapidly  in  Europe  than  the  diploids.  A  survey 
of  the  angiosperm  flora  of  Schleswig-Holstein  has  shown  that  the  per- 
centage of  polyploid  types  is  twice  as  great  among  northern  species  as 
among  southern  ones,  and  that  whereas  diploids  predominate  on  lime- 
poor  soils  the  polyploids  constitute  95  ])or  cent  of  the  species  found  on 


244  FUNDAMENTALS  OF  CYTOLOGY 

lime-rich  soils.  In  Scandinavia  the  polyploid  types  among  the  Ericaceae 
are  usually  more  widely  distributed  in  northern  and  severe  habitats. 
In  certain  other  cases  it  has  been  found  that  polyploids  predominate  in 
desert  habitats.  In  other  genera  and  floras,  however,  it  has  been  shown 
that  there  is  no  correlation  between  polyploidy  and  extremity  of  habitat; 
indeed,  autopolyploids  may  be  less  frost  resistant,  in  part  because  of 
their  higher  water  content.  Studies  on  Viola,  Achillea,  Potentilla, 
Artemisia,  and  other  genera  growing  in  the  Pacific  slope  region  of  the 
United  States  have  shown  that  although  chromosomal  differences  are 
commonly  associated  with  different  ecological  requirements  and  thus 
affect  distribution,  there  is  no  rule  as  to  the  kind  of  region  in  which  a 
given  chromosomal  type  is  found.  Among  perennial  forage  grasses 
growing  in  California,  however,  the  drier  and  hotter  regions  contain  a 
distinctly  higher  proportion  of  polyploids,  probably  of  hybrid  origin. 

Experimental  Taxonomy. — The  kinds  of  facts  cited  in  the  preceding 
section  should  make  it  evident  that  cytotaxonomj^  is  a  part  of  a  larger 
field  of  investigation.  This  more  comprehensive  field,  known  as  experi- 
mental taxonomy,  not  only  adds  cytological  characters  to  those  ordinarily 
cited  in  manuals,  but  appeals  to  all  other  sources  that  might  yield 
information  bearing  upon  its  problems.  The  criticism  from  the  geneticist 
that  many  species  listed  in  the  manuals  may  be  nothing  more  than 
intraspecific  Mendehan  forms  is  being  met  by  modern  experimental 
taxonomists  in  their  attempts  to  test  their  provisional  hypotheses  by 
suitable  breeding  experiments.  Similarly,  the  criticism  from  the  physiol- 
ogist and  ecologist  that  certain  supposed  species  may  be  merely  local 
variants  associated  with  a  special  habitat  is  being  answered  by  the  use 
of  data  froin  the  field  of  experimental  ecolog3^  Plants  of  the  kinds  in 
question  are  grown  in  different  soils  and  in  different  climatic  situations 
in  order  to  distinguish  more  surely  between  physiological  variations  and 
the  characters  trul}^  indicative  of  relationship.  This  often  requires 
observation  extending  over  a  period  of  years.  Furthermore,  observation 
must  also  be  extended  in  space,  for  a  knowledge  of  the  geographical 
distribution  of  species  and  other  taxonomic  units  is  often  essential  to  an 
understanding  of  the  relative  age  and  advancement  of  related  kinds  of 
organisms.  Taxonomists  have  long  been  aware  of  this  and  have  made 
use  of  collections  from  widely  separated  localities  in  formulating  their 
conclusions.  Now  that  cytological  data  are  being  sought  in  a  similar 
manner,  cytologists  share  with  taxonomists  the  benefits  of  this  broader 
observational  foundation. 

It  is  obvious  that  conclusions  based  on  such  a  variety  of  data  can  be 
reached  only  very  slowly,  but  that  when  attained  they  should  be  far 
more  dependable  than  concepts  reached  by  shorter  routes.  In  the  mean- 
time we  shall  have  to  make  use  of  provisional  schemes  of  relationship 


CYTOLOGY  AND   TAXONOMY 


245 


devised  for  iininediate  i)urpo.ses,  reineinl)ering  that  they,  like  other 
concepts  based  on  growing  evidence,  are  always  subject  to  further 
improvement.  It  is  beyond  the  scope  of  this  book  to  discuss  experi- 
mental taxonomy  in  detail.  Our  purpose  is  to  show  the  bearing  of 
cytology  upon  biological  investigation  in  a  related  field  and  to  cite  certain 
instructive  examples  of  the  results  obtained. 

The  taxonomy  of  Crepis,  a  genus  of  composites,  has  been  intensively 
investigated  for  many  years  with  the  aid  of  cytogenetics  and  geographical 
studies.  The  literature  on  the  subject  is  one  of  considerable  size,  but 
the  results  of  the  investigations  may  be  indicated  by  statements  selected 


EUCREPIS-PHYLOGENY  AND  CHROMOSOME  NUMBER 


HIERACIUM   18 


^ATRISARBA  8S? 
MONTICOLA  55?      LeAReiGERA  89? 
SCOPULORUM  447  I      L ACUMINATA  33.44.55? 
OCCIDENTALIS  2J.44\\    LcRACILIS  J2.55? 

\\ 
AMPWIOIPLOIO    HYBRIDS  22 

PTEROTHECOIDES  8,    ;    LEONTODONTOIOES  10 

PULCHRA  8, 
CRAAJATENSIS  S 
PALAESTINA  8. 
MuaifLORA    8 
DIOSCORIDIS    8. 
TUBAEFORMIS  8. 
ARCOLICA  8. 


ANOCRSONII  22 
CLAUCA  22 
RUNCINATA  22 


.SUrrRENIANA  S 
NEOIECTA  6  8 
PARVIELORA  8 
CAPILLARIS  6 
TECTORUM  8 
NICAEENSIS  8 


LACTUCA  18.16 


PRENANTHCS  18 


-INTERSPECIFIC  HYBRIDIZATION  AND 
AMPHIOIPLOIDY  (5+4X2=18) 


5-PAIRED  AND  4-PAIRED  SPECIES 


-ORIGIN  OF  8-CHROMOSOME  SPECIES 


10 -CHROMOSOME  PROGENIAL  STOCK 


PRE-EUCREPIS 

Fig.  174. — Right,  diagram  illustrating  hypothetical  origin  of  the  genus  Crepis  and 
its  relatives.  Left,  diagram  illustrating  the  phylogeny  of  the  subgenus  Eucrepis.  (After 
E.  B.  Bnhcock.) 


fi-om  recent  summaries.  The  genus  comprises  more  than  200  species 
distributed  widely  in  the  Northern  Hemisphere  and  Africa.  These 
evidently  constitute  a  natural  group  with  a  common  origin  and  center  of 
distribution.  The  subgenera  Catoyiia,  Eucrepis,  and  Barkhausia  are 
characterized  in  this  order  by  increasing  morphological  specialization 
with  decreasing  size  and  length  of  life  cycle.  The  genus  includes  a 
remarkable  range  of  morphological  types,  and  with  such  e\ddence  of 
progressive  evolution  there  is  associated  an  orderly  and  progressive 
modification  in  chromosome  number  and  morphology. 

The  subtribe  of  which  Crepis  is  a  member  seems  to  have  had  a  common 
origin  in  a  stock  with  10  somatic  chromosomes  (Fig.  174).  The  general 
trend  of  modification  within  the  Crepis  karyotype  has  been  from  nearly 


246 


FUNDAMENTALS  OF  CYTOLOGY 


uniform  chromosomes  with  more  or  less  median  kinetochores  to  shorter, 
distinctly  different  types  of  chi-omosomes  with  nonmedian  kinetochores. 
Since  morphologically  similar  species  usually  have  similar  karyotypes, 
the  genus  is  especiallj^  well  suited  to  cytotaxonomic  study.  A  change 
in  the  number  of  chromosomes  from  10  to  8,  the  most  prevalent  diploid 
number  in  the  genus,  and  from  this  to  still  lower  numbers  has  occurred. 
This  was  probably  accomplished  in  a  series  of  reciprocal  translocations 
by  which  all  the  essential  portions  of  certain  chromosomes  were  trans- 
ferred to  other  chromosomes,  the  chromosomes  losing  these  portions  then 
being  eliminated.  Following  this  was  the  formation  of  amphidiploids  by 
hybridization  and  chromosomal  doubling,  giving  a  series  of  species  having 
more  than  twice  as  many  chromosomes  as  the  primitive  ancestral  stock. 


Degree  of  separation 

Hybrids   fertile, 
second   generation 
vigorous 

Hybrids  partially 
sterile,  second 
generation   weak 

Hybrids  sterile 
or  none 

^"^^^internal 
External    ^^\^ 

In  different 
environment 

Distinct    subspecies 
ECOTYPES 

Distinct    species 
ECOSPECIES 

Distinct 
species  complexes 
CENOSPECIES 

In  the  same 
environment 

Local  variations  of 
one  species 
BIOTYPES 

Species   overlap- 
ping in  common 
territory   (with 
hybrid  swarms) 

Fig.  175. — Table  showing  the  concept  of  species  and  the  terms  employed  by  certain  experi- 
mental taxonomists.      {After  J.  Clausen,  D.  D.  Keck,  a?id  W.  M.  Hiesey.) 

Many  interspecific  crosses  in  the  breeding  plot  have  aided  in  determining 
the  degree  of  relationship  between  the  various  species. 

Five  processes  of  genetic  change  are  regarded  as  significant  in  the 
evolution  of  Crepis.  Of  primary  importance  has  been  (1)  the  structural 
transformations  of  the  chromosomes  revealed  in  their  number  and 
morphology,  for  these  produce  initial  intraspecific  sterility  which  makes 
possible  the  accumulation  of  further  intersterilitj^  along  with  morpho- 
logical and  physiological  divergence.  This  and  (2)  gene  mutation  prob- 
ably account  for  most  of  the  progressive  specialization  within  the  genus. 
To  these  causes  of  change,  (3)  interspecific  hybridization  is  secondary 
in  importance,  while  (4)  polyploidy  and  (5)  apomixis  have  played 
definite  though  relatively  unimportant  roles. 

A  second  series  of  investigations  illustrating  the  methods  and  results 
of  experimental  taxonomy  is  that  being  carried  out  in  the  Pacific  Coast 
region  of  the  United  States.  Following  the  lead  of  Turesson  in  Europe, 
American  botanists  are  investigating  the  species  of  numerous  genera  with 


CYTOLOGY  AND  TAXONOMY  247 

special  reference  to  the  influence  of  climate  and  ecological  habitat. 
Perennial  species  including  many  climatic  races  in  the  Pacific  Coast 
region  have  been  placed  in  gardens  at  three  localities  with  very  different 
climates:  near  sea  level  on  the  San  Francisco  peninsula,  at  4600  feet 
elevation  near  Yosemite  National  Park,  and  at  10,000  feet  elevation  on 
the  Sierra  Nevada.  In  this  way  it  is  possible  to  observe  the  effects  of 
different  climates  upon  genetically  uniform  material  as  well  as  the 
liehavior  of  races  from  different  regions  when  brought  into  the  same 
environment.  The  ecological  reactions  and  chromosome  numbers  of  the 
various  races  of  a  species  or  species  group  are  compared  with  the  purpose 
of  determining  whether  the  differentiation  of  intraspecific  races  has  a 
visible  cytological  basis.  In  pursuing  such  studies  the  investigators 
have  found  useful  the  terminology  indicated  in  Fig.  175.  They  are, 
however,  quick  to  admit  that  one  cannot  formulate  definitions  covering 
all  differences  between  taxonomic  units,  since  species  are  in  all  stages  of 
evolution. 

