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RELATION  OF  PHENOLIC  COMPOUNDS 
TO  GERMINATION  OF  PEACH  SEEDS 


By 

JAMES  BRUCE  AITKEN 


A  DISSERTATION  PRESENTED  TO  THE  GRADUATE  COUNCIL  OF 

THE   UNIVERSITY  OF   FLORIDA 
IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY 


UNIVERSITY  OF  FLORIDA 

August,  1967 


AGRI. 

CULTURAL 
LI9RARY 


;  (/ 


V 


->;•■• 


^iiiliii 


ACKNOWLEDGEMENT 

The  author  wishes  to  express  his  sincere  appreciation  and  gratitude 
to  Dr.  R.  H,  Biggs,  Associate  Biochemist,  Department  of  Fruit  Crops,  and 
Chairman  of  the  student's  Supervisory  Committee,  for  his  most  valuable 
assistance  and  guidance  during  the  course  of  research  and  the  prepara- 
tion of  this  manuscript. 

Appreciation  is  extended  to  Dr.  A.  H.  Krezdorn,  Chairman,  Depart- 
ment of  Fruit  Crops;  Dr.  T.  E.  Humphreys,  Associate  Biochemist,  Depart- 
ment of  Botany;  Dr.  C.  H.  Hendershott,  Associate  Professor  of  Fruit 
Crops;  and  Dr.  D.  0.  Spinks,  Professor  of  Soils,  Department  of  Soils, 
for  their  constructive  criticism  and  invaluable  assistance  in  the  pres- 
entation of  this  manuscript. 

The  author  also  wishes  to  express  his  gratitude  to  Mr.  J.  K.  Peter, 
laboratory  technician,  for  his  assistance  in  conducting  portions  of  the 
research. 

For  her  help  in  the  preparation  of  this  manuscript  and  also  for  her 
interest  and  encouragement  during  the  course  of  this  study,  the  author 
wishes  to  express  his  sincere  appreciation  to  his  wife,  Patricia. 


11 


TABLE  OF  CONTENTS 

Page 

ACKNOWLEDGEMENT ii 

LIST  OF  TABLES iv 

LIST  OF  FIGURES vi 

INTRODUCTION 1 

REVIEW  OF  LITERATURE 3 

Germination  Inhibitors  4 

Physiology  of  Seed  Germination 12 

Physiology  of  Peach  Seed  Germination  17 

MATERIALS  AND  METHODS  21 

EXPERIMENTAL  RESULTS .^ 31 

DISCUSSION 67 

SUMMARY  AND  CONCLUSIONS  73 

APPENDIX:   GAS  CHROMATOGRAMS  OF  STANDARDS 75 

LITERATURE  CITED 81 

BIOGRAPHICAL  SKETCH  90 


iii 


LIST  OF  TABLES 
Table  Page 

1.  Influence  of  thiourea  concentrations  on  germination  and 

per  cent  of  production  of  abnormal  seedlings  from 

'Okinawa'  peach  seeds  32 

2.  Effect  of  thiourea  concentration  and  embryo  excision  on 

germination  of  'Okinawa'  peach  seeds  12  days  after 

start  of  imbibition  and  on  abnormal  seedling  production 

32  days  after  start  of  imbibition 33 

3.  Per  cent  germination  of  'Okinawa'  peach  seeds  7  and  20 

days  after  start  of  imbibition  as  influenced  by  cyanide  ...   34 

4.  Per  cent  germination  of  'Okinawa'  peach  seeds  7  and  12 

days  after  start  of  imbibition  as  influenced  by 

mandelonitrile 36 

5.  Per  cent  germination  of  'Okinawa'  peach  seeds  7  and  12 

days  after  start  of  imbibition  as  influenced  by 

benzaldehyde 37 

6.  Relative  retention  time  and  possible  identity  of  com- 

ponents separated  by  gas  chromatography  of  the  propyl 

esters  of  the  acidic  fraction  from  an  ethanolic 

extract  of  peach  seeds 40 

/ 

7.  Paper  chromatographic  separation  of  the  inhibitory 

complex  from  dormant  peach  seeds 42 

8.  Influence  of  acids,  bases  and  heat  on  the  inhibitory 

complex  from  peach  seeds  after  paper  chromatography  43 

9.  Solubility  of  the  inhibitor-complex  in  various  organic 

solvents  as  determined  by  the  alfalfa  bioassay 44 

10.  Mean  per  cent  germination  of  dormant  'Okinawa'  peach 

seeds  30  days  after  start  of  imbibition  as  influenced 

by  benzaldehyde  and  mandelonitrile  concentrations  57 

11.  Mean  per  cent  germination  of  dormant  'Okinawa'  peach 

seeds  30  days  after  start  of  imbibition  as  influenced 

by  benzoic  and  p-hydroxybenzoic  acid  concentrations  58 


IV 


12.  Comparison  of  retention  times  of  p-hydroxybenzoic  acid 

and  L-mandelic  acid  as  influenced  by  various  acetylation 
procedures ^^ 

13.  Comparison  of  peak  areas  of  p-hydroxybenzoic  acid  and 

L-mandelic  acid  as  influenced  by  various  acetylation 

procedures ^^ 


LIST  OF  FIGURES 
Figure  Page 

1.  Influence  of  benzaldehyde  and  mandelonitrile  on  peach 

seed  germination 38 

2.  Gas  chromatogram  of  the  propyl  esters  of  the  acidic 

fraction  from  an  ethanol  extract  of  peach  seeds. 

Time  is  in  minutes 39 

3.  Gas  chromatograms  of  a  known  composite  sample  of  an 

etheral  solution  of  benzaldehyde-mandelonitrile: 

(a)  initial  solution;  (b)  after  addition  of  a 

solution  of  sodium  bisulfite;  and  (c)  after  addition 

of  potassium  cyanide 47 

4.  Changes  in  benzaldehyde-mandelonitrile  content  of 

peach  seeds  as  measured  at  various  intervals  after 

start  of  imbibition  under  the  designated  treatments  48 

5.  Change  in  the  content  of  mandelonitrile  in  peach 

seeds  at  various  intervals  after  start  of  imbibition 

under  the  designated  treatments 49 

6.  Change  in  the  content  of  benzaldehyde  in  peach  seeds 

at  various  intervals  after  start  of  imbibition  under 

the  designated  treatments  50 

7.  Germination  of  peach  seed  as  influenced  by  embryo 

excision,  and  thiourea  treatments  as  determined 

periodically  after  the  start  of  imbibition 52 

8.  Relative  inhibitory  activity,  as  measured  by  the 

alfalfa  bioassay,  of  the  inhibitory  complex  in  an 
ethanolic  extract  of  peach  seeds  chromatographing 
between  R^'s  0.6  to  0,8 54 

9.  Germination  of  peach  seeds  as  influenced  by  5°  C 

of  varying  durations 55 

10.  Gas  chromatogram  of  diazomethane-solvent  control. 

Retention  time  is  in  minutes 60 

11.  Gas  chromatograms  of  L-mandelic  acid(a)  and 

p-hydroxybenzoic  acid(b)  treated  for  30  minutes 

with  diazomethane.   Retention  time  is  in  minutes 61 


vx 


12.  Gas  chromatograra  of  an  ethanol  extract  from  peach 

seeds  treated  for  30  minutes  with  diazomethane. 

Retention  time  is  in  minutes 63 

APPENDIX:   Gas  chromatograms  of  standards. 

13.  Gas  chromatograms  of  the  propyl  esters  of  benzoic 

acid(a)  and  mandelic  acid(b).   Retention  time  in 

minutes 76 

14.  Gas  chromatograms  of  the  propyl  esters  of  o-hydroxy- 

benzoic  acid(a)  and  p-hydroxybenzoic  acid(b) .   Reten- 
tion time  in  minutes 77 

15.  Gas  chromatograms  of  the  propyl  ester  of  2, 6-dihydroxy- 

benzoic  acid.   Retention  time  in  minutes 78 

16.  Gas  chromatograms  of  the  propyl  ester  of  2,4-dimethoxy- 

benzoic  acid.   Retention  time  in  minutes 79 

17.  Gas  chromatograms  of  the  propyl  ester  of  o-hydroxy- 

cinnamic  acid.   Retention  time  in  minutes  80 


VI 1 


INTRODUCTION 

The  phenomena  of  seed  dormancy  have  interested  researchers  for  many 
years.   Little  by  little  the  details  are  being  unfolded  in  various  plant 
species.   Seed  dormancy  may  result  from  such  sources  as  mechanical  re- 
striction, immature  embryo,  or  chemical  inhibition. 

Various  chemicals,  e.g.,  thiourea,  have  been  found  which  will  termi- 
nate seed  dormancy,  but  in  many  cases  this  results  in  the  production  of 
abnormal  seedlings  (36,  37,  76,  97) .   However,  reports  in  the  literature 
show  that  abnormalities  may  be  due  to  temperature  (80) . 

The  breakdown  of  amygdalin  in  germinating  peach  seeds  possibly 
presents  a  fruitful  area  for  investigating  seed  dormancy.  Amygdalin  is 
hydrolyzed  to  mandelonitrile  and  glucose  by  prunsin  (104).   Mandelo- 
nitrile  is  further  hydrolyzed  by  emulsin  to  cyanide  and  benzaldehyde 
(104).   It  has  been  shown  previously  that  benzaldehyde  strongly  affects 
growth  (46) .   Phenolic  compounds  have  been  isolated  from  many  plant 
tissues  (39,  82,  92,  93,  94)  and  their  influence  on  certain  biochemical 
systems  within  the  plant  has  been  investigated  (38,  74,  78,  79,  83,  95, 
105) .   Recent  evidence  would  indicate  that  they  are  involved  in  plant 
growth  and  development  (78,  105).   Therefore,  phenolic  compounds  could 
play  a  prominent  role  in  controlling  dormancy  of  peach  seed. 

With  this  knowledge  at  hand,  research  was  undertaken  to  determine 
the  role  of  phenolic  compounds  in  peach  seed  germination.   It  was  recog- 
nized that  this  role  could  be  stimulatory,  inhibitory,  both,  or  neither. 


Also  various  means  of  terminating  dormancy  were  compared  with  regard  to 
their  influence  upon  certain  of  the  phenolic  compounds.   In  order  to 
conduct  this  investigation,  several  new  techniques  were  established  for 
isolating  and  aiding  in  the  identification  of  certain  of  the  phenolic 
compounds . 


REVIEW  OF  LITERATURE 

In  many  plants  the  phenomena  of  seed  dormancy,  regardless  of  cause, 
have  a  survival  benefit.   The  temi  "dormancy"  as  applied  to  viable  seeds 
is  generally  restricted  to  those  which  fail  to  germinate  in  a  reasonable 
length  of  time  when  subjected  to  an  adequate  moisture  supply,  a  temper- 
ature within  the  range  of  18-30°  C  and  the  normal  gaseous  composition  of 
the  atmosphere.   Dormancy  can  be  due  to  various  causes.   It  may  be  due 
to  the  immaturity  of  the  embryo,  impermeability  of  the  seed  coat  to 
water  and/or  gases,  prevention  of  embryo  growth  by  mechanical  restric- 
tions, special  requirements  for  temperature  or  light,  endogenous  factors 
which  inhibit  germination,  age  of  seed,  and,  in  certain  cases,  immaturity 
of  the  embryo.   These  factors  have  been  discussed  in  several  classic 
reviews  on  seed  germination  (16,  17,  24)  and  in  some  excellent  reviews 
in  the  last  several  years  (64,  96,  102,  108).   This  review  will  be  par- 
ticularly concerned  with  endogenous  factors  which  control  seed  dormancy 
since  this  is  the  type  of  dormancy  we  are  dealing  with  in  the  case  of 
peach  seeds  (11,  14,  36). 

Viable  seed  that  fail  to  germinate  when  exposed  to  conditions 
generally  considered  favorable  for  germination  can  be  induced  to  germi- 
nate in  most  cases  by  the  correct  exposure  to  certain  environmental 
factors.   Very  often  the  environmental  cue  for  the  resumption  of  growth 
is  attained  from  climatic  components,  e.g.,  low  temperature  of  a  given 
duration,  alternating  periods  of  moisture  stress,  daylength,  etc.   These 


environmental  components  precondition  tho  socvis,  so  th;\l  gormination 
occurs  when  they  are  supplied  with  adequate  moisture,  a  warm  temperature 
and  atmospheric  gases  of  the  normal  composition.   After-ripening  may  be 
defined  as  physiological  changes  occurring  in  any  part  of  the  seed  which 
enable  the  seed  to  germinate  and  the  seedling  to  grow  normally  (64) . 
The  necessity  for  a  period  of  after-ripening  may  be  due  to  several 
factors.   In  the  case  of  the  immature  embryo,  further  developmental 
changes  may  be  required  before  germination  (115).   In  other  seeds, 
chemical  changes  must  occur  in  the  embryo  before  they  germinate  (64) . 
In  still  others,  chemical  changes  must  occur  in  the  integuments  and/or 
other  tissues  associated  with  the  embryo  (96). 