Some  of  the  results  of  these  studies  are  as  follows.  Three  complexes 
of  climatic  races  (in  Sisyrinchium  heUum,  Potentilla  glandulosa,  and 
Penstemon  procerus)  have  become  differentiated  without  change  in 
chromosome  number.  In  two  other  complexes,  involving  several  other 
species  of  Potentilla,  the  chromosomes  vary  in  number  and  degree  of 
irregularity  in  behavior,  and  the  plants  are  evidently  apomictic,  yet 
climatic  races  have  been  successfully  developed  as  in  P.  glandulosa. 
In  six  complexes  (in  Zaiischneria,  Viola,  Aster,  Artemisia,  Achillea,  and 
Horkelia)  there  are  differences  in  chromosome  number  that  prevent  free 
interbreeding  and  are  usually  correlated  with  differences  in  morphology. 
Such  chromosomal  groups  can  be  recognized  as  taxonomic  species,  and 
they  usually  occupy  different  climatic  regions. 

These  data  and  many  others  have  led  to  the  conclusions  stated  in  the 
following  quotation. 

This  survey  and  the  one  conducted  by  Turesson  in  Euroj^e  indicate  that  the 
genetic-physiologic  (Hfferentiation  of  a  plant  group  is  correlated  with  the  climatic 
zones  it  occupies.  This  follows  from  the  fact  that  the  same  kinds  of  environ- 
ments are  occupied  by  races  that  have  similar  patterns  of  reaction,  even  though 
they  belong  to  unrelated  genera  or  families.  This  is  found  to  hold  irrespective 
of  whether  or  not  the  regional  forms  differ  in  chromosome  number. 

The  usual  pattern  of  differentiation  is  purely  genetic,  with  relatively  few 
major  steps  involved;  but  superimposed  upon  this  one  often  finds  a  cytological 
differentiation,  with  one  or  two  changes  in  chromosome  number  across  the 
California  transect.  However,  the  effects  of  increases  in  chromosome  number 
must  have  been  far  overshadowed  by  the  selective  influence  of  the  environment 
in  determining  the  appearance  and  reactions  of  plants.  From  these  consider- 
ations it  appears  that  it  is  the  genes  in  the  chromosomes,  and  not  the  number  of 
chromosomes,  which  determine  the  climatic  adaptation. 


248 


FUNDAMENTALS  OF  CYTOLOGY 


From  the  point  of  view  of  fitness  to  the  environment  it  is  evident  that  the 
ecologically  important  unit  is  not  the  species,  but  the  regional  climatic  race,  or, 
to  adopt  Turesson's  term,  the  ecotype.  Several  of  these  may  combine  to  form  a 
species,  or  a  single  ecotype  may  develop  an  isolating  genetic  barrier  to  form  a 
monotypic  species.  .  .  .  Such  monotypic  species  occui)y  a  narrow  climatic 
belt  and  show  little  variation  and  adaptability.  However,  it  makes  little 
difference  whether  a  given  area  is  populated  by  a  series  of  ecotypes  belonging  to 
one  species,  or  by  a  series  of  monotypic  species  belonging  to  one  species  complex, 


Fig.  176. — Diagram  representing  relationships  in  the  genus  Layia  as  indicated  by  the 
combined  results  of  taxonomic,  ecological,  and  cytological  studies.  Circles  represent  species 
with  the  chromosome  numbers  shown;  shaded  connections  show  degree  of  genetic  affinity; 
width  of  solid  black  lines  represents  degree  of  chromosome  pairing  in  interspecific  hybrids. 
The  dotted  lines  indicate  major  morphological  breaks  in  the  genus.  {After  J.  Clausen, 
D.  D.  Keck,  and  W.  M.  Hiesey.) 

or  by  a  combination  of  both.  The  evolutionary  past  and  future  differ,  however, 
in  the  three  instances. 

Evolutionary  processes  have  left  plants  arranged  in  groups  of  various  order 
and  separation,  such  as  populations,  ecotypes,  species  and  species  complexes. 
These  groups  indicate  stages  in  evolutionary  differentiation,  and  they  have 
evolved  only  where  there  is  a  diversity  of  environments. 

There  are  many  mechanisms  by  which  living  things  can  increase  their  heredi- 
tary variation,  but  regional  differentiation  requires  the  discriminating  selection 
offered  by  unlike  environments.  We  have  no  evidence  that  the  direct  influence 
of  environment  produces  fundamental  hereditary  changes  in  species,  but  major 
alterations  in  environments  provide  new  habitats  and  refuges  for  the  products 


CYTOLOGY  AND  TAXONOMY  249 

of  nature's  continual  experimentation  among  all  the  plant  species  that  populate 
a  given  area.     (Clausen,  Keck  and  Hiesey.) 

The  above  investigations  on  western  plants  include  detailed  studies  of 
interspecific  relationships  within  certain  genera,  the  taxonomic,  ecological, 
cytological,  and  genetical  evidence  all  being  brought  to  bear  upon  the 
problem.  One  of  these  genera  is  Layia,  of  which  the  analysis  to  date  is 
summarized  graphically  in  Fig.  176.  Inspection  of  this  diagram  may 
serve  better  than  a  long  description  to  suggest  the  complexity  of  problems 
of  this  kind  and  the  amount  of  time  and  labor  required  for  their  solution. 
One  must  choose  between  solving  them  quickly  and  solving  them  well. 

Conclusions.^ — Cytology  has  contributed  to  taxonomy  in  rwo  impoi- 
tant  ways.  First,  it  has  added  a  new  category  of  characters  to  those 
commonly  employed  in  classification.  Obviously,  chromosomes  are 
not  very  useful  in  the  field;  nevertheless,  the.y  are  available  and  should 
always  be  a  part  of  anj^  thorough  taxonomic  analysis.  They  do  not 
always  prove  valuable,  but  when  they  do  they  compensate  well  for  the 
effort  expended  upon  them.  In  numerous  cases  they  have  enabled 
workers  to  decide  whether  a  plant  type  newly  observed  in  the  field  is  a 
Mendelian  variant,  a  heteroploid  derivative  of  a  familiar  species,  or  a 
stable  and  fertile  interspecific  hybrid.  In  other  words,  visible  characters 
of  the  chromosomes  frequently'  indicate  the  invisil)le  genie  constitution 
primarily  responsible  for  the  external  characters. 

Cytology's  second  contribution  to  taxonomy  lies  in  the  clues  it  gives  to 
the  origin  of  the  species  and  other  taxonomic  units.  Evolution  involves 
the  origin  of  heritable  variations,  a  selective  process  operating  among  the 
\'ariants,  and  some  isolating  factor  that  permits  a  variant  to  become 
modified  independently  of  neighboring  tj^pes.  Darwin  began  with  the 
x'ariants;  now  cytology,  with  genetics,  is  revealing  the  inner  causes  of 
the  variations.  It  is  also  revealing  some  of  the  internal  reasons  for  the 
selection  of  certain  variants  to  the  exclusion  of  others,  for  cytological 
phenomena,  especiallj^  at  meiosis,  often  show  why  certain  chromosome 
coml^inations  are  viable  and  stable  while  others  are  not.  An  internal 
cause  of  isolation  is  also  evident  in  cytological  behavior  which  either 
prevents  successful  crossing  with  related  types  or  leads  to  the  sterility 
of  hybrids  in  case  the.y  are  formed.  Finally,  cytological  studies  some- 
times indicate  clearly  in  what  order  a  series  of  differing  types  should 
be  read. 

C.ytology  itself  has  gained  greatl.y  from  its  association  with  taxonomy. 
Cytologists  have  the  satisfaction  of  seeing  their  subject  given  wider 
usefulness  in  its  application  to  a  biological  problem  of  the  first  rank. 
The.v  are  gaining  a  greater  familiarity  with  the  work  of  other  biologists 
less  confined  to  the  laboratory  than  they,  and  through  this  the.y  are 
de\('lo}^ing  a  deeper  appreciation  of  the  significance  of  their  own  subject. 


SUGGESTED  READING 

I.  Some  General  Works  on  Cytology 

Cameron,  Gladys.     Essentials  of  Tissue  Culture  Technique.     New  York,  1935. 

Chambers,  R.  The  micromanipulation  of  living  cells.  In  The  Cell  and  Proto- 
plasm.    Lancaster,  Pa.,  1940. 

CowDRY,  E.  v.,  ed.     General  Cytology.     Chicago,  1924. 

.     Special  Cytology.     2d  ed.     New  York,  1932. 

Doncaster,  L.  An  Introduction  to  the  Study  of  Cytology.  Cambridge  (England), 
1920. 

Geitler,  L.     Grundriss  der  Cytologie.     Berlin,  1934. 

Gray,  J.     A  Text-book  of  Experimental  Cytology.     Cambridge  (England),  1931. 

Guillermond,  a.,  G.  AIangenot,  and  L.  Plantefol.  Traite  de  cytologie  vegetale. 
Paris,  1932. 

KtJSTER,  E.     Die  Pflanzenzelle.     Jena,  1935. 

Pfeiffer,  H.  H.     Experimentelle  Cytologie.     Leiden,  1940. 

Sharp,  L.  W.     An  Introduction  to  Cytology.     3d  ed.     New  York,  1934. 

White,  P.  R.     Plant  tissue  cultures.     BioL  Rev.  16:  34-48,  1941. 

Wilson,  E.  B.     The  Cell  in  Development  and  Heredity.     3d  ed.     New  York.  1925. 

II.  The  Organism  and  the  Cell 

Allen,  C.  PL  Regeneration,  development  and  genotype.  Anier.  Naturalist  76 : 
225-238,  1942. 

Baitsell,  G.  a.  a  modern  conception  of  the  cell  as  a  structural  unit.  Biol.  Sym- 
posia 1:  67-86,  1940. 

Bertalanffy,  L.  von  (Trans,  by  J.  H.  Woodger).  IModern  Theories  of  Develop- 
ment.    Oxford,  1933. 

Burr,  H.  S.,  and  F.  S.  C.  Northrop.  The  electro-dynamic  theory  of  life.  Quar.  Rev. 
Biol.  10 :  322-333,  1935. 

Child,  C.  ]\I.  Cellular  differentiation  and  external  environment.  In  The  Cell 
and  Protoplasm.     Lancaster,  Pa.,  1940. 

Gilchrist,  F.  G.  The  nature  of  organic  wholeness.  Qiiar.  Rev.  Biol.  12:  251-270, 
1937. 

Harrison,  R.  G.  Cellular  differentiation  and  internal  environment.  In  The  Cell 
and  Protoplasm,     Lancaster,  Pa.,  1940. 

KoFOiD,  C.  A.  Cell  and  organism.  In  The  Cell  and  Protoplasm.  Lancaster,  Pa., 
1940. 

Luyet,  B.  J.     The  case  against  the  cell  theory.     Science  91:  252-255,  1940 

RoHDE,  E.  Der  plasmodiale  Aufbau  des  Tier-  und  Pflanzenkorpers.  Zcit.  f.  Wiss. 
Zool.  120 :  32.5-535,  1923. 

Sinnott,  E.  W.     The  cell  and  the  problem  of  organization.     Science  89 :  41-46,  1939. 

.     The  problem  of  internal  differentiation  in  plants.     Amer.  Naturalist  76: 

253-268,  1942. 

Weiss,  P.  The  problem  of  cell  individuality  in  development.  Biol.  Symposia 
1:  96-108,  1940. 

251 


252  FUNDAMENTALS  OF  CYTOLOGY 

III.  Structural  Components  of  Protoplasts 

Beams,  H.  W.,  and  R.  L.  King.     The  effect  of  centrifugation  on  plant  cells.     Bol. 

Rev.  5:  132-154,  1939, 
Ellinger,  p.     Fluorescence  microscopy  in  biology.     Cambridge  Phil.  Soc.  Biol.  Rev., 

15 :  323-350,  1940. 
Franck,  J.     Some  functional  aspects  of  photosynthesis.     Sigma  Xi  Quar.  29 :  81-105, 

1941. 
Frey-Wyssling,  a.     Submikroskopische  Morphologic  des  Protoplasmas  und  seiner 

Derivate.     Proto^lasma  Monog.  15,  1938. 
Gates,  R.  R.     Nucleoli  and  related  nuclear  structures.     Bot.  Rev.  8:  337-409,  1942. 
Guilliermond,    a.    (Trans,   by  Lenette  R.   Atkinson).     The  Cytoplasm  of  the 

Plant  Cell.     Waltham,  Mass.,  1941. 
Heidenhain,  M.     Plasma  und  Zelle.     Jena,  1907,  1911. 
HusKiNS,   C.  L.     Structural  differentiation  of  the  nucleus.     In   A  Symposium  on 

the  Structure  of  Protoplasm.     Seifriz,  ed.     Ames,  Iowa,  1942. 
Kirkman,  H.,  and  A.  E.  Severinghaus.     A  review  of  the  Golgi  apparatus.     Anat. 

Record  70,  71,  1938. 
Knaysi,  G.     Elements  of  Bacterial  Cytology.     In  press,  1943. 
Knudson,  L.     Permanent  changes  of  chloroplasts  induced  by  X  rays  in  the  gameto- 

phyte  of  Polypodium  aureum.     Bot.  Gazette  101:  721-758,  1940. 
Lundegardh,  H.     Zelle  und  Cytoplasma.     Berlin,  1922. 
Maximow,  a.  a.,  and  W.  Bloom.     A  Textbook  of  Histology.     2d  ed.     Philadelphia 

and  London.  1934. 
Meyer,  A.     Morphologische  und  physiologische  Analyse  der  ZeUe  der  Pflanzen  und 

Tiere.     Jena,  1920-1921,  1926. 
MoBius,  M.     Die  Farbstoffe  der  Pflanzen.     Berlin,  1927. 

.     Pigmentation  in  plants,  exclusive  of  the  algae.     Bot.  Rev.  3 :  351-363,  1937. 

Nahm,  Laura  J.     The  problem^  of  Golgi  material  in  plant  cells.     Bot.  Rev.  6 :  49-72, 

1940. 
Newcomer,  E.  H.     Mitochondria  in  plants.     Bot.  Rev.  6 :  85-147,  1940. 
Rice,  Mabel  A.     The  cytology  of  host-parasite  relations.     Bot.  Rev.  1:  327-354, 

1935. 
SoROKiN,  H.     The  distinction  between  mitochondria  and  plastids  in  living  epidermal 

cells.     Amer.  Jour.  Bot.  28:  476-485,  1941. 
Tischler,  G.     Allgemeine  Karyologie.     Berlin,  1921-1922,  1934. 
Trombetta,  Vivian  V.     The  cytonuclear  ratio.     Bot.  Rev.  8:  317-336,  1942. 
Uber,  F.  M.      Microincineration  and  ash  analysis.     Bot.  Rev.  6 :  204-226,  1940. 
Weier,  T.  E.     The  structure  of  the  chloroplast.     Bot.  Rev.  4:  497-530,  1938. 
Zirkle,  C.     The  plant  vacuole.     Bot.  Rev.  3 :  1-30,  1937. 
Zscheile,  F.  p.     Plastid  pigments.     Bot.  Rev.  7:  587-648,  1941. 

IV.  Protoplasm 
Becker,  W.  A.      Vitale  C^ytoplasma-  und  Kernfilrbungen.     Protopla.^ma  26:  43&-487, 

1936. 
Bensley,  R.  R.     Chemical  structure  of  cytoplasm.     Science  96:  389-393,  1942. 
Freundlich,  H.     Some  mechanical  properties  of  sols  and  gels  and  their  relation  to 

protoplasmic   structure.     In   A   Symposium   on   the   Structure   of   Protoplasm. 

Seifriz,  ed.     Ames,  Iowa,  1942. 
Frey-Wyssling,  A.     Submikroskopische  ^^lorphologie  des  Protoplasmas  und  seiner 

Derivate.     Protoplaftma  Monog.  15,  1938. 


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CJoKTNER,  R.  A.     Outlines  of  Biochemistry.     New  York,  1929. 

GuLicK,  A.     The  chemistry  of  the  chromosomes.     Bot.  Rev.  7:  433-457,  1941. 

Heilbrunn,  L.  V.  Protoplasm  and  colloids.  In  The  Cell  and  Protoplasm.  Lan- 
caster, Pa.,  1940. 

KiESEL,  A.     Chemie  des  Protoplasmas.     Proioplasma  ]\Ionog.  4,  1930. 

KusTER,  E.     Vital  staining  of  plant  cells.     Bot.  Rev.  5:  351-370,  1939. 

Marsl.-vnd,  D.  a.  Protoplasmic  streaming  in  relation  to  gel  structure  in  the  cyto- 
plasm. In  A  Symposium  on  the  Structiu"e  of  Protoplasm.  Seifriz,  ed.  Ames, 
Iowa,  1942. 

Meyer,  K.  H.  Protein  and  protoplasmic  structure.  In  A  Symposium  on  the  Struc- 
ture of  Protoplasm.     Seifriz,  ed.     Ames,  Iowa,  1942. 

Mover,  L.  S.  Proteins  and  protoplasmic  structure.  In  A  Symposium  on  the  Struc- 
ture of  Protoplasm.     Seifriz,  ed.     Ames,  Iowa,  1942. 

ScARTH,  G.  W.  Structure  and  differentiation  of  cytoplasm.  In  A  Symposium  on 
the  Structure  of  Protoplasm.     Seifriz,  ed.     Ames,  Iowa,  1942. 

ScHMiTT,  F.  O.  The  ultrastructure  of  protoplasmic  constituents.  Physiol.  Rev. 
19 :  270-302,  1939. 

Seifriz,  W.     The  structure  of  protoplasm.     Bot.  Rev.  1:  18-36,  1935. 

.     Protoplasm.     New  York,  1936. 

.     Protoplasmic  streaming.     Bot.  Rev.  9:  49-123,  1943. 

Sponsler,  O.  L.  Molecular  structure  in  protoplasm.  In  The  Cell  and  Protoplasm 
Lancaster,  Pa.,  1940. 

,  and  Jean  D.  Bath.      Molecular  structure  in  protoplasm.      In  .V  Symposium 

on  the  Structure  of  Protoplasm.     Seifriz,  ed.      .\mes,  Iowa,  1942. 

V.  Division  of  the  Protoplast 

Becker,  W.  A.     Recent  investigations  in  vivo  on  the  division  of  plant  cells.      Bot. 

Rev.  4:446-472,  1938. 
Bernal,  J.  D.     Structural  units  in  cellular  physiology.     In  The  Cell  and  Protoplasm. 

Lancaster,  Pa.,  1940. 
C.\ROTHERS,  E.  Eleanor.     Components  of  the  mitotic  spindle  with  especial  reference 

to  the  chroinosomal  and  interzonal  fibers  in  the  Acrididae.     Biol.  Bull.  71:  469- 

491,  1936. 
Cleveland,  L.  R.,  with  collaboration  of  S.  R.  Hall,  E.  P.  Sanders,  and  J.  Collier. 

The  wood-feeding  roach  Cryptocercus,  its  protozoa,  and  the  symbiosis  between 

protozoa  and  roach.     Mem.  Artier.  Acad.  Arts  Sci.  17:  No.  2:  185-342,  1934. 
Dan,   K.,   T.   Yanagita,  and   M.  Sugiyama.     Behavior  of  the  cell  surface  din'ing 

cleavage.     Protoplasma  28 :  66-81,  1937. 
Fankhauser,  G.     The  development  of  fragments  of  the  fertilized  Triton  egg  with  the 

egg  nucleus  alone  (gyno-merogony).     Joiir.  Exp.  Zool.  75:  413—469,  1937. 
IL\RVEY,  Ethel  B.     Parthenogenetic  merogony  or  cleavage  without  nuclei  in  Arbacia 

punctidata.     Bi^l.  Bull.  71:  101-121,  1936. 
Kater,  J.  McA.     Amitosis.     Bot.  Rev.  6:  164-180,  1940. 
Lewis,  F.  T.     The  significance  of  cells  as  revealed  by  their  polyhedral  shapes,  etc. 

Proc.  Amer.  Acad.  Arts  Sci.  68:  251-284,  1933. 
IjEwis,  W.  H.     The  relation  of  viscosity  changes  of  protoplasm  to  ameboid  locomotion 

and  cell  division.     In  A  Symposium  on  the  Structure  of  Protoplasm.     Seifriz, 

ed.     Ames,  Iowa,  1942. 
Marvin,  J.  W.     The  shape  of  compressed  lead  shot  and  its  relation  to  cell  shape. 

Amer.  Jour.  Bot.  26:  280-288,  1939. 


254  FUNDAMENTALS  OF  CYTOLOGY 

ATatzke,  E.  B.     Volume-shape  relationships  in  lead  shot  and  their  b(>aring  on  cell    / 

shapes.     Amer.  Jour.  Bot.  26:  288-295,  1939. 
ScHRADEB,  F.     The  present  status  of  mitosis.     Atner.  Naturalist  74:  25-33,  1940. 

VI.  Cell  Walls  of  Plants 

Anderson,  D.  B.     The  structure  of  the  walls  of  the  higher  plants.     Bot.  Rev.  1 : 

52-76,  1935. 
Bailey,  I.  W.     The  walls  of  plant  cells.     In  The  C'ell  and  Protoplasm.     Lancaster, 

Pa.,  1940. 
,  and  T.  Kerr.     The  visible  structure  of  the  secondary  wall  and  its  significance 

in  physical  and   chemical  investigations   of  tracheary   cells  and  fibers.     Jour. 

Arnold  Arboretwn  16:  273-300,  1935. 
Berkley,  E.  E.     Shrinkage  and  cell  wall  structure  of  cotton  fibers.     Amer.  Jour.  Bot. 

29:  416-423,  1942. 
Eames,  a.  J.,  and  L.  H.  MacDaniels.     An  Introduction  to  Plant  Anatomy.     New 

York,  1925. 
Farr,  Wanda  K.     Formation  of  microscopic  cellulose  particles  in  colorless  plastids 

of  the  cotton  fiber.     Contrib.  Borjce  Thompson  Inst.  12:  181-194,  1941. 
Hock,   C.   W.     Microscopic  structure  of  the  cell  wall.     In  A  Symposium  on  the 

Structure  of  Protoplasm.     Seifriz,  ed.     Ames,  Iowa,  1942. 
Kerr,  T.,  and  I.  W.  Bailey.     The  cambium  and  its  derivative  tissues,  X.     Structure, 

optical  properties  and   chemical   composition  of  the  so-called   middle  lamella. 

Jour.  Arnold  Arboretum  15:  327-349,  1934. 
Livingston,  L.  G.     The  nature  and  distribution  of  plasmodesmata  in  the  tobacco 

plant.     Ainer.  Jour.  Bot.  22:  75-87,  1935. 
Martens,  P.     L'origine  des  espaces  intercellulaires.     La  Cellule  46:  357-388,  1937. 
Meeuse,  A.  J.  D.     On  the  nature  of  plasmodesmata.     Protoplasma  35:  143-151, 

1941. 

.     Plasmodesmata.     fioi.  ie^r.  7:  249-262,  1941. 

Preston,  R.  D.     The  wall  of  the  conifer  tracheid  as  a  single  spiral  complex.     Proc. 

Leeds  Phil.  Sac.  {Sci.  Sec.)  3:  546-552,  1939. 
Wisselingh,  C.  van.     Die  Zellmembran.     Berlin,  1924. 

VII.  The  Chromosomes 

Buck,  J.  B.     Growth  and  development  of  the  salivary  gland  chromosomes  in  Sciara. 

Proc.  Nat.  Acad.  Sci.  23:  423-428,  1937. 
.      Micromanipulation   of   salivary   gland   chromosomes.     Jour.    Heredity  33 : 

3-10.     1942. 
Cleveland,   L.    R.     Longitudinal   and   transverse   division   in   two    closely   related 

flagellates.     Biol.  Bull.  74:  1-24,  1938. 
Geitler,  L.     Chromosomenbau.     Protoplasma  Monog.  14,  1938. 
Kaufmann,   B.   p.      Chromosome   structure   in   relation   to   the   chromosome   cycle. 

Bot.  Rev.  2:  529-553,  1936. 
Metz,  C.  W.     Internal  structure  of  salivary  gland  chromosomes  in  Sciara.     Jour. 