In  contrast  to  seeds  which  will  not  germinate  until  subjected  to 
certain  environmental  factors  before  being  placed  under  favorable  germi- 
nation conditions  of  moisture,  warm  temperature  and  atmospheric  gases, 
some  seeds  will  gem;inate  readily  without  preconditioning.   However,  it 
is  interesting  to  note  that  the  latter  will  also  lose  their  readi.ioss  to 
germinate  if  subjected  to  stress  conditions  much  the  same  as  seeds  that 
require  factors  to  terminate  dormancy  (65,  98).   This  phenomenon  is 
referred  to  as  secondary  dormancy.   Secondary  dormancy  can  be  induced 
in  certain  seeds  by  subjection  to  high  or  low  temperatures,  high  COg 
levels  or  continuous  light. 

Germination  Inhibitors 
A  large  number  of  substances  are  capable  of  inhibiting  germination. 
Those  compounds  which  are  generally  toxic  to  living  organisms  will  also, 
at  toxic  concentrations,  prevent  germination  simply  by  killing  the  seed. 
However,  these  compounds  have  been  of  little  value  in  determining  the 


underlying  causes  of  dormancy.   Compounds  which  prevent  germination 
without  killing  the  seeds  are  by  far  the  more  valuable  in  determining 
the  mechanism  of  dormancy. 

The  simplest  type  of  inhibition  is  caused  by  non-toxic  chemicals 
in  high  concentration  and  this  has  been  shown  to  be  due  to  high  osmotic 
pressures  (16).   These  high  osmotic  conditions  may  be  obtained  by  in- 
organic salts,  sugars,  or  other  substances.  An  example  of  such  inhibi- 
tion is  the  inability  of  seed  within  some  mature  fruit  to  germinate. 
The  large  quantities  of  soluble  solids  present  in  the  flesh  create  high 
osmotic  conditions  around  the  seed  and  prevent  germination.   The  thresh- 
old of  osmotic  pressure  which  prevents  germination  differs  with  the 
species.  As  soon  as  the  seeds  are  removed  from  the  high  osmotic  environ- 
ment and  placed  in  water,  they  will  germinate  (64). 

A  more  complex  type  of  inhibition  is  that  caused  by  substances 
which  are  known  to  interfere  with  certain  metabolic  pathways.   Since 
germination  cannot  occur  without  active  metabolism,  any  substance  that 
would  alter  normal  metabolism,  would  probably  alter  the  germination 
pattern  of  seed.   Compounds  such  as  cyanide  (4),  dinitrophenol  (68), 
azide  (68),  fluoride  (24),  hydroxylamine  (24),  and  others  (4,  24)  which 
are  respiratory  inhibitors,  have  inhibited  germination  at  concentrations 
approximating  those  which  inhibit  metabolic  processes.   Therefore,  it 
seems  that  inhibition  of  germination  by  this  class  of  compounds  is  a 
result  of  their  effect  on  metabolism  (64),  but  only  in  the  case  of 
cyanide  (4)  have  these  chemicals  been  implicated  in  natural  seed 
dormancy . 

Another  class  of  compounds  that  inhibited  germination  are  auxins 
(54).   An  example  of  such  a  case  would  be  the  use  of  low  concentrations 


of  2,4-dichlorophenoxyacetic  acid  (2,4-D)  to  inhibit  germination.  Al- 
though auxins  have  been  shown  to  be  necessary  for  growth  of  isolated 
embryonic  tissues  and  to  increase  at  the  time  of  germination  or  shortly 
before  (32,  51);  however,  there  has  been  no  convincing  evidence  that 
they  are  directly  involved  in  the  dormancy  mechanism  (25,  42,  55).   Only 
in  a  few  instances  (14,  25,  37)  have  auxins  been  shown  to  stimulate 
germination  and  these  instances  were  cases  where  the  dormant  state  of 
the  seed  was  altered  by  pretreatments.   This  is  in  contrast  to  the  in- 
fluence of  auxins  on  fruit  growth  and  development  (59,  60,  61). 

On  the  other  hand,  growth  inhibitors  are  of  general  occurrence  in 
dormant  seeds,  and  there  is  abundant  evidence  for  their  involvement  in 
the  physiological  mechanisms  of  dormancy. 

Evidence  for  the  involvement  of  growth  inhibitors  in  seed  dormancy 
is  the  demonstration  that  they  are  often  present  in  dormant  seeds  and 
that  the  application  of  such  materials  can  impose  dormancy  on  seeds  in 
certain  cases.   Nutrile  (70)  was  the  first  to  show  this.   He  applied 
coumarin  to  lettuce  seeds  and  showed  they  required  preconditioning  again 
before  they  would  germinate.   These  experiments  were  substantiated  by 
Evenari  (25) . 

Many  phenolic  compounds  have  been  found  to  inhibit  germination. 
These  have  a  widespread  occurrence  and  distribution  in  plants  and 
fruits  and  thus  it  is  thought  that  tliey  may  occur  as  natural  germination 
inhibitors  (92).   It  was  suggested  by  van  Sumere  (92,  93)  that  the 
phenolic  compounds  may  be  classified  along  with  coumarins  as  dormancy 
inducing  agents.   Coumarin,  ferulic  acid  and  other  phenolic  compounds 
have  been  found  to  occur  in  the  skin  as  well  as  the  cortical  tissue  of 


potato  and  Hemberg  (42,  43)  suggested  that  the  rest  period  of  the 
potato  may  be  due  to  an  abundance  of  growth  inhibiting  substances  in  the 
periderm.   Koves  and  Varga  (53)  surveyed  the  dry  fruits  of  several 
species  with  reference  to  inhibitory  substances.   Inhibitors  were  found 
in  all  fruits  and  those  that  have  been  chemically  identified  were 
phenolic  acids  or  their  depsides  and  polydepsides.   Numerous  benzoic  and 
cinnamic  acid  derivatives  such  as  high  molecular  weight  tannic  acids, 
protocatechuic,  caffeic  and  chlorogenic,  ferulic,  p-coumaric  and  p- 
oxybenzoic  acids  had  a  lesser  activity  (53) .   Salicylic  acid,  and  in 
some  cases  unidentified  cinnamic  acid  derivatives,  had  strong  activity. 
Most  of  these  inhibitors  were  washed  out  or  destroyed  as  the  fruit  re- 
mained on  the  tree  for  a  prolonged  period  of  time. 

Although  the  phenolic  substances  range  in  structure  from  simple 
phenols  to  complex  compounds,  such  as  lignin,  it  seems  that  the  most 
important  phenols,  insofar  as  growth  regulation  is  concerned,  are  the 
•   monocyclic  aromatic  compounds  (82) .   In  recent  years  attention  has  been 
given  to  the  role  of  hydroxycinnamic  and  hydroxybenzoic  acids  in  plant 
growth  and  development.   The  biosynthesis  of  these  acids  in  higher 
plants  has  received  renewed  attention  recently  (22,  82,  94).   The  major 
pathway  for  the  formation  of  these  compounds  undoubtedly  involves 
phenylalanine  via  shikimic  acid.   The  inter-conversion  of  the  hydroxy- 
benzoic  acids  gave  rise  to  many  derivatives  (39,  45,  50).   p-Hydroxy- 
benzoic  acid  and  caffeic  acid  have  been  isolated  from  plants  and  shown 
to  be  active  as  growth  regulators  (103,  105).   Other  phenolic  compounds 
that  have  shown  lesser  activity  include  salicylic,  gallic,  ferulic, 
caffeic,  vanillic,  protocatechuic,  chlorogenic,  p-oxybenzoic,  and  pr 
coumaric  acids  (53,  64,  92). 


Another  possible  function  of  the  phenolic  acids  in  seed  genr.::  na- 
tion may  be  their  role  in  the  synthesis  and  degradation  of  indoleacetic 
acid  (lAA)  (74,  S3).   Pilet  (78,  79)  reported  that  the  mono-hydroxyben- 
zoic  acids  increased  the  iii  vitro  destruction  of  lAA.   Of  these,  p-hy- 
droxybenzoic  acid  had  the  greatest  effects,  causing  stimulatory  growth 
of  stem  sections  at  low  concentrations  and  inhibiting  elongation  at  higher 
concentrations.   Many  other  naturally  occurring  phenolic  acids  were 
studied  by  Zenk  and  Muller  (116)  as  to  their  influence  on  the  destruc- 
tion of  exogenously  applied  lAA.   By  growth  experiments  with  IAA-l--'-'^C 
and  determination  of  the  -^^CO^   evolved,  it  was  shown  that  monophenols 
stimulate  the  decarboxylation  of  lAA  under  conditions  where  growth  was 
suppressed  (95).   When  Mn++  was  present,  this  decarboxylation  was  enhanced. 
To  add  to  the  complexity  of  the  relation  of  phenols  to  growth,  Gordon  and 
Paleg  (38)  have  shown  that  phenols,  under  conditions  leading  to  their 
oxidation,  reacted  with  tryptophan  to  form  lAA . 

Probably  the  most  active  and  most  widely  used  gemiination  inhibitor 
is  coumarin.   Coumarin  is  characterized  by  an  aromatic  ring  and  an  un- 
saturated lactone  structure.   No  single  group  in  the  coumarin  molecule 
has  been  shown  to  be  the  cause  of  its  inhibitory  action.   Reduction  of 
the  unsaturated  lactone  ring  or  substitution  by  hydroxyl,  methyl,  nitro, 
chloro  and  other  groups  in  the  ring  system  reduced  the  inhibitory  activi- 
ty (63,  70). 

The  flavonoid,  naringenin,  which  has  been  isolated  from  peach  buds 
by  Hendershott  and  Walker  (44),  has  an  action  similar  to  coumarin  on 
lettuce  seeds.   Phillips  (77)  demonstrated  that  it  will  impose  dormancy 
on  lettuce  seeds  that  can  be  reversed  by  light  or  by  aj^'plication  of 
gibberellins. 


Recently,  several  new  compounds  have  been  isolated  which  exhibited 
growth  regulatory  properties.   One  group  of  compounds  which  show  a 
marked  elongation  effect  on  rice  and  lettuce  is  related  to  helmintho- 
sporol  (84).   'Dormin',  a  terpenoid  compound  has  shown  a  marked  influence 
on  the  regulation  of  bud  growth  in  some  woody  plants.   It  appears  that 
the  structure  of  'dormin'  and  'abscisin  II'  are  the  same  (15,  71). 
Eagles  and  Wareing  showed  that  an  inhibitor  v  dormin')  concentrated  from 
an  extract  of  birch  leaves  could  completely  arrest  apical  growth  when 
applied  to  the  leaves  of  seedlings.   Evidence  was  also  found  for  high 
levels  of  'dormin'  in  birch  leaves  under  short  days,  with  the  emergence 
from  dormancy  presumably  resulting  from  an  interaction  between  'dormin' 
and  growth-promoting  substances  (20,  21).   A  recent  finding  in  the  study 
of  dormancy  regulation  in  peach  seeds  was  that  an  inV.ibitor  isolated 
from  the  seed  integuments  chromatographed  identical  to  'dormin'  (57,  58). 
However,  Daley  (18)  has  shown  that  several  inhibitors  are  present  in 
peach  seed  cotyledons  and  that  several  chromatographed  in  the  zone 
labeled  'dormin'  by  Lipe  and  Crane  (58). 

Bennet-Clark  and  Kef ford  (8)  first  described  a  complex  o^  inhibitory 
substances  that  appeared  on  paper  chromatograms  of  plant  extracts  running 
ahead  of  lAA  when  developed  in  a  solvent  of  i sopropanol/ammoni a/water . 
This  inhibitory  area,  possessing  R^  values  of  0.6  to  0.8,  has  been  clas- 
sified as  the  beta-inhibitor  complex  (48,  49).   This  inhibitory  complex 
has  been  shown  to  be  widespread  in  plants  and  has  been  related  to  both 
dormancy  and  correlative  growth.   For  instance,  Varga  (100)  has  reported 
that  the  juice  of  lemons,  strawberries  and  apricots  contains  inhibitors 
which  appear  to  correspond  to  the  beta-in'iibitor  complex.   Lipe  (57) 
found  that  the  inhibitors  in  'Lovell'  peach  seeds  are  similar  to  the 


beta-inhibitor  complex.   Elution  and  rechroraatography  of  the  beta-in- 
hibitor-complex has  yielded  both  acidic  and  neutral  substances  (56). 
Recently,  the  beta-inhibitor-complex  concentrated  as  acidic  compounds 
from  extracts  of  dormant  maple  buds  was  shown  to  be  a  complex  of  phenolic 
substances  (86).   It  includes  coumarin  and  salicylic,  ferulic,  p-  and 
o-coumaric,  m-oxybenzoic  acid  (93,  108)  and  'dormin'  (15). 