Heredity  26:  491-501,  1935. 
.     Structure  of  salivary  gland  chromosomes.     Cold  Spring  Harbor  Symposia 

on  Quant.  Biol.  9:  23-39,  1941. 
Nebbl,  B.  R.     Structure  of  Tradescantia  and  Trillium  chromosomes  with  particular 

emphasis  on  number  of  chromonemata.      Cold  Spring  Harbor  Symposia  on  Quant. 

Biol.  9:  7-12.  1941. 
Painter,  T.  S.     The  structure  of  salivary  gland  chromosomes.      Biological  Symposia 

1:215-230,  1940. 


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Sax,   K.      An  analysis  of  X-ray  induced  cliromosomal  abcrnitions  in  Tradcscantia. 

Genetics  25:  41-68,  1940. 
ScHULTZ,  J.     The  evidence  of  tiic   nucleoprotein  nature  of  (he  nene.     Cold  Spring 

Harbor  Symposi-a  on  Quant.  Biol.  9:  55-65,  1941. 
Sparrow,   A.    H.     The  structure  and  development  of  the  chromosome  spirals  in 

microspores  of  Trillium.     Canad.  Jour.  Res.  20:  257-266,  1942. 
Warmke,  H.  E.      Cliromosome  continuity  and  individuality.     Cold  Spring  Harbor 

Symposia  on  Quant.  Biol.  9:  1-6,  1941. 

VIII.  Meiosis 

Darlington,  C.  D.     Recent  Advances  in  Cytology.     2d  ed.     New  York,  1937. 
KxJWADA,  Y.     Studies  of  mitosis  and  meiosis  in  comparison,  I.  Cylologia  11 :  217-244, 

1940. 
Marshak,  a.     The  effect  of  X-rays  on  chromosomes  in  different  stages  of  meiosis. 

Jour.  Gen.  Physiol.  19:  179-198,  1935. 
Nebel,  B.  R.      Chromosome  structure.     X.     Genetics  21:  605-614,  1936. 
Oehlkers,    F.     Die    zytologischen    Grundlagen    des    genetischen    "crossing-over." 

Ber.  Deu.  Bot.  Gesell.  55:  (96)-(118),  1937. 
S.-vx,  K.,  and  L.  'SI.  Humphrey.     Structure  of  meiotic  chromosomes  in  microsporo- 

genesis  of  Tradescantia.     Bot.  Gazette  96:  353-362,  1934. 
ScHRADER,  F.     The  structure  of  the  kinetochore  at  meiosis.      Chromosonia  1:  230- 

237,  1939. 
Wilson,  G.  B.,  and  C.  L.  Huskins.     Chromosome  and  chromonema  length  during 

meiotic  coiling  in  Trillium  erectum  L.     Ann.  Botany  3 :  257-270,  1939. 

IX.  Reproduction  in  Animals 

Agar,  W.  E.     Cytology.     London,  1920. 

Calkins,  G.  X.     The  Biology  of  the  Protozoa.     Philadelphia  and  X'^ew  York,  1926. 

,  and  F.  M.  Summers.     Protozoa  in  Biological  Research.     New  York,  1941. 

Cleveland,  L.  R.,  with  collaboration  of  S.  R.  Hall,  Elizabeth  P.  Sanders,  and 

Jane  Collier.     The  wood-feeding  roach  Cryptocercus,  its  protozoa,  and  the 

symbiosis  between  protozoa  and  roach.     Mem.  Arner.  Acad.  Arts  Sci.  17:  185- 

342,  1934. 
Cleveland,  L.  R.     Origin  and  development  of  the  achromatic  figure.     Biol.  Bull. 

74:41-55,  1938. 
Doncaster,  L.     An  Introduction  to  the  Study  of  Cytologj'.     Caml^ridge  (England) 

1920. 
Gray,  J.     A  Textbook  of  Experimental  Cytology.     Cambridge  (P^ngland),  1931. 
Hegner,  R.  W.     The  Germ-cell  Cycle  in  Animals.     New  York,  1914. 
Jennings,    H.    S.     Chromosomes   and   cytoplasm   in   protozoa.     In    The    Cell   and 

Protoplasm.     Lancaster,  Pa.,  1940.  • 

KoFOiD,  C.  A.     Cell  and  organism.     In  The  Cell  and  Protoplasm.     Lancaster,  Pa., 

1940. 
Wenrich,    D.    H.     Chromosomes  in   protozoa.      The   Collecting  N^et    (Woods   Hole) 

15 :  Xo.  6,  1940. 
Wilson,  E.  B.     The  Cell  in  Development  and  Heredity.     3d  ed.     Xew  York,  1925. 

X.  Reproduction  in  Angiosperms 

Coulter,  J.  AT.,  and  C.  J.  Chamberlain.     Morphology  of  Angiosperms.     X^ew  York, 

1909. 
Ernst,  A.      Bastardierimg  als  Ursache  der  Apogamie  im  Pflanzenreich.     Jena,  1918. 


256  FUNDAMENTALS  OF  CYTOLOGY 

Frost,  H.  B.     Xucellar  embryony  and  juvenile  characters  in  clonal  varieties  of  Citrus. 

Jour.  Heredity  29:  423-432,  1938. 
GusTAFSON,   F.   G.     The  cause  of  natural  parthenocarpy.     Amer.  Jour.   Bot.  26: 

135-138,  1939. 

.     Parthenocarpy:  natural  and  artificial.     Bot.  Rev.  %:  b^^-QbA.     1942. 

Maheshwari,   p.     a  critical  review  of  the  types  of  embryo  sacs  in  Angiosperms. 

New  Phytologist  36:  35^417,  1937. 
Schnarf,  K.     Embryologie  der  Angiospermen.     Berlin,  1929. 
.      Contemporary  understanding  of  embryo-sac  development  in  Angiosperms. 

Bot.  Rev.  2 :  565-585,  1936. 
ScHURHOFF,  P.  N.     Die  Zytologie  der  Bliitenpflanzen.     Stuttgart,  1926. 
Stebbins,  G.  L.  Jr.     Apomixis  in  the  Angiosperms.     Bot.  Rev.  7:  507-542,  1941. 
Webber,  J.  M.     Polyembryony.     Bot.  Rev.  6 :  575-598,  1940. 

XI.  Reproduction  in  Plants  Other  than  Angiosperms 

Allen,  C.  E.     Haploid  and  diploid  generations.     Afner.  N'aturalist  71 :  193-205,  1937. 
BuLLER,  A.  H.   R.     The  diploid  cell  and  the  diploidization  process  in  plants  and 

animals,  with  special  reference  to  the  higher  fungi.     Bot.  Rev.  7:  335-431,  1941. 
Chamberlain,  C.  J.     Gymnosperms.     Structure  and  Evolution.     Chicago,  1935. 
Cutter,  V.  M.,  Jr.     Nuclear  behavior  in  the  Mucorales.     Bull.  Torr.  Bot.  Club  69: 

480-508,  592-516,  1942. 
Eames,  a.  J.     Morphology  of  Vascular  Plants.     Lower  Groups.     New  York,  1936. 
FiTZPATRicK,  H.  M.     The  Lower  Fungi.     Phycomycetes.     New  York,  1930. 
Fritsch,  F.  E.     The  Structure  and  Reproduction  of  the  Algae.     New  York,  1935. 
Gaumann,  E.  a.  (Trans,  and  revised  by  C.  W.  Dodge).     Comparative  Morphology 

of  Fungi.     New  York.     1928. 
Geitler,  L.     Reproduction  and  life  history  in  diatoms.     Bot.  Rev.  1:  149-161,  1935. 
Hall,  R.  P.     Cytoplasmic  inclusions  in  Phytomastigoda.     Bot.  Rev.  2:  85-94,  1936. 
Knaysi,  G.     Cytology  of  bacteria.     Bot.  Rev.  4:  83-112,  1938. 
Martin,  G.  W.     The  Myxomycetes.     Bot.  Rev.  6:  356-388,  1940. 
ScHNARF,  K.     Embryologie  der  Gymnospermen.      Berlin,  1933. 
Smith,  G.  M.     The  Fresh-water  Algae  of  the  United  States.     New  York,  1933. 
.     Nuclear  phases  and  alternation  of  generations  in  the  Chlorophyceae.     Bot. 

Rev.  4:  132-139,  1938. 
Steil,    W.    N.     Apogamy,    apospory,    and    parthenogenesis    in   the    Ptoridophytes. 

Bot.  Rev.  5 :  433-453,  1939. 
SvEDELlus,  N.     Alternation  of  generations  in  relation  to  reduction  division.     Bot. 

Gaz.  83:362-384,  1927. 
Taylor,  W.  R.     Phaeophycean  life-histories  in  relation  to  classification.     Bot.  Rev. 

2 :  554-563,  1936. 

XII.   Cytology  and  Mendelian  Heredity 

.\llen,  C.  E.     The  genetics  of  bryophytes.     Bot.  Rev.  1:  269-291,  1935. 

.     The  genotypic  basis  of  sex-expression  in  Angiosperms.     Bot.  Rev.  6 :  227- 

300,  1940. 

Babcock,  E.  B.,  and  R.  E.  Clausen.  Genetics  in  Relation  to  Agriculture.  2d  ed. 
New  York,  1927. 

Bridges,  C.  B.  Cytological  and  Genetic  Basis  of  Sex.  In  Sex  and  Internal  Secre- 
tions (E.  Allen,  ed.),  2d  ed.,  Baltimore,  1939. 

Crane,  M.  B.,  and  A.  J.  C.  Lawrence.  The  Genetics  of  Garden  Plants.  London, 
1934. 


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Davenport,  C.  B.     Sex  linkajfe  in  man.     Genetics  15:  401-444,  1930. 
GuLiCK,  A.     What  are  the  genes?     Quar.  Rev.  Biol.  13:  1-18,  140-168,  1938. 
LoEHWiNG,  W.  F.     Physiological  aspects  of  sex  in  angiosperms.     Bot.  Rev.  4:  581- 

625,  1938. 
Louis-jMarie,  p.     Heredite.     Inst,  agricole  d'Oka,  La  Trappe,  Quebec,  1936. 
Rhoades,  M.  ]\I.,  and  B.  McClintock.     The  cytogenetics  of  maize.     Bot.  Rev.  1: 

292-325,  1935. 
Rousseau,   J.     Notions   ek'mentairos   de   genetiquc.     Bull.   jard.    bot.    Montreal.    2, 

1941. 
ScHULTZ,  J.     The  evidence  of  the  nucleoprotein  nature  of  the  gene.      Cold  Spring 

Harbor  Symposia  on  Quant.  Biol.  9:  55-65,  1941. 
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Xin.  Chromosomal  Aberrations 

Blakeslee,  a.  F.  New  .Jimson  weeds  from  old  chromosomes.  Jour.  Heredity  25 : 
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Cleland,  R.  E.  Some  aspects  of  the  cyto-genetics  of  Oenothera.  Bot.  Rev.  2 : 
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Dobzhansky,  T.     Position  effect  of  genes.     Biol.  Rev.  11:  364-384,  1936. 

DuGGAR,  B.  M.,  ed.     The  Biological  Effects  of  Radiation.     New  York,  1936. 

GoLDscHMiDT,  R.  Chromosomes  and  genes.  In  The  Cell  and  Protoplasm.  Lan- 
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GooDSPEED,  T.  H.  Induced  chromosomal  alterations.  In  Tlu;  Biological  Effects 
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Kaufmann,  B.  P.  Induced  chromosomal  breaks  in  Drosoijhila.  Cold  Spring 
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McClintock,  B.  The  production  of  homozygous  deficient  tissues  with  mutant 
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somes.    Genetics  23:  315-376,  1938. 

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Genetics  26:  542-571,  1941. 

Oliver,  C.  P.     Radiation  in  genetics.     Quar.  Rev.  Biol.  9:  381-408,  1934. 