Many  of  the  previously  mentioned  phenolic  compounds  have  been  found 
to  occur  in  various  plant  tissues,  especially  in  fruits  (64,  108).   ?or 
example,  Varga  (100)  and  Koves  and  Varga  (53)  have  shown  that  many 
phenolic  compounds  such  as  salicylic,  ferulic,  caffeic,  chlorogenic, 
p-coumaric,  protocatechuic  and  p-oxybenzoic  acids  are  present  in  fruits. 
Along  these  same  lines,  it  is  interesting  to  note  that  peach  juice  is 
injurious  to  peach  seed  germination  (85),   It  has  been  suggested  that 
the  inhibition  of  seed  germination  in  fruit  was  generally  not  due  to  a 
single  compound  but  was  due  to  the  synergistic  action  of  several  com- 
pounds that  might  be  present  within  the  fruit  or  the  seed  itself  (lOS). 

The  activity  of  endogenous  inhibitors  may  not  be  solely  directed 
at  the  prevention  of  germination  per  se,  but  may  also  influence  some  of 
the  other  factor's  controlling  dormancy.   Black  and  Warding  (10)  reported 
that  the  removal  of  the  embryo  from  intacx  seed  reduced  the  light  re- 
quirement for  germination  of  seed  of  the  Betula  spp.   They  also  suggested 
that  the  inhibitor  in  the  seed  coat  increased  the  oxygen  requirement  of 
the  embryo.   Wareing  and  Foda  (109,  110)  found  that  leaching  the  embryo 
of  Xanthium  seed  removed  the  inhibitor  and  that  maintaining  the  seed  in 
a  pure  oxygen  atmosphere  cause.'  a  reduction  in  the  inhibitor  within  30 
hours.   Elliott  and  Leopold  (23)  showed  that  the  inhibitors  from  Avena 
seeds  inhibited  alpha-amylase  activity. 


11 

Villiers  and  Wareing  (106,  107,  lOS)  reported  that  chilling  Fraxinus 
excelsior  seeds  had  no  effect  on  the  activity  of  the  inhibitor  but  that 
dormancy  was  overcome  during  chilling  by  production  of  a  growth  stirau- 
lator  in  the  embryo  tissues.   Flemion  and  De  Silva  (31)  also  demo.   rated 
with  peach  seeds  that  with  the  bioassay  they  were  using  they  coul^  I'ind 
little  correlation  between  growth  inhibitors  and  the  termination  of 
dormancy. 

The  promotive  effects  of  oxygen  on  germination  of  seeds  and  the 
parallel  effects  of  light  led  Paech  (73)  to  suggest  ^r.a'c  dorr.iancy  was 
regulated  by  phenolic  substances  in  the  seed  coat.   The  oxidative  activi- 
ties of  phenolic  compounds  could  trap  oxygen,  preventing  its  entry  into 
the  seed.   The  action  of  the  phenolics  could  be  blocked  by  oxygen  or 
light  through  the  photooxidation  of  the  phenolics  themselves. 

The  effects  of  gibberellin  in  breaking  the  dormancy  of  many  seeds 
indicated  that  it  could  possibly  be  the  stimulator  of  growth  if  it  were 
formed  during  the  period  in  which  donriancy  was  broken  (35)  .   Murakami 
(66)  has  shown  gibberellin  to  be  present  in  a  wide  diversity  of  seeds. 
As  seeds  of  Avena  f atua  emerged  from  dormancy  a  growth-promoting  sub- 
stance suggestive  of  gibberellin  was  formed  (67).   These  seeds  were  also 
brought  out  of  dormancy  if  soaked  in  gibberellin  solutic.-s.   Kahn  (47) 
reported  gibberellin  overcame  dormancy  of  lettuce  seed  regardless  of 
whether  it  was  imposed  by  hi^...  temperature,  by  far-red  light,  or  by 
osmotic  solutions. 

Recently,  a  mode  of  action  was  suggested  for  gibberellic  acid  (99). 
It  has  been  reported  that  gibberellic  acid  stimulated  alpha-araylase 
production  in  the  aleurone  layer  of    _  coat  of  cereals  which  in  turn 
increased  the  rate  of  starch  hydrolysis.   Th '  stimulation  of  alpha- 


12 

amylase  was  believed  to  be  due  to  the  direct  influence  of  gibberellic 
acid  on  messenger  RNA  polymerase,  an  enzyme  that  is  involved  in  producing 
the  alpha-amylase  enzyme  (101). 

Physiology  of  Seed  Germination 

The  actual  germination  of  a  seed  reflects  the  cumulative  effect  of 
interactions  between  many  factors  both  external  and  internal.   These 
factors  range  from  hereditary  traits  to  environmental  influences  during 
development  and  storage.   For  simplicity  of  this  review,  the  influencing 
factors  will  be  grouped  into  external  and  internal  factors.   Excellent 
reviews  have  been  published  on  the  physiology  of  seed  germination  (16, 
17,  24,  64,  96,  102). 
EXTERNAL  FACTORS: 

Among  the  external  factors  required  for  seed  germination  are  an 
adequate  supply  of  moisture,  a  suitable  temperature  range  and  composi- 
tion of  gases  in  the  atmosphere,  light,  and  sometimes  certain  chemicals. 
The  requirement  for  these  conditions  varies  according  to  the  species 
and  variety  and  is  determined  by  hereditary  factors  and  by  the  condi- 
tions which  prevailed  during  seed  formation.   Frequently  it  appears 
there  is  a  correlation  between  the  environmental  requirement  for  germi- 
nation and  the  ecological  conditions  occurring  in  the  habitat  of  the 
plant  and  the  seeds  (64). 

Water;   One  of  the  first  processes  which  must  occur  for  germination 
of  dry  seeds  is  the  uptake  of  water.   The  extent  of  this  uptake  is  deter- 
mined by  (a)  the  composition  of  the  seed  coat,  (b)  the  permeability  of 
the  seed  coat  to  water,  (c)  the  availability  of  water  (liquid  or  gaseous) 
in  the  environment,  and  (d)  soluble  solids  (64). 


13 

Gases:   Germination,  a  process  of  living  cells,  requires  an  expendi- 
ture of  energy.   Energy  requiring  processes  in  living  cells  are  usually 
supported  by  processes  of  oxidation,  in  the  presence  or  absence  of 
oxygen.   These  processes,  respiration  and  fermentation,  involve  an  ex- 
change of  gases,  an  output  of  carbon  dioxide  in  both  cases  and  the  uptake 
of  oxygen  for  respiration.   Consequently,  seed  germination  is  markedly 
affected  by  the  composition  of  the  ambient  atmosphere  (64). 

The  partial  pressure  of  oxygen  in  the  atmosphere  can  be  reduced 
considerably  without  greatly  interfering  with  the  rate  of  respiration. 
In  fact,  the  seeds  of  some  water  plants  germinate  better  under  lower 
oxygen  tensions  than  in  air.   Seeds  of  many  terrestrial  plants  can 
germinate  under  water  where  the  concentration  of  oxygen  often  corresponds 
to  a  partial  pressure  of  oxygen  very  much  less  than  that  of  the  atmos- 
phere (65). 

In  the  early  stages  of  germination  of  seeds  of  species  such  as 
Pi  sum  sativum,  respiration  is  largely  or  almost  totally  anaerobic  be- 
cause of  the  relative  impermeability  of  even  hydrated  seeds  of  such 
species  to  oxygen.   As  soon  as  the  seed  coats  are  ruptured,  aerobic 
respiration  replaces  the  anaerobic  oxidative  processes  (65). 

The  influence  of  carbon  dioxide  concentration  is  usually  the  re- 
verse of  that  of  oxygen.   Many  seeds  fail  to  germinate  when  the  carbon 
dioxide  tension  is  high.   There  seems  to  be  a  minimal  requirement  for 
carbon  dioxide  in  order  for  germination  to  occur  in  Atriplex  halimus 
and  Salsola  as  well  as  lettuce  whereas  some  other  species  of  Atriplex 
are  resistant  to  high  levels  of  carbon  dioxide  as  long  as  the  oxygen 
concentration  is  kept  constant  (7). 


14 

Temperature:   Different  kinds  of  seeds  have  specific  ranges  of 
temperature  within  which  they  germinate.   Very  low  and  very  high  temper- 
atures tend  to  prevent  the  germination  of  all  seeds.   A  rise  in  temper- 
ature does  not  necessarily  cause  an  increase  in  either  the  rate  or  the 
percentage  of  germination.   Therefore,  germination  is  not  characterized 
by  a  simple  temperature  coefficient  (107), 

Light;   Among  cultivated  and  non-cultivated  plants  there  is  con- 
siderable evidence  for  light  as  a  factor  influencing  germination.   For 
example,  lettuce,  tobacco  and  many  crucifers  require  light  to  germinate 
(33,  75,  77,  96).   Seeds  may  be  divided  into  those  which  germinate  only 
in  the  dark,  those  which  germinate  only  in  continuous  light,  those  which 
germinate  after  being  given  a  brief  illumination  and  those  which  are  in- 
different to  the  presence  or  absence  of  light  during  germination  (96), 

Studies  have  shown  that  different  spectral  zones  affected  germina- 
tion differently.   Light  of  wavelength  less  than  2900  A°  has  inhibited 
germination  of  all  seeds  tested  (33).   Between  2900  A°  and  4000  A°  the 
germination  of  some  seeds  is  inhibited  (33).   In  the  visible  range, 
4000  AO  -  7000  A°,  it  was  shown  that  light  in  the  range  of  5600  A°  - 
7000  A   and  especially  red  light,  usually  promoted  germination  (64,  75). 
If  seeds  exposed  to  red  light  were  followed  promptly  by  an  exposure  to 
far-red  light  (7350  A  ),  germination  was  partially  or  totally  inhibited 
(65),   An  excellent  review  of  the  phytochrome  system  and  its  relation  to 
germination  has  been  made  by  Siegelman  and  Butler  (87) , 
INTERNAL  FACTORS: 

The  changes  which  take  place  during  the  germination  process  are  to 
a  certain  extent  determined  by  the  type  of  seed  and  its  chemical  compo- 
sition.  The  composition  is  in  turn  influenced  by  environmental  condi- 


15 

tions  present  durin^  seed  formation  as  well  as  the  hereditary  factors  of 
the  species  involved. 

Once  the  germination  process  is  initiated,  there  is  mobilization 
and  translocation  of  compounds  from  storage  organs  to  the  actively  grow- 
ing meristematic  tissues  (64).   Studies  with  tree  peony  embryo  and  endo- 
sperm tissues  reveal  that  biochemical  changes  which  take  place  with 
germination  are  different  for  tissue  af xer-ripened  at  5°  C  from  those 
that  are  kept  in  the  greenhouse  at  21°  -  30°  C.   The  latter  can  be  con- 
si-dered  dormant  tissue  (5,  6,  27).   Major  biochemical  changes  in  organic 
acids,  amino  acids  and  sugars  were  noted.   These  typify  what  has  been 
found  with  many  seeds.   A  good  discussion  of  this  aspect  of  seed  germi- 
nation can  be  found  in  the  book  by  Mayer  and  Poljakof f-Mayber  (64). 

Since  phosphates  play  an  extremely  important  role  in  a  variety  of 
reactions  of  seeds,  some  discussion  of  the  metabolism  of  phosphorus- 
containing  compounds  would  be  in  order.   The  phosphates  are  required  for 
the  formation  of  nucleic  acids  which  in  turn  are  intimately  concerned 
with  protein  synthesis  and  the  hereditary  constitution  of  plant  cells. 
They  are  components  of  many  other  .;ey  compounds  including  phospholipids 
which  function  in  controlling  surface  p:.-operties  and  permeability  of 
cell  membranes.   Also,  the  various  phosphorylated  sugars  and  nucleotides 
are  very  closely  linked  with  the  energy-producing  processes  in  the  cell 
during  germination  (64). 

Phosphoi-us  pi'imar^ly  appears  in  seeds  as  organic  phosphorus,  v.'ith 
very  little  being  present  as  inorganic  orthophosphate.   Phytin  is  fre- 
quently present  and  may  constitute  up  to  S0%  of  the  total  phosphorus 
content  of   ..e  seed  (64).   Since  most  of  the  phosphate  :.3  present  in  the 
bovind  foi^,  o.'jaophosphate  may  be  the  limiting  factor  in  certain  of  the 


16 

reactions  of  the  germination  process.   With  this  in  mind  the  large  amount 
of  phytin  present  may  be  considered  as  a  reserve  of  inorganic  phosphate 
which  can  be  liberated  t.   germinatio.  proceeds  by  phosphatase  activity 
or  more  specii'  cally  phytase  activity.   Phytir,  is  also  present  in  the 
embryo,  disappearing  rapidly  during  germination.   The  phosphorus  is 
replenished  by  transport  from  the  endosperm  to  the  embryo  during  germi- 
nation (2) .   The  rate  of  phytin  hydrolysis  and  subsequent  transport  of 
phosphorus  to  the  growing  sites  pr^^sents  a  possible  limiting  facxor  for 
the  race  of  germination  and  subsequent  seedling  development. 

Recently,  reports  of  myo-inositol  acting  as  a  growth  factor  in 
plant  tissue  have  .^een  made  (3).   This  is  of  particular  interest  in  re- 
gard to  phytin  since  it  is  the  salt  of  phytic  acid  or  inositol  .exaphos- 
phate.   The  "neutral  fraction"  of  coconut  milk  contains  myo-inositol 
along  with  scyllo-inositol  and  sorbitol,  but  myo-inositol  was  regarded 
as  the  most  important,  as  far  as  activity  in  grov/th-stimulation  was 
concerned.   Myo-inositol  may  stiir.ulate  the  growth  of  seedlings  and  the 
germination  of  certain  seeds.   In  addition,  myo-i..ositol  has  stimulated 
growth  of  callus  in  cultures  of  elm  (Ulmus  campestris),  Norway  spi'uce 
(Picea  abies) ,  tobacco  (Xicotiana  tabacium) ,  Vinca  rosea,  and  carrot 
(normal  and  tumorous)  tissues  (3) . 

Studies  on  the  nucleotide  content  of  seeds  during  germination  dis- 
closed that  the  ATP  content  rose  initially  during  imbibition  and  then 
decreased  (54).   The  content  of  nicotinamide  adenine  dinucleotide  (NAD) 
and  nicotinamide  auonine  dinucleotide  phosphate  (NADP)  in  seeds  and 
sec "lings  rises  in  all  cases  during  germination.   During  the  early 
stages  of  germination  there  was  a  net  increase  in  the  RNA  of  peanut 
cotyledons  (102).   Some  of  this  RNA  synthesis  was  thought  to  be  associ- 


17 

ated  with  increased  numbers  of  mitochondria,  or  in  mitochondrial  function, 
and  the  ability  of  the  cells  to  form  chloroplasts.   However,  a  part  of 
the  increase  was  postulated  to  be  associated  with  the  appearance  of 
enzymes  required  for  metabolism  of  the  storage  materials  in  the  peanut. 
A  peak  was  reached  in  about  8  days  followed  by  a  more  or  less  parallel 
decline  in  RNA  content  and  enzymic  activities.   These  declines  were  con- 
comitant with  an  increase  in  RNase  activity. 

As  germination  proceeded  there  was  a  sharp  rise  in  carbon  dioxide 
evolution  and  a  gradual  rise  in  oxygen  uptake  of  pea  seeds.   However, 
after  24  hours  there  was  a  sharp  decrease  in  the  respiratory  quotient 
(SS) .   This  same  pattern  was  observed  for  wheat  for  both  carbon  dioxide 
evolution  and  oxygen  uptake  (67). 

The  energy  pathways  in  seeds  have  been  studied  in  some  detail. 
Both  glycolysis  and  the  organic  acid  metabolism  have  been  observed  in 
germinating  seeds  (68,  91).   Evidence  for  the  presence  of  the  pentose 
phosphate-shunt  pattern  of  metabolism  has  been  found  in  mung  beans  (13). 
In  seeds  containing  large  quantities  of  fats  and  oils,  the  tricarboxylic 
acid  pathway  of  metabolism  may  be  partially  replaced  by  the  glyoxylate 
pathway  of  metabolism  which  is  a  modified  form  of  the  tricarboxylic  acid 
cycle  (52,  62,  68,  114).   The  glyoxylate  pathway  functions  in  the  con- 
version of  fats  to  sugars. 

Physiology  of  Peach  Seed  Germination 
Peach  seeds  are  characterized  by  a  requirement  for  a  period  of  low 
temperature  for  natural  termination  of  dormancy.   Chemicals  have  been 
found  that  will  induce  germination  of  dormant  seed.   These  factors  and 
others  are  discussed  below. 


18 

ENVIRONMENTAL  FACTORS: 

The  optimum  temperature  of  5°  C  with  a  range  of  5-10  C  for  60-90 
days  has  been  found  best  suited  for  the  termination  of  dormancy  of  peach 
seeds  (12,  16,  19,  29).   The  duration  needed  varies  with  varieties. 
Some  varieties  require  fewer  hours  of  chilling  to  break  dormancy  than  do 
others  (12).   If  a  warm  temperature  treatment  immediately  follows  ex- 
posure of  seeds  to  low  temperatures,  the  growth  capacity  of  the  seeds 
will  be  greatly  reduced  (14,  80).   The  reduction  in  growth  capacity  can 
subsequently  be  restored  by  subjecting  the  seeds  to  additional  exposures 
to  low  temperatures. 

Observations  indicate  that  peach  seeds  are  indifferent  or  day- 
neutral  toward  the  influence  of  light  on  germination  (R.  H.  Biggs,  Un- 
published data). 

It  has  been  observed  that  the  amount  of  free  water  present  during 
germination  will  influence  the  process.   If  seeds  were  allowed  to  be  in 
contact  with  free  water,  as  in  a  petri  dish,  they  generally  became 
bloated  as  a  result  of  too  rapid  an  uptake  of  water.   However,  if  the 
seeds  were  placed  in  moist  vermiculite,  they  were  not  bloated  (36). 
This  has  been  shown  to  occur  with  other  types  of  seeds,  particularly  the 
legumes  (64) . 
CHEIIICAL  FACTORS: 

External:   Tukey  and  Carlson  (97)  showed  that  applications  of 
thiourea  to  dormant  'Lovell'  peach  seeds  induced  germination.   Evidence 
obtained  by  Garrard  (36)  indicated  that  both  the  sulfhydryl  and  the 
imido  group  are  requisite  to  the  activity  of  thiourea.   Mercaptoethanol, 
mercaptoethylamine,  and  urea  were  not  effective  either  alone  or  in  com- 
bination in  promoting  germination  of  'Okinawa'  peach  seed  (76).   The 


19 

induction  of  germination  of  dormant  peach  seeds  has  resulted  in  the 
formation  of  abnormal  seedlings  when  induction  was  by  means  of  thiourea 
or  seed  coat  excision  (2S,  36,  37,  97).   However,  it  has  been  recently 
sho\vn  that  the  temperature  during  germina"c;.on  plays  a  major  role  in  the 
development  of  abnormalities  in  the  seedlings  (SO)  and  that  warm  temper- 
atures during  treatments  with  chemicals  or  by  embryo  excision  was  re- 
sponsible for  increasing  the  severity  of  abnormalities  (9)  and  not  the 
treatments.   Thus,  it  is  possible  that  two  mechanisms  are  functioning 
within  the  embryo;  one  that  breaks  dormancy  and  initiates  germination, 
and  another  which  controls  the  development  of  the  epicotyl. 

Gibberellic  acid  has  been  found  to  induce  the  germination  of  dorraant 
peach  seeds  but  by  a  different  mode  of  action  than  that  of  thiourea  (76). 
Gibberellic  acid  can  decrease,  to  some  extent,  the  occurrence  of  leaf 
anomalies  on  peach  seedlings  and  stimulate  stem  elongation  (30).   It  is 
possible  that  gibberellic  acid  has  a  modifying  influence  on  both  germi- 
nation and  epicotyl  development. 

Internal :   Pollock  and  Olney  (72,  81)  have  studied  extensively  -.le 
i-est  period  of  seeds  of  sour  cherry,  Prunus  cerasus,  with  respect  to 
metabolic  changes  and  growth.   Their  results  showed  that  during  low 
temperature  treatment  to  terminate  donaancy,  nitrogen  and  phosphorus 
are  translocated  from  the  cotyledons  to  the  embryonic  axis  of  the  embryo. 
The  rate  of  translocation  of  nitrogen  was  equal  to  the  rate  of  cell 
division;  therefore,  the  nitrogen  content  per  call  seemed  to  remain   '  '' v 
constant.   The  rate  .^f  translocation  of  phosphorus  was  in  excess  of 
cell  division  and  the  phosphorus  concentration  in  the  cells  increased. 
The  experiments  indicated  that  the  translocated  phorphorus  was  incorpo- 
rated into  all  phosphate  compounds  in  the  cells.   In  fully  'curgid  seeds 


20 


kept  at  warm  temperatures,  phosphorus  tended  to  accumulate  as  inorganic 
phosphate  rather  than  in  organic  metabolites.   These  authors  suggested 
that  the  rest  period  may  be  associated  with  a  block  in  the  phosphate 
metabolism  of  the  cells.   This  hr -•  not  "aen  substantiated  at  th :  present 
time. 

Pollock  (SO),  using  'Elberta'  peach  seed,  suggests  that  the  causal 
agent  of  the  dwarfing  effect  in  seedlings  is  independent  of  the  growth 
inhibitor  content  of  the  seed.   The  physiological  and  anatomical  a._  .octs 
of  dwarfing  suggested  a  control  by  a  self-duplicating  system  loci:.lized 
in  a  limited  region  of  the  apical  meristem  and  transmitted  only  by  cell 
division.   This  system  was  temperature  sensitive  during  the  time  between 
the  first  visible  root  growth  and  shoot  elongation. 

Investigations  have  been  made  into  the  effect  of  the  degradation 
products  of  the  glucoside  amygdalin  within  the  seed.   Upon  imbibition, 
mandelonitrile  could  be  detected  in  the  seeds  (1);  it  was  assumed  to 
have  arisen  from  the  hydrolysis  of  amygdalin  to  mandelonitrile  and 
glucose  by  prunsin.   The  mande_onitrile  was  further  hydrolyzed  to 
cyanide  and  benzaldehyde  (104),  presumably  by  emulsin. 

The  presence  of  benzaldehyde  during  imbibition,  as  a  result  of  the 
degradation  of  ainygdalin,  suggested  that  possibly  .^enzoic  acid  and  some 
cf  its  derivatives  m.\y  be  formed  (25,  104).   An  alternative  to  this 
pathway  is  that  in  which  man^lelic  acid,  formed  from  r..-  ndelonitrile, 
undergoes  enzymatic  conversion  to  benzoic  acids  (40,  41,  S9). 

This  study  will  be  concerned  with  the  changes  in  phenolic  compounds 
during  the  breaking  of  iorraancy  and  subsequent  germination  of  the  seed. 


MATERIALS  AND  METIiODS 


All  seeds  were  obtained  from  the  I960  and  1966  crops  of  Prunuf 


persica  cv,  'Okinawa'.   This  m:.'^orial  was  chosen  for  several  reasons. 
Principally,  the  seeds  are  relatively  homozygous  in  respect  to  the  chill- 
ing requirement  to  temiinate  seed  dormancy  (9),  the  seeds  require  a  rel- 
atively shoi-t  period  of  low  temperature  stratification  to  overcome  the 
dormant  state  (9),  and  when  the  embryos  are  excised  they  germinate  readi- 
ly without  any  apparent  abnormalities  if  the  temperature  range  during 
germination  is  18-25°  C  (76). 

The  seeds  were  removed  from  the  endocarp  just  prior  to  each  experi- 
ment and  allowed  to  imbibe  water  from  moistened  vermiculixe.   Depending 
upon  the  nature  of  the  expei'iment,  the  time  in  moistened  vermiculite 
varied.   The  seeds  were  planted  in  seed  flats  containing  a  2:1  mixture 
of  perlite:  vermiculite.   Techniques  for  each  experiment  will  be  dis- 
cussed separately. 

Tests  for  interaction  of  thiourea  and  seed  coat  excision  on  germi- 
nation:  In  order  to  determine  the  most  effective  thiourea  concentration 
to  promote  the  greatest  amount  of  gerrainatioii  with  the  least  amount  of 
anomolous  growth,  a  range  of  concentrations  was  tested.   This  was  as 
follows:  0.0,  1.0,  3  x  lO"-*-,  10"   and  10""^  M  thiourea.   The  seeds  were 
kept  in  a  moist  medium  for  42  hours  and  then  followed  by  6  hours'  soaking 
in  the  respective  thiourea  concentrations.   Before  imbibition,  the  seeds 
were  surface  sterilized  for  3  minutes  with  a  1,000  ppm  raerthiolate  in 


21 


22 

25%  ethanol:  water  solution.   After  the  soaking  period,  the  seeds  were 
blotted  and  planted  in  flats  and  kept  in  the  dark  at  20°  C  for  16  days 
before  being  placed  in  a  greenhouse.   Each  treatment  was  replicated  3 
times  with  40  seeds  per  replication. 

A  second  experiment  was  designed  to  determine  if  any  interaction 
existed  between  thiourea  and  the  seed  coat  on  the  degree  of  anomalous 
development  of  the  subsequent  seedlings.   Thiourea  concentrations  of 
0.0,  10"  ,  and  3  x  10~  M  were  applied  to  intact  seeds  and  to  excised 
embryos  after  42  hours  imbibition.   After  a  6  hour  treatment  period, 
the  seeds  were  removed  from  the  solutions,  blotted,  and  planted  in  seed 
flats.   At  3  time  intervals  of  24,  48  and  72  hours,  seed  coats  were  re- 
moved from  samples  of  intact  seeds  treated  with  thiourea  and  the  excised 
embryos  replanted.   All  treatments  were  kept  in  the  dark  at  20°  C  for  10 
days,  except  for  brief  period  of  examination.   After  10  days  the  flats 
were  moved  to  the  greenhouse.   Each  treatment  was  replicated  3  times  with 

9  seeds  per  replication. 