Sax,  K.  Types  and  frequencies  of  chromosomal  aberrations  induced  by  X-rays. 
Cold  Spring  Harbor  Symposia  on  Quant.  Biol.  9:  93-103,  1941. 

Stadler,  L.  J.  The  comparison  of  ultraviolet  and  X-ray  effects  on  mutation.  Cold 
Spring  Harbor  Symposia  on  Quant.  Biol.  9:  168-178,  1941. 

SwANSON,  C.  P.  A  comparison  of  chromosomal  aberrations  induced  by  X-ray  and 
ultra-violet  radiation.     Proc.  Nat.  Acad.  Sci.  26:  366-373,  1940. 

XIV.  Chromosome  Numbers  and  Their  Alteration 

Blakeslee,   A.    F.      Effect  of  induced  polyploidy  in  plants.     Amer.  Xaturalist  75: 

117-135,  1941. 
Chen,  T.-T.     Polyploidy  and  its  origin  in  Paramecium.     Jour.  Heredity  31:  175- 

184,  1940. 
Clausen,  R.  E.     Polyploidy  in  Nicotiana.     A7ner.  Naturalist  75:  291-306,  1941. 
Dermen,  H.     Colchicine  polyploidy  and  technic.     Bot.  Rev.  6:  599-635,  1940. 
Emsweller,  S.  L.,  and  M.  L.  Ruttle.     Induced  polyploidy  in  floricidture.     Amer. 

Naturalist  75:  310-326,  1941. 


258  FUNDAMENTALS  OF  CYTOLOGY 

Fankhauser,  G.     Polyploidy  in  the  salamander,  P]nrycca  hislinenta.     Jour.  Hrrcriiti/ 

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GooDSPEED,  T.  H.,  and  P.  Avery.      Trisoniii-  and  other  typcss  ni  Xieotiana  .sylve.stri.s. 

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242,  1934. 
HusKiNS,  C.  L.     Polyploidy  and  nintations.     Amei-.  Naturalist  75:  329-344,  1941. 
Lindstrom,  E.  W.     Genetics  of  polyploidy.     Bot.  Rev.  2:  197-215,  1936. 
MtJNTZiNG,    A.     The    evolutionary    significance    of   autopolyploidy.     Hereditas    21: 

263-378,  1936. 
Nebel,  B.  R.,  and  M.  L.  Ruttle.     Colchicine  and  its  place  in  fruit  breeding.     X.Y. 

State  Agr.  Exp.  Sta.  Circ.  183,  1939. 
Randolph,  L.  F.     An  evaluation  of  induced  polyploidy  as  a  method  of  breeding  crop 

plants.     Amer.  Naturalist  75:  347-363,  1941. 
Stebbixs,    G.   L.    Jr.     The   significance   of   polyploidy   in    plant   evolution.     Amer. 

Naturalist  74:  54-66,  1940. 
Warmke,  H.  E.      Polyploidy  and  evolution.     Amer.  Naturalist  75:  344-346,  1941. 

XV.   Cytolegical  Aspects  of  Hybridity 

Aase,  Hannah  C.     Cytology  of  cereals.     Bot.  Rev.  1 :  467-496,  1935. 

Allan,   K.   H.      Wild  species  hybrids  in  the  phanerogams.      Bot.  Rev.  3:  593-615, 

1937. 
GooDSPEED,  T.  H.,  and  Muriel  V.  Bradley.     Amphidiploidy.     Bot.  Rev.  8:  271-316, 

1942. 
Jones,  W.  N.      Chimeras:  a  summary  and  some  special  aspects.     Bot.  Rev.  3:  545- 

562,  1937. 

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Mangelsdorp,  p.  C,  and  R.  G.  Reeves.     The  origin  of  Indian  corn  and  its  relatives. 

Texas  Agr.  Exp.  Sta.  Bull.  574,  1939. 
Raper,  K.  B.,  and  C.  Thom.     Interspecific  mixtures  in  the  Dictyosteliaceae.     Amer. 

Jour.  Bot.  28:69-78,  1941. 
Sax,  K.     The  cytological  analysis  of  species  hybrids.     Bot.  Rev.  1:  100-117,  1935. 
Thompson,  W.  P.     The  causes  of  hybrid  sterility  and  incompatibility.     Trans.  Roy. 

Soc.  Canada,  Ser.  Ill,  34,  1940. 
WiNGE,  O.     On  the  origin  of  constant  species-hybrids.     Svensk  Bot.  Tidskr.  26 :  107- 

122,  1932. 

XVI.  Role  of  Cytoplasm  in  Development  and  Heredity 

Demerec,  M.  Behavior  of  chlorophyll  in  inheritance.  Cold  Spring  Harbor  Sym- 
posia on  Quant.  Biol.  3:  80-86,  1935. 

Lehmann,  E.  Der  Anted  von  Kern  und  Plasma  an  den  reziproken  \'erschiedenheiten 
von  Epilobium-Bastarden.     Zeitschr.  Zilcht.  A  17:  157-172,  1932. 

MicHAELis,  p.  liber  die  Konstanz  des  Plasmons.  Zeitschr.  hid.  Abst.  Vererb.lA- 
435-459,  1938. 

Renner,  O.  Zur  Kenntnis  der  Plastiden-  und  Plasmavererbung.  Cytnlogia, 
Fujii  Jubilee  Vol.:  644-653,  1937. 

SiRKS,  M.  J.     Plasmatic  inheritance.     Bot.  Rev.  4:  113-131,  1938. 

Wettstein,  F.  von.  til)er  plasmatische  Vererbung,  sowie  Plasma-  und  Genwirkung. 
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Zeitschr.  Ind.  Abst.  Vererb.  73:  349-366,  1937. 
WiNGE,  O.,  and  O.  Laustsen.     On  a  cytoplasmic  effect  of  inbreeding  in  homozygous 

yeast.     Compt.  Rend.  Trav.  Lab.  Carlsbcrg,  Ser.  Physiol.  23:  17-38,  1940. 

XVII.  Cytology  and  Taxonomy 

Anderson,  E.     Cj'tologj'^  in  its  relation  to  taxonomy.     Bot.  Rev.  3:  335-350,  1937. 
B.\BCOCK,  E.  B.     Systematics,  cytogenetics  and  evolution  in  Crepis.     Bot.  Rev.  8: 

139-190,  1942. 
,  and  G.  L.  Stebbins,  Jr.     The  American  species  of  Crepis.     Carnegie  Inst. 

Wash.  Pub.  504,  1938. 
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Naturalist  76:  337-363,  1942. 
Bowden,  W.   M.     Diploidy,  polyploidy,  and  winter  hardiness  relationships  in  the 

flowering  plants.     Ainer.  Jour.  Bot.  27 :  357-371,  1940. 
Clausen,  J.,  D.  D.  Keck,  and  W.  M.  Hiesey.     Experimental  taxonomy.     Carnegie 

Inst.  Wash.  Yearbooks  35-40,  1936-1941. 
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Jour.  Bot.  26:  103-106,  1939. 
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75:  231-250,  1941. 

Clausen,  R.  T.     On  the  use  of  the  terms  ''subspecies"  and  ''variety."     Rhodora 
43:  157-167,  1941. 

Cleland,  R.  E.     Analj'sis  of  wild  American  races  of  Oenothera  (Onagra).     Genetics 
25:  636-644,  1940. 

DoBZHANSKY,  T.     Gcnctics  and  the  Origin  of  Species.     2d  ed.     New  York,  1941. 

Du   RiETZ,    G.    E.     The  fundamental  units   of  biological   taxonomj-.     Svensk   Bot. 
ridskr.  24:  333-428,  1930. 

Gaiser,   Lulu.     Chromosome    numbers   in   angiosperms.     I,    II,    III.     Genetica   8: 
401-484;  Bibliog.  Genetica  6:  171-466;  Genetica  12:  162-260.      1926,  1930. 

GoLDSCHMiDT,  II.     The  Material  Basis  of  Evolution.     New  Haven,  1940. 

Goodspeed,  T.   H.     Nicotiana  phylesis  in  the  light  of  chromosome  number,  mor- 
phology and  behavior.     Univ.  Calif.  Publ.  Bot.  17:  369-398,  1934. 

Hall,  H.  M.,  and  F.  E.  Clements.     The  phylogenetic  method  in  taxonomy.     Car- 
negie Inst.  Wash.  Pub.  326,  1923. 

Huxley,  J.,  ed.     The  New  Systematics.     Oxford,  1940. 

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263-289,  1941. 

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Sknn,    H.    a.     Chromosome    number   relationships  in   the  Leguminosae.     Bibliog. 
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plant  groups.     Proc.  8th  Amer.  Sci.  Cong.  3:  287-294,  1940. 


INDEX 


Nmnbeis  in  bold-face  iiulicate  images  bearing  ilhistrations. 


Abies  (conifer),  37 

Accessorj'  fruits,  145 

Acetocarmine,  96 

Achillea  (angiosperm),  244,  247 

Achlya  (fungus),  162 

Achromatic  figure,  61,  67,  68,  166 

Acroblast,  120,  121 

Acrosome,  120,  121 

Activation,  126 

Adoxa  (angiosperm),  140 

Adsorption,  47 

Adventitious  embryo,  146,  242 

Aegilops  (angiosperm),  220 

Aesculus  (angiosperm),  222 

Agamic  complexes,  242 

Agglutination,  115 

Alchemilla  (angiosperm),  146 

Aleurone,  144 

Algae,  14,  15,  17,  156-162 

Alkaloids,  38 

Allele,  172 

Allelomorph,  172 

Allen,  154 

Allergy,  51 

AUium  (angiosperm),  67,  87,  99 

Alloheteroploid,  205 

AUomyces  (fungus),  162 

Allopolyploid  plants,  219 

Allosynapsis,  223 

Alternation  of  generations,  98,  136,  158 

Ameba,  46 

Ameiosis,  205 

Amino  acid,  51 

Amitosis,  59 

Amphiaster,  67,  68 

Amphidiploidy,  220-223 

Amphitene,  108 

Amyloplast,  31 

Anastomoses,  26 

Anderson,  243 

Androgenesis,  146 

Aneuploidy,  205,  212 

Angstrom  unit,  8n. 