Testing  chemicals  for  modification  of  germination  of  peach  seeds: 
Benzaldehyde,  benzoic  acid,  cyanide,  p-hydroxybenzoic  acid  and  mandelo- 
nitrile  in  a  series  of  concentrations  were  tested  on  seed  germination. 
Because  of  volatility  and  water  solubility  of  the  chemicals,  methods  of 
treatment  varied.   Each  treatment  was  replicated  3  times  with  a  random- 
ized block  design  and  observation  on  germination  were  taken  at  7  and  12 
days  after  the  start  of  seed  imbibition  in  all  cases.   Data  was  analyzed 
statistically  using  F  test  and  Duncan's  multiple  range  (90), 

In  the  cyanide  treatments,  the  concentrations  used  were  0,0,  1.0, 

-1        -9  -2 

10  ,  3  X  10   ,  and  10   M  made  with  potassium  cyanide.   Seeds  were 

allowed  to  imbibe  for  42  hours,  seed  coat  removed  and  the  embryos  placed 


23 


in  an  aqueous  solution  of  the  chemical  for  6  hours.   They  were  then 
planted  in  seed  flats  and  placed  in  a  growth  charaber  with  a  controlled 
temperature  of  20°  1  2°  C  and  a  12-hour  day  of  approximately  900  ft-C 
lig;ht  intensity. 

Benzoic  acid  and  p-hydrox;  ;;enzoic  acid  were  tested  at  concentrations 
of  0.0,  10"^,  3  X  10~2,  10"^,  and  10"^  M.   To  test  the  respective  concen- 
trations of  each  compound,  approximately  5  g-  of  dry  perlite  were  placed 
in  100  ml  beakers  and  the  perlite  saturated  with  the  solution  of  che.uical 
to  be  tested.   After  equilibration  of  the  mixture,  fully  turgid  seeds, 
attaining  this  condition  in  moist  vermiculite  in  48  hours  at  20°  C,  were 
placed  in  the  perlite  plus  chemical  media,  and  maintained  under  aerobic 
conditions.   After  5  days  in  the  media,  the  seeds  were  transferred  to 
flats  i:untaining  a  2:1  mixture  of  perlite:  vermiculite.   All  ;..  .?es  of 
the  experiments  were  conducted  in  growth  chambers  at  20°  ^  2°  C  with  a 
12-hour  day  of  900  ft-C.  light  intensity. 

Since  benzaldehyde  and  mandelonitrile  are  only  slightly  soluble  in 
water,  the  method  of  treatment  was  modified.   For  these  tests,  a  loga- 
rithmic range  of  quantities  of  the  material  per  unit  of  perlite  was  used. 
A  measured  amount  of  the  chemical  was  absorbed  onto  fine  perlite  and 
water  added  to  the  medium.   The  concentrations  noted  are  based  on  the 
amount  that  was  available  to  the  water  phase.   Five  grams  of  the  mixture 
were  used  per  container  per  ti'eatment  and  care  was  taken  to  maititain 
aerobic  conditions.   For  preparation  of  the  seed  before  treatment,  they 
were  allowed  to  imbibe  for  4S  hours,  embryos  excised  and  placed  in  the 
perlite-chemical  ...rixture.   The  embryos  were  left  in  the  media  for  5 
days  at  20   C.   Thei.  they  were  removed  fi'om  the  chemical  environments, 
planted  in  flats  and  placed  in  a  greenhouse  for  the  remainder  of  the 
observational  period. 


24 

Extraction  and  preparation  of  fractions  from  seeds  for  gas  chroma- 
tography:  The  isolation  of  the  fractions  was  made  from  seeds  that  were 
fully  turgid  after  4S  hours  in  moist  vermiculite  at  20   C.   The  seeds 
(10  g)  were  ground  in  a  Servall  Omni-mixer  at  15,000  rpm  for  3  minutes 
in  30  ml  of  80%  ethanol .   The  homogenate  was  filtered,  the  subsequent 
filtrate  dried  under  vacuum  and  the  residue  dissolved  in  0.1  M  tartaric 
acid.   This  aqueous  solution  was  partitioned  against  ethyl  ether  and  the 
ether  phase  separated  and  partitioned  against  an  aqueous  solution  of 
0.1  AI  sodium  bicarbonate.   The  aqueous  bicarbonate  phase  was  acidified 
with  tartaric  acid  to  pH  2.0  and  then  partitioned  again  with  100  ml 
ethyl  ether.   The  resulting  ether  solution  was  concentrated  under  nitro- 
gen gas.   The  ether-soluble  acidic  fraction  was  subjected  to  gas  chroma- 
tography before  and  after  treatment  with  acetylating  agents. 

Diazopropane  was  prepared  with  slight  modification  by  the  method 
of  Wilcox  (112).   Briefly,  N-propyl-N-nitrosourea  (  obtained  from  Dr. 
Merrill  Wilcox,  Agronomy  Department,  University  of  Florida)  was  reacted 
with  40%  KOH  in  water  and  trapped  in  peroxide-free  ethyl  ether.   The 
etheral  solution  was  stored  over  sodium  sulfate  in  a  polyethylene  bottle 
in  a  freezer.   To  acetylate  a  sample,  sufficient  amounts  of  the  solution 
were  added  so  that  a  straw-yellow  color  persisted  at  the  end  of  the  re- 
action period. 

Alternative  esterif ication  methods  with  diazomethane  and  diazobutane 
were  used  to  aid  in  the  identification  of  aromatic  acids.   The  diazo- 
methane reagent  was  prepared  as  outlined  by  Williams  (113).   Briefly, 
N-methyl-N ' -nitro-N' -nitrosoguanidine  (Aldrich  Chemical  Co.,  Milwaukee) 
was  added  to  20%  KOH  and  trapped  in  ethyl  ether.   The  diazobutane  was 
prepared  in  a  manner  similar  to  the  diazopropane  except  substituting  N, 


25 

X-butyl-N-nitrosourea  (obtained  from  Dr.  Merrill  Wilcox,  Agronomy 
Department,  University  of  Florida)  for  the  N-propyl-N-nitrosourea.   With 
both  diazomethane  and  diazobutane,  the  initial  esterif ication  period, 
30  minutes,  was  the  same  as  with  diazopropane. 

In  tests  where  esterif ication  was  slow  for  the  carboxyl  group  or 
where  acetylation  of  hydroxyl  groups  on  the  ring  was  slow  or  non-existent, 
0,7%  methanolic  boron  trifluoride  was  added  to  these  diazo-compounds  and 
the  reaction  was  allowed  to  proceed  at  room  temperature  for  3  hours. 

Standards  of  chemicals  and  fractions  of  extracts  were  dissolved  in 
ethyl  acetate  for  gas  chromatography.   Weights  and  volume  on  seed  and 
solvent  fractions  were  kept  so  that  quantities  could  be  expressed  as 
seed  equivalents.   Standards  had  a  final  concentration  of  1  mg  per  ml. 

Conditions  for  gas  chromatography:   Separation  of  compounds  of  the 
acidic  fraction  of  the  ethanol  extract  was  on  a  model  400  F  and  M  gas 
chromatograph  equipped  with  a  f lame-ionization  detector.   The  column 
consisted  of  1/4  inch  stainless  steel  tubing  6  feet  long  packed  with  8% 
S.E.  30  on  60-80  mesh  Chromosorb  W.  Helium  was  used  as  a  carrier  gas  with 
flow  rate  of  70  ml  per  minute.   Temperatures  for  the  system  were  as 
follows:  oven,  180°  C;  injection  port,  260   C;  and  detector,  250   C, 
except  as  noted  in  the  results. 

Identification  of  extracted  compounds  was  made  by  comparison  of 
their  retention  times  with  those  of  the  known  compounds.   Matched  reten- 
tion times  of  several  derivatives  of  knowns  to  those  of  identically  treat- 
ed unknowns  lent  greater  support  to  tentative  identification. 

Alfalfa  bioassay:  Peruvian  alfalfa  seed  were  separated  into  red  and 
yellow  seeds.  The  red  seeds  were  discarded  because  of  their  low  germina- 
tion capability  (111)  and  the  yellow  seeds  were  used  for  the  bioassay. 


26 

The  bioassay  was  conducted  in  petri  dishes  with  either  filter  paper 
disks  or  chromatography  paper  strips  as  a  moisture  holding  absorbent, 
depending  upon  the  test.   Generally  40-50  seeds  per  dish  were  used  for 
each  assayed  fraction.   Before  placing  the  seeds  on  the  moistened  paper, 
they  were  soaked  in  distilled  water  for  a  few  seconds  to  improve  the 
rate  of  imbibition  of  the  seeds.   Once  the  seeds  had  been  placed  in  the 
dishes,  the  dishete  were  placed  in  the  dark  at  20°  t  2°  C.   After  24 
hours,  observations  were  made  on  the  number  of  germinated  and  non-germi- 
nated seeds  per  dish,   A  seed  was  considered  to  have  germinated  upon 
protrusion  of  the  radicle. 

Inhibitor  characterization:   Bioassays  were  conducted  on  80% 
ethanolic  extracts  and  fractions  paper  chromatographed  in  isopropanol: 
ammonia:  water  (80:1:19,  v/v/v)  solvent  on  TOiatman  3  MM  chromatography 
paper.   Chromatograms  were  divided  into  sections  of  10  R^  units  and 
assayed,  using  the  alfalfa  seed  bioassay  (18) . 

Extracts  of  peach  seeds  were  also  subjected  to  acid  hydrolysis 
(pH  2)  with  acetic  acid,  alkaline  hydrolysis  (pH  10)  with  ammonium 
hydroxide,  dialysis  against  distilled  water  for  24  hours;  and  heating 
for  10  minutes  at  50,  75,  and  100*-"  C.   Changes  in  inhibitory  activity 
were  moiiitored,  using  the  alfalfa  bioassay. 

Solubility  of  components  of  the  inhibitor  complex  in  various  organic 
solvents  was  investigated.   Sections  of  the  paper  chromotograms  contain- 
ing the  inhibitory  zone  were  cut  into  strips  representing  the  equivalent 
of  a  0.5  g  seed  sample.   These  strips  were  steeped  in  various  solvents 
for  2  hours.   The  solvents  were  decanted  into  small  petri  dishes  con- 
taining a  Wliatman  No,  4  filter  paper  disk  and  the  residue  deposited  on 
the  paper  by  evaporation.   Distilled  water  (1.5  ml)  was  added  to  the 


27 

petri  dishes,  and  to  appropriate  controls,  and  then  bioassayed.   Redis- 
tilled solvents  of  water,  hexane,  acetonitrile,  ethyl  ether,  chloroform, 
methanol,  ethyl  acetate  and  carbon  disulfide  were  used  for  the  solubility 
studies . 

Measurement  of  benzaldehyde  and  mandclonitrile:   The  quantity  of 
benzaldehyde  and  raandelonitrile  present  in  seeds  under  various  treat- 
ments was  determined.   All  seeds  were  fully  turgid  since  they  were  placed 
in  moist  vermiculite  for  42  hours  at  20°  C  prior  to  treatment.   Treat- 
ment  1  was  seeds  steeped  in  3  x  10  "  M  thiourea  for  6  hours,  blotted 
and  kept  in  a  moist  medium  until  sampled.   Treatment  2  was  embryos  re- 
moved from  the  seed  coat  and  associated  tissue  after  48  hours  from  the 
start  of  the  experiment.   Treatment  3  was  the  control  of  intact  seeds. 
Seeds  in  each  treatment  were  kept  at  20°  C  and  a  4.8  g  sample  wet 
weight,  equivalent  to  approximately  3  g  dry  weight,  were  taken  at  the 
following  times  from  the  start  of  seed  imbibition:   48,  60,  72,  80,  88, 
96,  104,  112,  120,  132,  144,  156,  and  168  hours.   The  samples  were 
frozen  immediately  to  -70°  C  and  then  placed  in  a  freezer  at  -30°  C 
until  ground,  approximately  8  hours.   The  frozen  seeds  were  ground  in  a 
Wiley  mill  with  a  20-mesh  sieve.   The  mill  had  been  thoroughly  cooled  by 
passing  large  quantities  of  dry  ice  through  it  before  the  samples  were 
ground.   Also,  sufficient  amounts  of  powdered  dry  ice  were  passed 
through  the  mill  along  with  the  frozen  seeds  to  keep  the  grinding  head 
at  approximately  the  temperature  of  the  dry  ice.   The  ground  seeds  plus 
powdered  dry  ice  were  collected  together  and  added  to  ethyl  ether  at 
-70   C.   After  the  dry  ice  had  sublimed  from  the  ethyl  ether  (generally 
30-40  minutes  at  room  temperature)  the  solutions  were  allowed  to  warm 
to  approximately  -5°  C  before  they  were  placed  in  a  -30°  C  environment 


28 

for  3  hours.   This  warming  and  steeping  in  a  freezer  was  needed  to  obtain 
benzaldehyde  and  mandelonitrile  in  the  ether  phase.   This  etheral  solu- 
tion was  subjected  to  gas  chromatography  under  conditions  noted  with  the 
results.   Weight  and  volume  were  taken  quantitatively  so  the  data  could 
be  expressed  in  the  amount  of  chemical  per  seed  equivalent. 

Under  the  conditions  of  gas  chromatography,  benzaldehyde  and 
mandelonitrile  chromatographed  as  benzaldehyde  since  heat  caused  mandelo- 
nitrile to  decompose  to  HCN  and  benzaldehyde.   Therefore,  the  following 
series  of  reactions  were  used  to  separate  the  2  components.   Firstly, 
the  quantity  of  both  compounds  was  obtained  from  gas  chromatographic 
analysis  of  an  aliquot  of  an  extract.   Secondly,  the  quantity  of 
mandelonitrile  remaining  in  an  etheral  solution  was  determined  after 
quantitatively  removing  benzaldehyde  by  reacting  with  sodium  bisulfite. 
This  was  accomplished  by  solvent  partitioning  between  the  etheral  solu- 
tion and  aqueous  40%  sodium  bisulfite,   Thirdly,  quantitative  analysis 
was  again  done  on  the  ether  phase  after  407o  potassium  cyanide  was  added 
to  the  aqueous  sample  layered  under  ethyl  ether  and  the  mixture  shaken 
vigorously.   This  converted  the  sodium  bisulfite  addition  product  of 
benzaldehyde  to  mandelonitrile  which  allowed  it  to  pass  back  into  the 
ether  phase.   After  allowing  the  mixture  to  stand  for  5  minutes  in  the 
cold,  the  ether  phase  was  subjected  to  gas  chromatographic  analysis 
the  third  time.   Quantitative  determinations  were  made  using  the  area 
under  the  peak  as  a  measure  of  both  compounds  and  the  peak  area  of  the 
sample  after  addition  of  sodium  bisulfite.   The  latter  represents  that 
due  to  mandelonitrile.   The  difference  between  the  two  peak  areas  was 
assumed  to  be  that  due  to  benzaldehyde. 


29 

The  conversion  of  benzaldehyde  to  a  sulfite  derivative  soluble  in 
water  and  then  conversion  to  mandelonitrile  is  a  well-knov/n  reaction 
(26).   The  sodium  bisulfite  reacts  with  the  carbonyl  group  of  benz-  Ide- 
hyde  to  forni  the  sulfite  addition  product.   Addition  of  potassium  cyanide 
acts  as  a  base  and  neutralized  the  sodium  bisulfite  in  equilibrium  with 
the  bisulfite  compound  to  form  potassium  bisulfite;  the  simultaneously 
liberated  benzaldehyde  and  hydrogen  cyanide  then  combine  to  give  mandelo- 
nitrile (26). 

Chilling  study:   Detemiinations  were  made  of  the  inhibitor  complex 
benzaldehyde  and  mandelonitrile  after  periods  of  chilling.   Fully  turgid 
seeds,  attaining  this  condition  after  48  hours  in  moistened  vermiculite 
at  20°  C,  were  placed  at  4°  C  for  0,  16S,  336,  504,  and  672  hours.   At 
the  time  of  sampling,  one  sample  was  removed  and  extracted  immediately 
and  another  sample  was  placed  for  an  additional  40  hours  at  20°  C.   A 
control  lot  of  seeds  was  maintained  at  20  C  for  sampling  at  equivalent 
times.   At  each  time  of  sampling,  seeds  equivalent  to  5  g  dry  weight 
were  taken  in  duplicate.   The  quantity  of  benzaldehyde  and  mandeloni- 
trile in  the  seeds  was  determined  as  outlined  previously  and  the  level 
of  non-volatile  iiihibitors,  presumably  the  beta-inhibitory  complex  (8), 
was  assayed  as  follows.   A  sample  of  treated  seeds  was  subjected  to 
extraction  with  807o  ethanol  after  grinding,  as  previously  noted.   The 
solution  was  taken  to  near  dryness  by  vacuum  distillation,  keeping  the 
distilling  chambei'  at  less  than  5   C.   The  residue  was  redissolved  in 
807o  ethanol,  applied  to  chromatographic  paper  (Whatman  3  MM)  and  devel- 
oped in  isopropanol:  ammonia;  water  (80:1:19  v/v/v)  solvent,  using  de- 
cending  techniques.   The  inhibitory  zone,  as  determined  by  Rf,  were 
sectioned  from  the  chroraatograms,  and  solutes  eluted  from  the  paper  with 


glass  distilled  water.   The  eluates  were  then  diluted  in  such  a  way  that 
equivalent  seed  weights  in  the  solutions  were  1.0  g,  500  mg,  300  mg, 
100  mg  and  0  mg.   The  solutions  were  placed  in  small  petri  dishes  on 
Wliatman  No.  4  filter  paper  disk,  frozen  and  water  removed  by  sublimation 
under  vacuum.   After  again  moistening  the  filter  pads  with  1.5  ml  of 
H„0,  they  were  bioassayed  with  the  alfalfa  bioassay  using  40  seeds  per 
disk.   Inhibitory  levels  were  determined  by  calculations  from  a  dilution 
curve  based  on  relative  seed  weight. 


EXPERIMENTAL  RESULTS 

Thiourea  and  seed  coat  excision;   The  influence  of  thiourea  on 
gei'mination  of  dormant  'Okinawa'  peach  seed  and  on  anomalous  seedling 
development  is  shown  in  Table  1.   It  was  quite  evident  from  this  data 
that  thiourea  greatly  increased  the  per  cent  germination,  but  enhanced 
anomalous  development  in  the  seedlings.   In  both  cases,  the  higher  the 
concentration,  the  greater  the  effect.   The  data  indicates  further  that 
increases  in  germination  and  abnormal  growth  were  statistically  signifi- 
cant  with  concentrations  of  thiourea  stronger  than  10   M. 

In  determining  the  possible  interaction  between  thiourea  and  seed 
coat  on  germination  and  subsequent  seedling  growth,  the  most  striking 
finding  was  the  absence  of  abnormal  seedlings  in  any  of  the  treatments; 
yet  very  good  germination  was  obtained,  as  shown  in  Table  2.   The  length 
of  time  after  imbibition  and  thiourea  treatment  for  embryo  excision 
seemed  to  have  little  effect  on  germination. 

Influence  of  Benzaldehyde,  cyanide,  and  mandelonitrile  on  seed 
germination;   The  data  in  Table  3  indicates  that  cyanide  does  not  dras- 
tically reduce  germination,  except  in  very  high  concentrations  (1.0  M) . 
Data  taken  20  days  after  start  of  imbibition  showed  that  the  1.0  M  con- 
centration was  still  significantly  different  from  the  lower  concentra- 
tions used.   No  abnormalities  were  noted  in  the  seedlings  from  any  of 
the  tx-eatments,  and,  interestingly,  the  1.0  M  cyanide  did  not  kill  the 
seeds. 


31 


Table  1. — Influence  of  thiourea  concentrations  on  germination  and 
per  cent  of  production  of  abnormal  seedlings  from 
'Okinawa'  peach  seeds. 


32 


Thiourea 
concentration,  M' 


X 


Ale  an 
%  Kemiination^ 


Mean 

V  2 

%  abnormal'' ' 


0  (Control) 
3 


10 


10-2 


10 

3  X  10 

1.0 


-1 


5.8  a 

5.8  a 
26.8  ab 
45.8   be 
62.5    c 
58.3    c 


0.0  a 

0.0  a 
21.9   b 
40.1   b 
61.4   c 
79.0   c 


^Each  treatment  was  replicated  3  times  with  40  seed  per  replication. 

^Means  not  having  a  following  letter  in  common  are  significantly  differ- 
ent at  the  1%  level. 


"Percentage  based  on  the  total  number  of  seed  germinated. 


Table  2. — Effect  of  thiourea  concentration  and  embryo  excision  on 
germination  of  'Okinawa'  peach  seeds  12  days  after  start 
of  imbibition  and  on  abnormal  seedling  production  32 
days  after  start  of  imbibition. 


Treatments^ Mean 

Chemical  Seed  coat    %  germination^ %  atypical  seedling 

Control       Intact 

Excised;   42  hrs 

72  hrs 

96  hrs 

120  hrs 

10"% 

Thiourea      Intact 

Excised;   42  hrs 

72  hrs 

96  hrs 

120  hrs        100.0 


0.0 

a 

100.0 

e 

92.6 

cd 

100.0 

e 

100.0 

e 

81.5 

b 

96.3 

de 

92.6 

cde 

100.0 

e 

3  X  10"% 

Thiourea      Intact 

Excised;   42  hrs 

72  hrs 

96  hrs 

120  hrs 


88.9 

c 

96.3 

de 

100.0 

e 

100.0 

e 

100.0 

e 

0. 

0 

0. 

0 

0. 

0 

0. 

0 

3. 

,7 

7. 

,4 

0, 

.0 

0, 

.0 

0, 

.0 

3, 

.7 

3 

.7 

0 

.0 

0 

.0 

0 

.0 

3 

.7 

^Seeds  were  imbibed  42  hours,  then  treated  with  chemicals  for  6  hours 
before  planting. 

^Each  treatment  consisted  of  3  replications  of  9  seed  each. 

^Means  not  having  a  following  letter  in  common  are  significantly  differ- 
ent at  the  1%   level. 

^Hours  after  start  of  imbibition. 


34 


Table  3. — Per  cent  germination  of  'Okinawa'  peach  seeds  7  and  20  days 
after  start  of  imbibition  as  influenced  by  cyanide^. 


Cyanide 

M^ 

Mean  " 

%   germination^ 

concentration, 

7  davs 

20  davs 

1.0 

10-1 

3  X  10 


-2 


-2 


10 


Control 


0.0  a 

88. 9   b 

100.0   c 

100.0   c 

100.0   c 


0.0  a 
94.4   b 

100.0   b 
94.4   b 

100.0   b 


^Seeds  were  imbibed  for  42  hours,  then  treated  with  the  designated 
cyanide  concentrations  for  6  hours. 

Each  concentration  consisted  of  3  replications  with  6  seed  per 
replication. 

"Means  not  having  a  following  letter  in  common  are  significantly 
different  at  the  57o  level. 


u^ 


The  treating  of  samples  of  excised  embryos  with  various  concentra- 
tions of  mandelonitrile  and  benzaldehyde  resulted  in  the  inhibition  of 
germination  with  some  of  the  stronger  concentrations  (Tables  4  and  5) . 
Mandelonitrile  at  1.4  to  140.0  mg/g  completely  inhibited  germination, 
while  all  other  concentrations  except  0.42  mg/g  inhibited  only  slightly. 
Data  taken  5  days  after  removing  the  seeds  from  the  chemical  shov;ed 
that  1.4  mg/g  exhibited  very  little  inhibitory  influence.   Concentrations 
of  4.2  to  140.0  mg/g  were  still  strongly  inhibitory  (Table  4).   Concen- 
trations of  benzaldehyde  of  11.0  and  110.0  mg/g  completely  inhibited 
germination,  while  a  concentration  of  3.3  mg/g  resulted  in  only  16.77o 
germination.   Concentrations  lower  than  3.3  mg/g  had  no  measurable  in- 
fluence (Table  5).   Five  days  after  removal  of  seeds  from  the  benzaldehyde 
media,  germination  occurred  to  an  appreciable  extent  in  the  3.3  mg/g 
treatment  but  11.0  and  110.0  mg/g  were  still  inhibitory.   As  found  with 
cyanide,  the  anomalous  growth  patterns  were  not  present  on  seedlings 
produced  from  seeds  treated  with  either  mandelonitrile  or  benzaldehyde. 
The  influence  of  the  benzaldehyde  and  mandelonitrile  on  seed  germination 
is  portrayed  graphically  in  Figure  1. 

Aromatic  acids  investigation;   Tentative  identification  of  the  com- 
ponents isolated  from  the  propyl  esters  of  the  acidic  fraction  of  an 
ethanol  extract  of  peach  seeds  was  made  by  comparing  the  retention  times 
on  gas  chromatograms  with  those  of  known  compounds.   A  gas  chromatogram 
of  the  fractions  is  shown  in  Figure  2. 

The  phenolic  compounds  tentatively  identified  were  benzoic,  mandelic, 
o-hydroxycinnamic,  2, 6-dihydroxybenzoic,  o-hydroxybenzoic,  p-hydroxyben- 
zoic  and  2,4-dimethoxybenzoic  acids  (Table  6),   The  gas  chromatograms  of 
the  standards  for  the  known  compounds  listed  above  can  be  found  in  the 
Appendix. 


36 


Table  4. — Per  ceiit  germination  of  'Okinawa'  peach  seeds  7  and  12  days 

after  start  of  imbibition  as  influenced  by  mandelonitrile^'^. 


Mandelonitrile 

Mean  %   germination 

mg/g  of  perlite 

7  days 

i:.'.  days 

140.0                         0.0  a  0.0  a 

14.0                         0.0a  0.0a 

4.2                         0.0  a  0.0  a 

1.4                          0.0  a  83.3   b 

0.42  100.0    c  100.0    c 

0.14  94.4   be  100.0    c 

0.042      ■'  94.4   be  100.0    c 

0.014  94.4   be  100.0   c 

Control  88.9   b  88.9   be 


X 


Seeds  were  allowed  to  imbibe  for  2  days,  then  placed  in  perlite 
containing  mandelonitrile  for  5  days.   The  indicated  quantity  of 
mandelonitrile  was  applied  to  the  perlite. 