Animal  pole,  127 

Ant,  130 

Antagonism,  50 

Anther,  114 

Anthoceros  (liverwort),  32,  33,  34,  155 

Anthocyanin  pigments,  38 

Antipodal  cells,  139,  140,  145 

Antirrhinum  (angiosperm),  223 


Aphid,  99,  130 

Apical  body,  153,  154 

Apogamy,  146,  152 

Apomixis,  145 

Apospory,  140,  152,  l.")('> 

Apothecium,  165 

Apple,  209 

Aquilegia  (angiosperm),  222 

Arbacia  (sea  urchin),  70 

Aristotle,  1 

Arrhenatherum  (angiosperm),  56,  72 

Artemia  (crustacean),  210 

Artemisia  (angiosperm),  244,  247 

Artificial  parthenogenesis,  131 

Ascaris  (threadworm),  28,  47,  114 

Ascogenous  hyphae,  166 

Ascogonium,  164,  165 

Ascomycetes,  164,  165,  166,  202 

Ascus,  164,  165 

Aster,  28,  67 

Aster  (angiosperm),  247 

Astral  mitosis,  68 

Asynapsis,  217 

Autogamy,  133,  135 

Autoheteroploidy,  205,  214 

Autopolyploidj'  in  maize,  206 

Autosome,  186 

Autosynapsis,  223 

Auxospore,  161 

Axial  filament,  120,  121 

Axial  gradient,  20 


B 


Babcock,  245 

Backcross  test,  170 

Bacteria,  25 

Bailey,  37,  75,  77,  78,  80 

Barbulanympha  (protozoan),  133 

Bar-eye  mutation,  201 

Barkhausia  (angiosperm),  245 

Basidiomycetes,  163,  164 

Bauer,  95 

Beams,  41,  120 

Bean,  36,  41 

Becker,  66,  75 

Bee,  130,  131 

Belling,  117 

von  Berg,  220 

Bergner,  99,  212 

Beroe  (ctenophore),  129 

Biology,  1 

Biotype,  246 


261 


262 


FUNDAMENTALS  OF  CYTOLOGY 


Birds,  123,  186 

BiscuteUa  (angiosperni),  243 

Bivalent  chromosomes,  103 

Black  body,  176 

Blakeslee,  99,  212,  213 

Blastoderm,  128 

Blastomere,  127,  128,  129 

Blastula,  127 

Blepharoplast,  133    150,  151,  153 

Bloch,  59 

Blood,  7,  45 

Bloodroot,  139 

Blue-green  algae,  162 

Bordered  pit,  76,  77 

Bound  water,  49 

Bouquet,  108 

Bowen,  31,  39,  40,  120,  121 

Bower,  152 

Box  elder,  143 

Brassica  (angiosperni),  223 

Breaks  (chromonemata  by  X  rays),  88 

Bridges,  181 

Brieger,  110 

Brochymena  (insect),  120 

Brown,  R.,  3 

Brown  midrib,  202 

Brownian  movement,  61 

Bryophytes,  153-156 

Bryopsis  (green  alga),  19 

B-type  chromosome,  92 


Cabbage,  9,  223 

Calendula  (angiosperm),  221 

Calkins,  134 

Cambium,  75 

Canti,  3 

Capillitium  filaments,  18 

Carbohydrate,  50 

Carex  (angiosperm),  237 

Carotene,  30 

Carpospores,  160 

Carrel,  7 

Cartilage,  15,  16 

Cat,  15,  99 

Catenation,  196 

Catonia  (angiosperm),  245 

Cell,  3 

Cell  division,  4,  56-74 

Cell  plate,  65,  66,  75 

Cell  sap,  38 

Cell  shape,  73 

Cell  theory,  21 

Cell  waU,  75-83 

Cells  and  organism,  11-21 

Cellulose,  41,  50,  77-82 

Cenospecies,  246 

Central  spindle,  67 

Centrifugation,  28,  31,  40,  41,  45,  47 

Centriole,  28,  67,  68,  120,  121,  12(i,  133 

Centrolecithal  egg,  128 

Centrosomal  blepharoijlast,  157 

Centrosome,  28 

Centrosphere,  28 


Cephalolaxus  (conifer),  37 
Cerebrntulus  (worm),  122 
Chamberlain,  149,  150 
Chambers,  45 
Chiasma,  105,  111 
Chimera,  225 
Chironomus  (insect),  95 
Chitin,  77 

Chlatnydomonas  (green  alga),  158 
Chlorophyll  inheritance,  230-232 
Chloroplast,  29,  30 
Chondriosome,  3,  34,  35,  36,  39,  41 
Chromatid,  59,  103 
Chromatid  bieak,  88,  89 
Chromatin,  26,  51 
Chromatophore,  31 
Chromocenter,  27,  87,  95 
Chromomere,  85,  92 
Chromonema,  2(i,  27,  85 
Chromonema  cycle,  64,  90 
Chromonema  number,  87—90 
Chromoplasts,  31 
Chromoproteins,  51 
Chromosome,  84—101 

aberrations,  193-203 

arms  of,  56 

break,  88,  89 

B-type,  92 

complement,  97-100 

constrictions,  85,  80 

continuity,  100 

doubling,  210 

in  heredity,  172 

heteromorphic,  116,  182,  183,  186 

historical,  4 

homologous  i)airs,  99 

human,  99,  188 

in  hybrids,  216-220 

individuality,  100 

inversion  in,  194,  241 

knob,  92 

map,  180,  181,  185 

matrix,  84,  85 

in  Mendelian  heredity,  168-192 

morphology,  85,  238 

movement,  71 

multivalent,  210 

nucleolus  organizer,  85,  86,  92 

number,  9,  97-99,  204-215,  235 

pairing,  in  somatic  cells,  99 

reduction  of,  102-117,  220 

satellites,  85,  86 

set  of,  97 

sex,  186 

splitting  of,  90,  103 

structure,  87-97 

tetrad,  103 

trivalent,  116,  207 
Cilia,  22,  150 
Citrus  (angiosperm),  242 
Cladophora  (green  alga),  17,  158 
Clausen,  246,  248-249 
Cleavage,  69,  119,  126,  127-130 
Cleland,  199 
Cleveland,  93,  133 


INDEX 


203 


Climatic  races,  243,  247 
Clytia  (coelenterate),  129 
Coagulation,  45-47 
Coal-tar  dyes,  5 
Codium  (green  alga),  82 
Coenocyte,  5,  13,  16 
Coenocytic  embryo,  15 

Coiling  of  chromonemata,  86,  90,  91,  93,  110 
Colchicine,  20G 
Collagen,  51 
Colloidal  system,  40 
Colonial  algae,  14,  15 
Color  bUndness,  190 
Columbine,  222 

Complement,  of  chioniosomes,  97-100 
Component,  46 
Conant,  57 
Conifer,  99,  148 

Conjugate  division,  of  nuclei,  163,  165 
Conjugation,  134,  135 
Connective  tissue,  15,  16 
Contractile  vacuole,  17,  36,  157 
Cooper,  138,  141 
Correlation,  20,  82 
Cotton,  79,  222 
Cow,  99 
Crayfish,  67 
Creighton,  183 

Crepis  (angiosperm),  agamic  complexes,  242 
amphidiploid,  222 
chromosome  complement,  98,  99 
embryo  sac,  144 
monoploid  sporophyte,  212 
species  and  chromosomes,  238,  245 
syngamy,  142 
Crossing  over,  105,  116,  117,  175,  176,  177,  178- 

179 
Crossover  unit,  178 
Crystal,  38,  42,  43 
CrystalUzation,  49 
Cucumber,  74 
Cucurbita  (angiosperm),  19 
Cutin,  77 

Culleria  (brown  alga),  160 
Cyanophyceae,  162 
Cycad,  148,  149,  150 
Cytaster,  70,  126 
Cytogenetics,  9,  168 
Cytokinesis,  57,  65-66,  69-70 
Cytoplasm,  cell  constituent,  11,  22-25 
in  heredity,  227-233 
in  syngamy,  142,  232 
Cytosome,  22 
Cytotaxonomy,  9,  234-249 


D 


Daltonism,  190 

Dangeard,  37 

Darlington,  207 

Darwin,  249 

Datura  (angiosperm),  99,  143,  199,  212,  213 

Deficiency,  193 

Deletion,  182    183,  184,  193,  194 

Desynapsis,  217 


Development,  12 
Diakinesis,  107,  110 
Diatoms,  31,  161 
Dictyota  (brown  alga),  18,  160 
Differentiation,  17-20 
Diffused  stage,  111 
Digby,  152 

Digitalis  (angiosperm),  218,  223 
Dikaryon,  163,  164,  166,  106 
Dikaryophase,  163,  164,  165 
Dikaryotic  hybrids,  224 
Dikinetic  chromatid,  88 
Dioecism,  136,  189 
Dioon  (cycad),  149,  150 
Diospyros  (angiosperm),  81 
Diplodinium  (protozoan),  17 
Diploid,  97 
Diplohaplont,  158 
Diploidization,  163,  164 
Diplont,  158 
Diplosis,  102,  127,  143 
Diplotene,  109,  110,  111,  116 
Diptera,  94,  99 
Disjunction,  102,  103,  106 
Dominance,  170 
Double  refraction,  48 
Draparnaldia  (green  alga),  32 
Drosophila  (fruit  fly),  bar  eye,  201 

chromosome  complement,  99,  100 

chromosome  map,  181 

crossing  over,  175 

deletion,  184 

dupUcation,  201 

geographic  strains,  241 

gynandromorph,  190 

heteropyknosis,  87 

inversion,  241 

linkage,  176 

linkage  map,  179,  181,  186 

recombination,  175,  176 

salivary  chromosomes,  95,  201 
Druse,  42 

Duplication  of  genes,  201 
Dwarf  character,  169 
Dyad,  103,  113,  114 


E 


Echinoderm  egg,  69,  127 

Ecology  and  chromosomes,  9,  243,  244,  247 

Ecospecies,  246 

Ecotype,  246,  248 

Ectocarpus  (brown  alga),  160 

Ectoplasm,  22 

Egg  apparatus,  139,  140 

Einset,  139 

Elastin,  51 

Elater,  18,  155 

Electron  microscope,  7 

Embryo,  142 

p:mbryo  sac,  139,  140,  142 

Embryogeny,  in  angiospernis.  143,  144 

in  animals,  18,  119 

in  ferns,  152 

in  gymnosperms,  150 


264 


FUNDAMENTALS  OF  CYTOLOGY 


Embryonic  characters,  228 

Empetraceae,  237 

Emulsion,  47 

End  piece,  121 

Endocarp,  145 

Endomixis,  135 

Endoplasm,  23 

Endosperm,  15,  142,  144,  148 

Energic  stage,  25 

Enzyme,  38,  51,  79 

Epilobium  (angiospenii),  229 

Epithelium,  74 

Equational  division,  64,  65,  103,  106 

Ergastic  matter,  41,  42,  43 

Ergosterol,  50 

Ericaceae,  237,  244  • 

Erythrocyte,  7 

Euchlaena  (angiosperm),  218,  219,  238,  239, 

Euchlaena-Zea  hybrids,  218,  241 

Euchromatic,  86,  92 

Euchromocenters,  87 

Eucrepis  (angiosperm).  245 

Eudorina  (green  alga),  15 

Euphorbia  (angiosperm),  146 

Euploidy,  204 

Exine,  137 

Exocarp,  145 

Experimental  taxonomy,  244-249 

Eyespot,  31 


Factor,  169 

(See  also  Gene) 
Fankhauser,  211 
Farmer,  152 
Fats,  43,  50 
Ferns,  150-153 
Fertilization,  5,  123-127 

{See  also  Syngamy) 
Fesfuca  (angiosperm),  242 
Feulgen  reaction,  54,  62,  96 
Fiber  tracheid,  80 
Fibroblast,  7 
Fibrous  molecule,  48 
Ficus  (angiosperm),  38 
Fission,  132 
Fixation,  8,  35,  53,  84 
Flagellum,  22 
Flavone,  39 
Flower,  136 
Fluorescence,  32 
Fly,  99 

Follicular  epithelium,  122 
Forage  grasses,  244 
Four-o'clocks,  170 
Fowl,  179 
Frey-Wyssling,  48 
Fritillaria  (angiosperm),  140 
Frog,  131 
Fruit  tissue,  145 
Fucoxanthin,  30 
Fucus  (brown  alga),  68,  1(10 
Funaria  (moss),  230 
Fundulus  (fish),  36 


Fungi,  162-166 

Furrowing  (cytokinesis),  69 


G 


Galactose,  50 

Gamagrass,  238,  239,  241 

Gametic  meiosis,  119,  123,  159,  160 

Gametic  number,  98 

Gametophyte,  136 

Gastrula,  127 

Gatenby,  120 

Gaudens  comi^lex,  200 

Gel,  46 

Gelatin,  51 

Gelation,  46,  69,  71 

Generative  cell,  137,  138,  141 

Gene,  169 

location,  177-186 

mutation,  201,  202 

nature  of,  201 
Genome,  86,  97 
Genotype,  171 

Geographic  distribution  and  polyploidy,  242 
Gerassimova,  142 
Germ  ceU,  118 
Germinal  disc,  123 
Germinal  vesicle,  122 
Ginkgo  (gymnosperm),  148 
Gland  cell,  40 
GHadin,  51 

Globular  molecule,  49 
Glossy  leaf,  178,  179 
Glucose,  50 
Glucose  residue,  77 
Glucoside,  38 
Glutenin,  51 
Glycerides,  50 
Glycogen,  42,  50 
Glycoproteins,  51 
Gnetales,  148 
Goblet  cell,  40 
Goldfish,  35 

Golgi  material,  39,  40,  120,  121 
Grana,  32 
Graner,  110 
Green  pod,  173 
Greenleaf,  207 
Gr^goire,  27 
Gregory,  240 
Griffen,  97 
Growth,  12,  19 
Growth  patterns,  13-16 
Growth  period  in  oocyte,  111,  122 
Gymnosperms,  148-150 
Gynandromorph,  190 
Gynogenesis,  146 
Gyres,  90,  91 


H 


Hair,  49 

Half-chromatid,  61 
Half-chromatid  break,  88,  89 
Haliciislis  (green  alga),  82 


INDEX 


265 


Hall,  133 

Haploid  {see  JM(jiioploi<l) 

Haploinicts,  224 

Haplont,  156 

Haplosis,  102 

Heard,  94 

Heartwood,  77 

Heat  rigor,  35 

Heat  treatment,  205 

Hegner,  119 

Heidenhain,  3,  15,  21 

Heniaglobiii,  30 

Hemin,  30 

Heredity,  168 

Hermaphroditism,  136 

Heterochromatism,  85,  86,  92 

Heteromorphic  chromosome  pairs,  116,  182 

186 
Heteronema  (flagellate),  132 
Heterophytic,  136 
Heteroploidy,  204-21.5 
Heteropyknosis,  85,  86,  92,  188 
Heterothallism,  136,  163 
Heterozygosity,  172 
Hexosans,  50 
Hexose,  50 

Hieracium  (angiosperm),  242 
Hiesey,  246,  248,  249 
Hilum,  41 
Histone,  51 

History  of  cytology,  2-10 
Hofmeister,  4 
HoUingshead,  212,  222 
Holoblastic  cleavage,  128 
Holomastigot aides  (protozoan),  93 
Homolecithal  egg,  127 
Homophytic,  136 
Homothallism,  130 
Homozygosity,  172 
Hooke,  3 
Hordein,  51 

Horkdia  (angiosperm),  247 
Horse,  99 

Human  chromosomes,  99,  188 
Humjihrey,  113 
Hunter,  93 
Huskins,  109 
Huxley,  44 
Hyacinth,  62,  209 
Hyaloplasm,  22,  44 
Hybridity,  216-226 
Hydration,  46 
Hyperploidy,  205 
Hypoploidy,  205 


Idiograni,  238,  239 
Idiosome,  120 
Immunity,  51 
Impatiens  (angiosperm),  27 
Incinerated  tissues,  52,  77 
Infusoria,  132,  135 
Inorganic  salts,  50 
Integument,  139 


Intercellular  substance,  70,  76 

Interkinesis,  112 

Interjihase,  64 

Intino,  137 

Iris  (angiosperm),  66,  209 

Isoelectric  point,  54 


Janssens,  111,  178 
Jimson  weed,  143 
Jungers,  66 


85,  87,  92,  100 


Karyogamy,  155,  163 
Karyokinesis,  57 
Karolymph,  26,  52 
Karyotype,  238 
Kaufmann,  100 
Keck,  246,  248,  249 
Keratin,  51,  77 
Kerr,  75,  78,  80 
Kinetochore,  6 
King,  41 
Kinomere,  85 
Kinoplasm,  25 
Kinosome,  85 
Mebahn,  157,  161 
ICnudson,  29 
Koos,  144 
Kowalski,  26 
Kretschmer,  158 
Kuhn,  27 


Lagging  chromosomes,  175,  220 

Laminaria  (brown  alga),  160 

Larval  characters,  228 

Latex,  38 

Layia  (angiosperm),  248,  249 

Lecithin,  51 

van  Leeuwenhoek,  135 

Leguminosae,  237 

Leptotene,  108,  109 

Lethal  gene,  200 

Lettuce,  139 

Leucoplast,  31 

Levan,  210 

Levulose,  50 

Lewis,  74 

Lichen,  225 

Lignin,  77,  79,  80 

Lilium  (angiosperm),  114,  14(1.  141 

Lillie,  124 

Linkage,  174-185 

Lipide,  43,  51 

Liquid-crystal  state,  48 

Liver  cells,  35 

Liverworts,  18,  1,53-156 

Loefer,  132 

Longlej-,  218,  239 

Lorbeur,  189 

Lycopene,  31 

Lycopersicum  (angiosperm),   \  U',,  207 


2GG 


FUNDAMENTALS  OF  CYTOLOGY 


M 

MacCaidle,  35 

McClintock,  92,  110,  115,  197 

McCuUagh,  236 

Mackensen,  184,  185 

Macromeres,  127 

Macronucleiis,  17,  132 

Madia  (angiosperni),  223 

Maianthemum  (angiosperni),  26 

Major  coil  or  spiral,  93,  94 

Male  sterility,  230 

Malvaceae,  237 

Mannose,  50 

Marsilia  (water  fern),  73 

Martens,  76 

Maternal  inheritance,  231 

Matrix,  in  cartilage,  14 

of  chromosome,  26,  59,  86,  100 
Matthiola  (angiosperni),  228 
Maturation  divisions,  123 
May  apple,  114 
Megaspore,  136,  139 
Megasporocyte,  139 
Megasporogenesis,  138-140 
Meiocyte,  102 
Meiosis,  102-117,  220 
Melandrium  (angiosperni),  188 
Membranes,  11,  22,  24,  45 
Mendehan  heredity,  168-192 
Menoidium  (flagellate),  133 
Mentha  (angiosperni),  222 
Meristem,  65,  75 
Meroblasrtc  cleavage,  128 
Metabolic  gradient,  20 
Metabolic  stage,  25 
Metaphase,  61,  62 
Metaxenia,  146 
Metz,  94,  97 
Meyer,  42 
Miastor  (fly),  119 
Micelles,  48,  77 
Michaelis,  229 
Micromanipulation,  6,  7,  45 
Micromeres,  127 
Micron,  8?!. 
Micronucleus,  17,  132 
Micropyle,  139 
Microscope,  2,  6 
Microspore,  136,  138 
Microsporocyte,  137 
Microsporogenesis,  137-138 
Microtome,  6 
Middle  lamella,  75,  76,  78 
Middle  piece,  120,  121 
Minor  coil  or  spiral,  93,  94 
Minouchi,  99 
Mint,  222 

Mirabilis  (angiosperni),  170,  231 
Misdivision,  214 
Mitosis,  57-69 
Mobile  proteins,  49 
Modified  plants,  197 
Monkey,  99,  188 
Monoecisra,  136 


Monoploid,  97,  211,  212 
Monoploid  hybrids,  224 
Monopolar  spindle,  72 
Monosomic  plants,  214 
Monotropa  (angiosperni),  143 
Morgan,  134,  176,  178,  181,  190 
Morula,  14 
Mosses,  153-156,  230 
Motorium,  133 
Mucor  (fungus),  162 
Multiple  fruits,  145 
Multivalent  chromosomes,  210 
Muscle,  16,  22,  23,  48 
Mushroom,  18,  163 
Mutation  theory,  199 
Myofibril,  22,  23 
Myxomycete,  15,  18,48 


N 


Nasturtium  (angiosperni),  31 

Navashin,  98 

Nebenkeni,  120 

Nemalion  (red  alga),  161 

Nereis  (annelid  worm),  124,  125 

Nerve  cells,  22,  24,  26 

Neurofibrils,  22,  24,  26 

Neuroglia,  24 

Neuromotor  apparatus,  17,  133 

Neurospora  (fungus),  166 

Neutral  red,  38 

Newt,  211 

Newton,  207 

Nickel-particle  technique,  45 

Nicotiana  (aiigiosperm),  207,  212,  222 

Nondisjunction,  174 

Nonnucleate  egg  fragments,  70 

Nucellus,  139,  145 

Nuclear  membrane,  26 

Nucleic  acid,  52,  87 

Nuclein,  51 

Nucleocytoplasmic  hybrid,  229 

Nucleolus,  27,  52,  98,  100 

Nucleolus  organizer,  85,  86,  92 

Nucleoproteins,  51 

Nucleus,  3,  11,  25-28 

Nymphaea  (angiosperni),  235 


O 


Oak,  143 

O'Brien,  36 

Octoploidy,  209 

Oedogonium  (green  alga),  32,  140,  156.  157,  158 

Oenothera  (angiosperni),  199,  200 

Ohta,  99 

Oidia,  163,  164 

Oil,  42,  43,  50 

Onion,  57,  87,  99 

Ontogeny,  12 

Oocytes,  or  ovocytes,  122 

Oogenesis,  122,  123 

0()gonia,  118 

Oogonium  (of  plants),  157 

Opossum,  188 


ISDEX 


267 


Orange,  146 

Organic  acids,  38 

Organism  and  cell,  11-21 

Organismal  theory,  21 

Organization,  12 

Organs,  20 

Oryzein,  51 

Oscillatoria  (blue-green  alga),  162 

Osraiophilic  platelets,  41 

Overton,  169 

Ovule,  139 

Ovum,  123 

Iiromorphology  of,  129 


Pachytene,  99,  108,  109,  110 

Paeonia  (angiospemi),  112,  198 

Painter,  96,  97,  185,  188,  201 

Pancreas,  40 

Papaver  (angiosperm),  219 

Paracrystalline  state,  48 

Paramecium  (infusorian),  132,  134,  13o,  229 

Parenchyma,  15 

Parthenocarpy,  146 

Parthenogenesis,  130,  145,  146 

Parthenote,  130 

Pathological  tissue,  9,  36,  43 

Pea,  69,  99,  169,  173,  218 

Pecan.  143 

Pectin,  76,  77 

Pectization,  46 

Pdlionia  (angiosperm),  42 

Peniophora  (fungus),  166 

Penstemon  (angiosperm),  247 

Pentosan,  50 

Pentose,  50 

Peony,  112,  198 

Peperomia  (angiosperm),  140 

Peptization,  46 

Perforatorium,  121 

Pericarp,  145 

Perisperm,  145 

PeriNdtelline  space,  114 

Persimmon,  81 

Petunia  (angiosperm),  210 

Peziza  (fungus),  164 

pH,  38,  53 

Phaseolus  (angiosperm),  27 

Phase,  in  colloids,  46 

Phenotype,  171 

Phospholipide,  51 

Photosynthesis,  30 

Phragmoplast,  65,  66 

Phragmosome,  58,  69 

Phycoerythrin,  30 

Phycomyces  (fungus),  162 

Phycomycetes,  162 

Physcomilrium  (moss),  230 

Physiological  gradient,  20 

Physiology,  5,  9 

Picea  (conifer),  78 

Pigment,  30,  38 

Pisum  (angiosperm),  09,  99,  169,  173,  218 

Pits,  76,  77 


Plantayo  (angiosperm),  235,  236,  238 

Plasma  membrane,  11,  23 

Plasmagel,  22 

Plasmasol,  22 

Plasmodermal  blepharoplast,  157 

Plasmodesms,  20,  80,  81 

Plasmodial  tissues,  13,  15 

Plasmodium,  16 

Plasmone,  230 

Plastid,  29-34 

of  algae,  158,  159 

as  cell  organ,  11 

as  cellulose  former,  82 

and  chondriosome,  35,  36 

development,  33,  35 

function,  30,  31 

inheritance,  34,  230-232 

mutation,  29 

origin,  34 

primordia,  36 

relation  to  Golgi  material,  40 

in  spermatogenesis,  153 

in  sporogenesis,  155,  156 

in  syngamy,  158,  159 

X-ray  effects,  29 

in  zygospore,  158,  159 
Plum,  222 

Poa  (angiosperm),  242 
Podophyllum  (angiosperm),  114 
Podostemon  (angiosperm),  140 
Polar  bodies,  114,  122,  140 
Polar  fusion  nucleus,  142 
Polar  nuclei,  137,  140 
Polarity,  19,  20 
Polarized  light,  32,  41,  78 
Pole  plasm,  119 
Pollen  grain,  37,  138 
Pollen  tube,  138,  141 
Pollination,  140 
Polocyte,  114,  122,  123 
Polyembryony,  146 
Polypeptide  chain,  48 
Polyploidy,  98,  156,  204-215,  219,  256 
Poly  podium  (fern),  29 
Polysiphonia  (red  alga),  1(10 
Polyspory,  218,  220 
Polytrichum  (moss),  154,  lo.'),  156 
Portidaca  (angiosperm),  138 
Position  effect,  194 
Postnuclear  cap,  120 
Postnuclear  granules,  120 
Postreduction,  106 
Potamobius  (crustacean),  67 
Potato,  31,  209 