^Each  treatment  was  replicated  3  times  with  6  seed  per  replication. 

^Means  not  having  a  following  letter  in  common  are  significantly 
different  at  the  1%  level. 


37 


Table  5. — Per  cent  germination  of  'Okinawa'  peach  seeds  7  and  12  days 
after  start  of  imbibition  as  influenced  by  benzaldehyde'^' y. 


Benzaldehyde  Mean  %  germination^ 

mg/g  of  perlite  7  days  12  days 

110.0  0.0   a  0.0   a 

11.0  0.0   a  0.0   a 

3.3  16.7      b  94.4      b 

1,1  100.0        c  100.0        c 

0.33  100.0        c  100.0        c 

0.11                                       •            100.0        c  100.0        c 

0.033  100.0         c  100.0        c 

0.011  100.0         c  100.0         c 

Control  100.0   c  100.0   c 

Seeds  were  allowed  to  imbibe  for  2  days,  then  placed  in  perlite  contain- 
ing benzaldehyde  for  5  days.   The  indicated  quantity  of  benzaldehyde 
was  applied  to  the  perlite. 

^Each  treatment  was  replicated  3  times  with  6  seed  per  replication, 

z 

Means  not  having  a  following  letter  in  common  are  significantly 

different  at  the  1%  level. 


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Gas  chroraatogram  of  the  propyl  esters  of  the  acidic 
fraction  from  an  ethanol  extract  of  peach  seeds. 
Time  is  in  minutes.   (See  Table  6  for  gas  chromato- 
graph  parameters.) 


40 


Table  6. — Relative  retention  time  and  possible  identity  of  components 
separated  by  gas  chromatography  of  the  propyl  esters  of  the 
acidic  fraction  from  an  ethanolic  extract  of  peach  seeds^. 


Peak  No. 


Relative  retention  timc'^ 


Possible  identity  of  acids'' 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 


1.00 
1.37 


1. 
2, 
2, 
2, 
3, 
4, 
5, 
7, 


91 
13 
50 
89 
59 
99 
89 
11 


10.21 
19.29 


Benzoic 
Succinic 

Malic  or  mandelic? 

o-hydroxycinnamic 

Fumaric  ? 

2, 6-dihydroxybenzoic 

o-hydroxy benzoic 

p-hydroxybenzoic 

2, 4-diraethoxybenzoic 

? 
Citric 

? 


-  Instrument;   F  &.  M  model  400,  flame  detector. 

Column;       87o  S.E.  30  on  60-80  mesh  chromasorb  W,  acid  washed, 

silane  treated;  1/4"  0,D.  stainless  steel  6'  in  length. 

Carrier  gas;  He.   Outlet  flow  rate;  70  ml/min. 

Oven  temp;    180°  C.  Injection  port  temp;  260°  C. 
Detector  temp;  250°  C. 

Range  and  attenuation;  10  x  8.   Chart  speed;  1/4"  per  rain. 

-  Relative  retention  time  is  based  on  benzoic  acid. 


Possible  identity  based  on  matched  retention  times, 


41 

Inhibitor  chai-acterization;   Using  the  Peruvian  alfalfa  bioassay, 
it  was  found  that  extraction  of  8  g  wet  weight  of  dormant  peach  seeds 
with  80%  ethanol  yielded  a  strongly  inhibitory  complex  (0%  germination). 
Specific  gravity  measurements  indicated  that  the  inhibitory  influence 
was  due  to  factors  other  than  osmotic  ones.   Paper  chromatography  of  the 
ethanol  extract,  using  an  isopropanol:  ammonia:  water  (80:1:19  v/v/v) 
solvent  system,  yielded  a  strong  inhibitory  complex  between  R^ ' s  0.6  and 
0.8  when  bioassayed  with  the  alfalfa  seed  test  (Table  7). 

From  tests  on  the  influence  of  acids,  bases  and  heat  on  the  stabil- 
ity of  the  inhibitory  complex,  the  data  on  per  cent  germination  from  the 
alfalfa  bioassays  (Table  8)  would  seem  to  indicate  that  the  inhibitory 
complex  was  reasonably  stable  since  none  of  the  treatments  destroyed  the 
inhibitory  capacity  of  the  extract. 

Comparison  of  various  organic  solvents  (Table  9)  for  the  solubil- 
ization of  the  inhibitory  complex  showed  that  the  more  polar  solvents 
(water  and  alcohol)  serve  as  suitable  solvents  for  the  inhibitor.   The 
data  indicated  also  that  the  inhibitor  may  be  only  partially  soluble 
in  acetonitrile. 

Isolation  and  characterization  of  benzaldehyde  and  mandelonitrile 
from  peach  seeds:   Benzaldehyde  and  mandelonitrile  were  isolated  and 
characterized  from  peach  seeds  by  several  techniques.   Crushed  peach 
seeds  evolve  an  aroma  similar  to  that  of  benzaldehyde  and  mandelonitrile. 
Co-chromatography  of  the  pure  chemicals  and  the  extract  components  from 
peach  seeds  by  chromatography  yielded  identical  R „ ' s  and  retention  times, 
respectively.   Ultra-violet  fluorescence  (3200  A°  and  2537  A°)  of  benz- 
aldehyde and  a  fraction  from  an  ethanol  extract  from  peach  seeds  on 
paper  chromatograms  were  identical.  Also,  benzaldehyde  and  the  extracted 


42 


Table  7. — Paper  chromatographic  separation  of  the 
inhibitory  complex  from  dormant  peach 
seeds. 


Rf  Value-^  %  Germination^ 


0,0  -  0,1  92,5 

0,1-0,2  85,0 

0,2-0.3  87,5 

0,3-0,4  85.0 

0.4  -  0.5  55,0 

0,5  -  0,6  0,0 

0.6  -  0,7  0,0 

0.7  -  0,8  0.0 

0.8-0.9  85.0 

0,9  -  1.0  80.0 


^Solvent  system:  Isopropanol:  ammonia:  water 
(80:1:19  v/v/v) . 

^Bioassayed  with  the  alfalfa  seed  test. 
Control  =  90%. 


43 


Table  8. — Influence  of  acids,  bases  and  heat  on  the 
inhibitory  complex  from  peach  seeds  after 
paper  chromatography. 


Test  %  Germination^ 

Acid  hydrolysis  (pH  2)  0.0 

Alkaline  hydrolysis  (pH  10)  0.0 

y 

Dialysis,  inside  tubing  0.0 

outside  tubing  0.0 

Heating  for  10  minutes: 

50°  C  0.0 

75°  C  0.0 

100°  C  0.0 

Extract  control  0,0 

Water  control  90.0 


Bioassayed  with  the  alfalfa  seed  test.   Seed 


equivalent  of  the  extract  was  0.5  g  dry  weight, 

Dialysis  was  conducted  with  seamless  cellulose 
tubing  against  distilled  water  for  24  hours. 


Table  9. — Solubility  of  the  inhibitor-complex  in 
various  organic  solvents  as  determined 
by  the  alfalfa  bioassay. 


44 


%  Germination 


Solvent 


Eluate^ 

Chromatogram 

Section^ 
(Rj  0.6-0.8) 

0.0 

80.0 

92.5 

0.0 

30,0 

0.0 

85.0 

0.0 

90.0 

0.0 

0.0 

82.5 

87.5 

0.0 

92.5 

0.0 

Water 

Hexane 

Acetonitrile 

Ethyl  ether 

Chloroform 

Methanol 

Ethyl  acetate 

Carbon  disulfide 


^Elution  fraction  from  chromatogram  section  of  R^  0.6- 
0.8. 

^Chromatogram  section  containing  the  inhibitory  complex 
after  eluting  with  the  respective  solvent. 


45 


component  reacted  similarly  to  aldehyde  indicators.   Component  of  an 
ethanol  extract  and  benzaldehyde  formed  a  sodium  bisulfite  addition 
product  which  then  generated  mandelonitrile  on  treatment  with  KCN. 

Ultra-violet  fluorescence  (3200  A°  and  2537  A°)  of  mandelonitrile 
and  a  fraction  from  an  ethanol  extract  from  peach  seeds  on  paper  chroma- 
tograms  were  identical.   The  extract  components  and  mandelonitrile  form 
benzaldehyde  and  cyanide  when  subjected  to  high  temperatures  (200-250°  C) , 
Mandelonitrile  and  components  of  the  extract  reacted  alike  when  tested 
with  hydrocyanin  indicators. 

Quantitative  detemiinations  of  benzaldehyde  and  mandelonitrile: 
Benzaldehyde  and  mandelonitrile  were  determined  using  procedures  estab- 
lished in  identifying  the  2  compounds.   Briefly,  this  was  gas  chromatog- 
raphic analysis  of  the  ethereal  extract  before  making  a  sodium  bisulfite- 
addition  product,  after  the  reaction  to  assay  the  level  of  decrease  and 
again  after  converting  the  benzaldehyde  to  mandelonitrile  by  KCN.   A 
chromatogram  of  the  composite  of  both  compounds  is  shown  in  Figure  3a. 
After  treating  with  sodium  bisulfite,  the  peak  is  reduced  (Figure  3b) 
and  increased  after  the  subsequent  addition  of  potassium  cyanide 
(Figure  3c) . 

Comparison  of  the  influence  of  thiourea  treatment,  and  embryo  exci- 
sion, as  compared  to  a  non-germinating  control  of  intact  seeds,  on  the 
rate  of  release  of  benzaldehyde  and  mandelonitrile  both  collectively  and 
individually  is  shown  in  Figures  4,  5  and  6,   The  graphs  indicated  that 
intact  seeds  and  thiourea-treated  seeds  had  peak  times  of  production  of 
benzaldehyde  and  mandelonitrile  at  about  the  same  time,  72  hours,  while 
the  excised  seeds  had  a  delay  in  the  maximum  period  of  production  by  16 
hours.   The  thiourea-treated  seeds  had  a  second  peak  of  production  at 


Fig.  3.   Gas  chromatograms  of  a  known  composite  sample  of  an  etheral 

solution  of  benzaldehyde-mandelonitrile:  (a)  initial  solution; 
(b)  after  addition  of  a  solution  of  sodium  bisulfite;  and  (c) 
after  addition  of  potassium  cyanide. 

Gas  chromatograph  parameters. 


Instrument: 
Column: 


Carrier  gas: 
Temperatures: 

Range  and 
Attenuation: 
Chart  Speed: 


F  &,  M  model  400,  flame  detector. 
8%  S.E.  30  on  60-80  mesh  Chromosorb  W,  acid- 
washed,  silane  treated;  1/4"  0,D.  stainless 
steel  6'  in  length. 

Helium.   Outlet  flow  rate:   70  ml/min. 
Oven,  100°  C;  Injection  port,  150°  C;  Detector, 
160°  C. 

10  X  8 

1/4"  per  minute. 


47 


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51 

120  hours  after  the  start  of  imbibition.   Relatively  larger  amounts  of 
mandelonitrile  than  benzaldehyde  were  in  the  extracts  from  the  seeds. 

The  effect  of  the  3  treatments  on  peach  seed  germination  is  shown 
in  Figure  7.   The  excised  seeds  attained  100%  germination  at  approxi- 
mately 132  hours  or  about  44  hours  after  the  peak  in  production  of  benz- 
aldehyde and  mandelonitrile.   However,  those  seeds  treated  with  thiourea 
required  a  much  longer  period  of  time  after  the  peak  production  time  in 
order  to  attain  nearly  1007o  germination.   This  was  true  even  when  the 
time  from  the  second  peak  at  120  hours  was  considered. 

Quantitative  determination  of  benzaldehyde,  mandelonitrile  and  the 
inhibitory  complex  of  seeds  subjected  to  various  degrees  of  chilling: 
Gas  chromatographic  determination  of  the  quantities  of  benzaldehyde  and 
mandelonitrile  in  peach  seeds  at  weekly  intervals  during  the  chilling 
period  indicated  only  trace  amounts  were  present.   Calculations  indicated 
that  the  tissue  level  of  both  chemicals  ,was  below  1.0  ug/gra  dry  weight 
of  tissue.   Only  trace  amounts  of  benzaldehyde  and  mandelonitrile  were 
detected  by  gas  chromatography  on  seeds  placed  at  20°  C  for  40  hours 
after  removal  from  various  intervals  of  chilling. 