Potenfilla  (angiosperm),  242,  247 
Prereduction,  106 
Primary  mutant,  199 
Primary  trisomic  types,  213 
Primary  wall,  75,  76,  78 
Prime  types,  199 
Primordial  germ  cell,  118 
Primula  (angiosperm),  222 
Principal  piece,  121 
Prometaphase,  61 
Pronucleus,  125 


268 


FUNDAMENTALS  OF  CYTOLOGY 


Prophase,  59 
Proplastid,  33,  41 
Protamine,  51 
Protein,  42,  51 
Protoplasm,  44-55 

elasticity  of,  45,  47 

fibrillar  theory  of,  47 

streaming  of,  46 
Protoplasmic  growth  patterns,  13,  14,  15— IG 
Protoplast,  3,  11 
Protozoa,  17,  131-135 
Prunus  (angiosperm),  222 
Pseudoplasmodium,  16,  225 
Pseudopodiiim,  22 
Pteridophytes,  150-153 
Pyrenoid,  30,  32,  33 
Pyronema  (fungus),  164 
Pythium  (fungus),  162 


Q 


Quadripartition,  137,  138 
Quadrivalent  chromosomes,  207,  210 
Quartets,  102 


R 


Rabbit,  16,  129 
Randolph,  180,  206,  208 

Random  assortment,  105 

Ranunculaceae,  240 

Ra-phanus  (angiosperm),  223 

Raphanus-Brassica  hybrid,  223 

Raphides,  43 

Receptive  spot,  157 

Recessiveness,  170 

Reciprocal  crosses,  227-230 

Reciprocal  translocation,  195-200 

Recombination,  175,  176,  177-179 

Relic  coils,  90,  91 

Renner  complex,  200 

Reproduction,  in  angiosperms,  136-147 

in  animals,  118—135 

in  other  plants,  148-167 
Repulsion,  71 
Resin,  77 
Respiration,  30 
Reticulum,  26 
Rhizomorph,  163 
Rhizoplast,  133 
Rhizopus  (fungus),  162 
Rhoades,  230 
Rhoeo  (angiosperm),  199 
Rhopalodia  (diatom),  161 
Ribonucleic  acid,  87 
Riboviolascin,  30 
Riccardia  (liverwort),  154,  155 
Rings,  due  to  deletion,  194 

due  to  reciprocal  translocation,  196-200 
Robyns,  62 

Rosa  (angiospiTTii),  242 
Rotifers,  130 
Rubber,  38 
Rubus  (angiosperm),  242 


Rusts,  163 

Rye,  36,  143,  220 


Sakamiira,  93 

Salamander,  99,  108,  131,  211 

Salivary-gland  cliromosomes,  94-97,  184,  185,  241 

Saprolegnia  (fungus),  162 

Satellite,  85,  86 

Satina,  99,  212 

Sax,  112,  113,  199 

Scandinavian  flora,  244 

Schleswig-Holstein,  243 

Schmitz,  32 

Sciara  (fly),  72,  94 

Scinaia  (red  alga),  161 

Scott,  42 

Secondary  constriction,  85,  80,  100 

Secondary  trisomic  types,  214 

Secondary  wall,  75,  76,  78 

Secretion,  39,  40 

Seed  coat,  145 

Segmentation,  127 

Segregation,  170 

Seifriz,  7 

Selaginella  (pteridophyte),  33 

Semipermeability,  24,  38 

Semisterility,  196 

Sertoli  cells,  119 

Sex-chromosomes,  100,  186-191 

Sex-limited  characters,  175 

Sex-linkage,  190,  191 

Sharp,  L.  W.,  61,  87 

Sharp,  R.  G.,  17 

Sheep,  99 

Showalter,  155 

Silica  gel,  47 

Silk,  49 

Silver-line  system,  133 

Sinnott,  59 

Siparuna  (angiosperm),  80 

Sisyrinchium  (angiosperm),  247 

Slime  mold,  15,  18,  46,  225 

Smith,  G.  M.,  15,  30,  159 

Smith,  L.,  197 

Smith,  S.  G.,  109 

Sol,  46 

Solation,  45 

Solvation,  46 

Somatic  cell  division,  56 

Somatic  doubling,  205 

Sorokin,  36 

Space  lattice,  41,  77 

Sparrow,  90,  91 

Species  and  chromosomes,  235-249 

Specific  grax-ity,  28,  41 

Specificity  of  organisms,  51,  52 

Spermatia,  160 

Spermatid,  119 

Spermatocyte,  119 

Spermatogenesis,  in  animals,  119-122 

in  plants,  138,  141,  150,  151,  154 
S|jciinat()goiiia,   118 
SpiTnKi1..z(.i<l.s,  150,  151.  154 


IXDEX 


209 


Spermatozoor ,  121 
Spermiogenesis,  in  animals.  119-122 

in  bryophytes,  153 

in  ferns,  151 

in  gymnospt.  nis.   148,  149 

in  mosses,  154 
Sphacelaria  (brown  alga;,  5(j,  7.3 
Sphaerocarpos  (liverwort),  187,  189 
Spinal  cord,  24 
Spindle,  61,  62,  68 
Spindle-attachment  region,  8.") 
Spindle  elongation,  71 

.Sp'Vof/i/ro  (green  alga),  30,  34,  37,  1.56,  I.' 
Spirotrichonprnpha  (protozoan),  93 
.Spore-bearing  organs,  18 
Sporocyte,  138,  139 
Sporodinia  (fungus),  162 
Sporophyte,  136 
Sporophytic  budding,  146,  242 
Spruce,  78 
Squash,  3,  19 
Stains,  5,  8,  38,  52-54 
Stamen  hairs,  65.  66 
Stangeria  (cycad).  149 
Starch,  31,  41,  42 
.Statistical  methods,  8 
Sterility,  in  hybrids,  217-220 
Stern,  181 
Sterols,  50 

Stigeoclonium  (green  alga),  17 
Stocks,  167 
Stomates,  82,  207 
Strangeways,  3 
Stroma,  31 

Structural  hybrid,  196,  216,  224,  241 
Structure  proteins,  49 
Sturtevant,  181 
Stypocaulon  (brown  alga),  68 
Suberin,  77 
Sugar,  30,  31,  38 
Sugary  endosperm,  178,  179 
Surface  activit.\',  46 
Surface  film,  45 
Surface  tension,  70 
.Surface-volume  ratio,  13 
Swingle,  68 

.Synap,sis,  99,  103,  108.  11.5 
Sync.vtium.  15,  16 
Synergids.  139,  140,  142 
Syngamy,  in  algae.   1.56—161 

in  angiospenns.   140-143 

in  animals,  123-127 

in  a.sconiycetes,  164 

in  basidiomycetes,  163 

in  bryoph.vtes,  154-155 

in  ferns,  152 

iti  gymnosperms,  149 

in  hybrids,  217-220,  239,  241 


Taraxacum  (angiosperm),  242 

Taxonomy  and  cytolog.\ ,  23  1-24!) 

Telezynski,  27 

Telokinetic  chromosomes,  80 

Telophase,  63 

Tendons,  49 

Teosinte,  218,  219,  238,  239,  241 

Teosinte-maize  hybrid,  218 

Terminalization,  110 

Tertiary  split.  111 

Testcro.ss,  170 

Tetrad  chromosome,  103 

Tetrakaidecahedron,  74 

Tetraploidy,  20.5-209 

Tetrasomic  ratios,  208-210 

Tetraspores,  160,  161 

Theophrastus,  1 

Thixotropy,  48 

Thymonucleic  acid,  87,  96 

Tissue  culture,  3,  6,  7,  73 

Tissues,  20 

Tityus  (scorpion),  110 

Tobacco,  30,  31,  210,  222 

Tomato,  146,  207 

Tonoplast,  38 

Torus,  76 

Tractile  fibers,  62,  71 

Tradescantia  (angiosperm),  cell  division,  4 

cell  plate,  66 

chromosomes,  199 

geographic  distribution  and  polyp!oid\  .  243 

living  nucleus,  27 

meiosis,  113 

reciprocal  translocation,  199 

somatic  mitosis,  56,  72 

spiral  chromonemata,  93 
Translocation,  195-201 
Trichocyst,  133 
Tiichogyne,  160 

Trillium  (angiosperm),  63,  87,  90,  91,  93,  109 
Triple  fu.sion,  143 
Triploidy,  209 

Tripsacum  (angiosi)erm),  238 
Trisome,  174 
Trisomic  plants,  212 
Trisomic  ratios,  174 
Triticum  (angiosperm),  amidiidiplnidy,  223 

chromosomes  and  taxonomy,  237 

reciprocal  translocation,  197 
Triton  (salamander),  70 
Trilurus  (newt),  211 
Trivalent  chromosome,  115,  207 
Trochodendron  (angiosi>erm),  78 
Trophoplasm,  25 

Tube  cell,  or  nucleus,  137,  138,  141 
TuUp,  209 

Tunicate  ear,  178,  179 
Turesson,  246,  247 
Turgor,  36 


Tall  character,   KiO,  173 
Tannin,  38,  77 
Tajjetal  Plasmodium,  137 
Taiietum,  16,  137 


U 

Ulothrii  (green  alga),   156,  157 
Ultraviolet  liglit,  8.  .50,  96 
I'ndulating  membninc,  22 


270 


FUNDAMENTALS  OF  CYTOLOGY 


Vacuole,  11,  3G,  37,  38 

Valeriana  (angiospeini),  42 

Vaiicheria  (green  alga),  17 

Vegetal  pole,  127 

Vejdovsky,  120 

Velans  complex,  200 

Verbena  (angiospeini),  230 

Vestigial  wings,  176 

Viola  (angiosperm),  210,  237,  244,  247 

Virus,  43,  52,  203 

Viscosity,  44,  45 

Vital  dyes,  53 

Vitamin  A,  30,  50,  208 

Vitamin  C,  30,  207 

Vitamin  D,  50 

Vitamin  E,  50 

Vitelline  membrane.  123,  125 

Vitrification,  50 

Volume-surface  ratio,  13 

de  Vries,  199 


W 


Wall,  of  plant  cell,  75-83 
Warmke,  63,  188 
Water,  49 
Wax,  43,  50 
Webber,  160 
Weddle,  221 
Weier,  166 
Weismann,  6 
Westergaard,  188 
Wheat  (see  Triticum) 
Wheat-rye  hybrid,  220 
White  eye,  184 
Whitefish,  58 
Williams,  18 
Willow,  99 
Wilson,  135 
Wood,  77,  78,  79 


X 

X-chromosome,  180-19 1 

X-ray  analysis,  7,  77 

X-ray  effects,  on  chloroplasts,  29 

on  chromosomes,  88,  202 

on  mutation,  202 
Xanthophyll,  30 
Xenia,  146 
Xylan,  77 


Y-chromosonie,  186-191 
Yamanouchi,  68 
Yellow-green  leaf,  182,  183 
Yolk,  43,  122 
Yuasa,  161 


Zauschneria  (angiosperm),  247 

Zea  (angiosperm),  antipodal  cells,  145 

autopolyploidy,  206 

chromosomes,  92,  99,  110,  206 

crossing  over,  179 

cytotaxonomy,  238 

hybrids,  241 

idiogram,  239 

inversion,  196 

linkage  map,  179,  180 

nucleolus,  86 

reciprocal  translocation,  197 

tetraploidy,  206 

trivalent  chromosome,  115 

Zea-Euchlaena  hybrid,  218,  241 
Zein,  51 

Zoospores,  157,  158,  159 
Zygnema  (green  alga),  34,  56,  158 
Zygomycetes,  162 
Zygospore,  162 
Zygotene,  108,  109 
Zygotic  meiosis,  157,  158,  162 
Zygotic  number,  98