The  level  of  the  80%  ethanol  soluble  inhibitory  complex  of  peach 
seeds,  as  determined  by  the  alfalfa  bioassay,  does  not  decrease  during 
the  chilling  period  (Figure  8) .   Slight  week-to-week  fluctuations  were 
present  but  the  overall  analysis  showed  little  change  in  the  level. 
However,  the  per  cent  germination  of  seed  periodically  removed  from  the 
chilling  temperatures  and  placed  at  20°  C  indicated  that  504  hours  of 
chilling  was  sufficient  to  terminate  dormancy  in  over  807o  (Figure  9)  of 
the  population  of  the  seeds.      •    'i 


52 


loo- 


se - 


60  ;r 


c 
q 

03 


C7)  40- 


20 


EXCISED 

o o  —  o 


Oir 


THIOUREA 


INTACT(CONTROL) 

o  —  o o 


^ 


5 


-I , ^- 


100 


160 


220 


280 


Time,  hours 


Fig,  7,   Germination  of  peach  seed  as  influenced  by  embryo  excision, 
and  thiourea  treatments  as  detennined  periodically  after  the 
start  of  imbibition,   (Growth  of  excised  embryos  was  taken 
to  be  equivalent  to  germination  when  the  radicle  had  elongated 
to  2  mm. ) 


Fig.  8.   Relative  inhibitory  activity,  as  measured  by  the  alfalfa 

bioassay,  of  the  inhibitory  complex  in  an  ethanolic  extract 
of  peach  seeds  chromatographing  between  R^ ' s  0.6  to  0.8. 
(Hours  of  chilling  were  just  prior  to  extraction;  and  seed 
equivalents  were  A=  1.0  g.  B=  0.5  g,  C=  0.3  g,  D=  0.1  g  and 
E=  control . ) 


54 


CM 

o 

lO 

Q 

< 

Q 

U 

CD 

< 

LU 


Q 


U 


CD 


CO: 


LiJI 


a 


u 


CD 


< 


00 

CD 


«     UJ-- 

o 

Cl 

. 

1 

u 

1 

1 

..l„l     ' 

CD 
< 

o 
o 

o 

CO 

o 

CD 

o 

o 

C 

5 

UOII-BUILUJGB    °/o 


en 

c 


u 

o 

c/) 

D 

o 


r:-^!K-' 


loe- 


ss 


801'- 


o 

•i-> 

'  1 

03 

601- 

C 

i 

E 

[ 

L. 

^'^ 

CD 

U) 

40- 

o 

j ' 

o 

y 

Hours  of  chilling 


Fig.  9.   Germination  of  peach  seeds  as  influenced  by  5   C  of  varying 
durations. 


56 

Influence  of  chemicals  on  possible  stimulation  of  peach  seed  germi- 
nation:  The  influence  of  benzaldehyde,  mandelonitrile,  benzoic  acid  and 
p-hydroxybenzoic  acid  on  the  breaking  of  seed  dormancy  was  determined 
and  the  test  differed  from  that  for  inhibition  of  germination  in  that 
they  were  applied  to  intact  seeds.   Using  benzaldehyde  and  mandelonitrile 
at  various  concentrations,  there  was  no  evidence  for  a  stimulatory  effect 
on  seed  germination  (Table  10).   However,  the  2  chemicals  were  active 
in  inhibiting  the  weak  capacity  for  germination,  which  supported  the 
data  of  an  inhibitory  influence  as  shown  earlier. 

Benzoic  and  p-hydroxybenzoic  acids  were  used  on  intact  seed  at 

-T      -1 
concentrations  ranging  from  10    to  10   M.   The  data  in  Table  11  in- 

-1  -2 

dicates  that  p-hydroxybenzoic  acid  at  10    and  3  x  10   M  may  have 

slightly  stimulated  seed  germination  as  compared  to  the  control;  yet 
this  is  of  doubtful  significance  since  the  control  had  a  weak  capacity 
to  germinate  (compare  Tables  10  and  11).   Benzoic  acid  had  little  in- 
fluence on  germination  under  the  conditions  of  these  tests. 

L-mandelic  and  p-hydroxybenzoic  acid  determinations:   Gas  chroma- 
tograms  of  the  control,  peach  seed  extract,  L-mandelic  acid  and  p-hy- 
droxybenzoic acid  after  treating  with  diazomethane  for  30  minutes  are 
shown  in  Figures  10,  11,  and  12.   These  should  be  primarily  the  esiors 
of  the  aromatic  acids.   These  were  separated  on  the  gas  chromatograph 
at  an  oven  temperature  of  180°  C.   The  methoxy  and  butoxy  esters  were 
separated  by  gas  chromatography  after  acetylation  by  diazomethane  and 
diazobutane,  respectively.   Since  those  derivatives  were  more  volatile, 
an  oven  temperature  of  150°  C  was  used. 

Based  upon  the  comparison  of  retention  times  of  the  components  in 
the  extract  with  those  of  the  standards  (Table  12),  it  was  concluded 


57 


Table  10. — Mean  per  cent  germination  of  dormant  'Okinawa' 
peach  seeds  30  days  after  start  of  imbibition 
as  influenced  by  benzaldehyde  and  mandelo- 
nitrile  concentrations^. 


%  Germination 


Concentration,  M^       Benzaldehyde^     Mandelonitrile^ 


1.0  0,0  a  0.0  a 

10  0.0  a  0.0  a 

3  X  10~  16.7   b  0.0  a 

-2 

10  22.2   b  38.9   b 

0  33.4   b  33.4   b 


X 


Seeds  were  imbibed  for  2  days,  then  exposed  to  chemical 
concentrations  for  5  days. 

^Each  treatment  was  replicated  3  times  with  6  seed  per 
replication. 

Means  not  having  a  following  letter  in  common  were 
significantly  different  at  the  5%  level. 


58 


Table  11. — Mean  per  cent  germination  of  dormant  'Okinawa' 
peach  seeds  30  days  after  start  of  imbibition 
as  influenced  by  benzoic  and  p-hydroxybenzoic 


acid  concentrations^, 


%  Germination 


2 

Concentration,  My Benzoic^ p-Hydroxybenzoic 

lO"-"-  5,5  a             38.9   b 

3  X  10"^  0.0  a             38.9   b 

lO"^  16.7  a             11.2  a 

10""^  16.7  a             11.2  a 

0  16.7  a             16.7  a 


'^Seeds  were  in  moist  medium  for  2  days,  then  exposed  to 
chemical  concentrations  for  5  days. 

^Each  treatment  was  replicated  3  times  with  6  seed  per 
replication. 

^Means  not  having  a  following  letter  in  common  are 
significantly  different  at  the  5%  level. 


Fig.  10.   Gas  chromatogram  of  diazomethane-solvent  control.   Retention 
time  is  in  minutes.   (See  Table  6  for  parameters  of  gas 
chromatograph , ) 


60 


9SUOClSG^ 


00 


O 


00 


C\J 


0 


CD 


00 


O 


61 


If) 
c 
O 
Q. 
t/) 
<D 

q: 


_M 


J 


0       4 


8 


Time 


0       4        8       12 


Fig.  11.   Gas  chromatograms  of  L-mandelic  acid(a)  and 

p-hydroxybenzoic  acid(b)  treated  for  30  minutes 
with  diazomethane.   Retention  time  is  in  minutes. 
(See  Table  6  for  parameters  of  gas  chromatograph. ) 


Fig.  12.   Gas  chromatogram  of  an  ethanol  extract  from  peach  seeds  treated 
for  30  minutes  with  diazomethane.   Retention  time  is  in  minutes. 
(See  Table  6  for  parameters  of  gas  chromatograph. )   (M= 
L-mandelic  acid  and  pHBA=  p-hydroxybenzoic  acid.) 


63 


^i-v.-Z^ 


o 


CM 

en 


c\j 


CD 


CD 


00 


^ 


O 


GSUOdSG^ 


Table  12. — Comparison  of  retention  times  of  p-hydroxybenzoic 
acid  and  L-mandelic  acid  as  influenced  by  various 
acetylation  procedures. 


64 


Acetylation  procedure 


Retention  times  (minutes) 


Extract 


Standard 


1-diazomethane,  30  minutes; 
p-Hydroxybenzoic  acid 
L-Mandelic  acid 

2-diazomethane,  3  hours: 
p-Hydroxybenzoic  acid 
L-Mandelic  acid 


4.10 

4.10 

2.05 

2.05 

11.80 

11.80 

4.57 

4.57 

3-diazobutane,  3  hours: 

p-Hydroxybenzoic  acid 
L-Mandelic  acid 

Methyl  derivative 
Butyl  derivative 


12.60 

4,48 
6.70 


12.90 

4.48 
6.35 


^Refer  to  Materials  and  Methods  for  the  details  of  each 
procedure. 


65 


that  L-mandelic  acid  and  p-hydroxybenzoic  acid  were  present  in  peach 
seeds  after  48  hours'  imbibition.   However,  only  trace  amounts  of  L- 
mandelic  acid  could  be  detected  in  the  extract  (Table  13).   On  the  other 
hand,  sufficient  quantities  of  p-hydroxybenzoic  acid  were  present  for  an 
estimation  of  amounts  in  the  tissue.   Based  on  peak  area  comparison  of 
components  of  the  extract  with  the  standard,  it  was  estimated  that  ap- 
proximately 0.12  ug  of  p-hydroxybenzoic  acid  was  present  in  1  g  wet 
weight  of  seed  tissue  under  the  conditions  of  the  tests. 


Table  13. — Comparison  of  peak  areas  of  p-hydroxybenzoic 
acid  and  L-mandelic  acid  as  influenced  by 
various  acetylation  procedures. 


66 


Acetylation  procedure'^ 


9 

Peak  area,  mm 


Extract     Standard^ 


1-diazomethane,  30  minutes! 
p-Hydroxybenzoic  acid 
L-Mandelic  acid 

2-diazoraethane,  3  hours: 
p-Hydroxybenzoic  acid 
L-Mandelic  acid 


45.0 

540.0 

Approx .  5.0 

597,0 

49.0 

342.0 

Approx.  5.0 

675.0 

3-diazobutane,  3  hours: 
p-Hydroxybenzoic  acid 
L-Mandelic  acid 

Methyl  derivative 
Butyl  derivative 


30.0 

Approx.  5.0 
Approx.  5.0 


545.0 

996.0 
812.0 


Refer  to  Materials  and  Methods  for  the  details  of  each 
procedure. 

^Each  standard  represents  2  ug  of  the  respective  compound. 


DISCUSSION 

A  quantitative  method  was  devised  to  determine  the  amount  of  benz- 
aldehyde  and  raandelonitrile  in  peach  seeds.   The  method  was  designed  to 
quantitatively  obtain  the  2  volatile  components  in  an  ethereal  solution 
so  it  could  be  analyzed  by  gas  chromatography.   Grinding  of  the  frozen 
tissue  and  extraction  at  a  low  temperature  prevented  enzymatic  release 
and  destruction  of  benzaldehyde  and  mandelonitrile.   Keeping  the  ethereal 
solution  cool  and  immediate  analysis  prevented  loss  by  volatilization. 
Also,  the  analysis  by  gas  chromatograph  was  done  before  and  after  reac- 
tion with  aqueous  sodium  bisulfite  and  again  after  reacting  the  benz- 
aldehyde-sulf ite  addition  product  with  potassium  cyanide  to  form  mandelo- 
nitrile.  The  chemical  reactions  used  are  well  known  (26),  and  the  deter- 
mination of  pure  benzaldehyde  and  mandelonitrile  by  the  technique  de- 
scribed was  shown  to  be  quantitative. 

Using  this  procedure,  the  quantity  and  rate  of  release  of  mandelo- 
nitrile and  benzaldehyde  were  studied  in  relation  to  germination.   The 
data  indicated  that  a  lag  time  existed  in  excised  seeds  for  the  maximum 
release  of  mandelonitrile  and  benzaldehyde.   The  intact  seeds  and  those 
treated  with  thiourea  differed  only  in  magnitude  and  the  thiourea-treated 
seeds  exhibited  a  secondary  peak  at  120  hours. 

The  maximum  period  of  gemiination  of  the  excised  seeds  was  about 
48  hours  after  the  maximum  period  of  release  of  benzaldehyde  and  mandelo- 
nitrile whereas  the  greatest  period  of  germination  of  seeds  treated  with 
thiourea  occurred  about  160  hours  after  the  second  peak  of  release  of 

67 


68 

benzaldehyde  and  mandelonitrile.   Thus,  there  was  no  indication  that 
either  benzaldehyde  or  mandelonitrile  was  correlated  with  an  inhibition 
of  germination  under  conditions  of  these  tests.   Yet,  when  concentrations 
of  benzaldehyde  and  mandelonitrile  are  present  in  tissues  at  concentra- 
tions of  11.0  and  4.2  mg/g  respectively,  they  would  be  affecting  the 
system.   Thus,  the  2  compounds  may  have  a  temporary  influence  on  germi- 
nation, and  it  could  be  possible  under  certain  circumstances  a  factor 
contributing  to  dormancy. 

Determinations  were  also  made  of  the  content  of  mandelonitrile  and 
benzaldehyde  present  in  peach  seeds  at  weekly  intervals  during  chilling. 
Only  trace  amounts  were  observed  at  the  various  times  of  sampling.   Thus, 
it  seems  that  the  majority  of  mandelonitrile  and  benzaldehyde  was  re- 
leased between  about  72  and  96  hours  after  the  start  of  imbibition. 

This  was  the  first  reported  instance  of  the  detection  and  measure- 
ment of  mandelonitrile  in  peach  seeds.   It  may  be  significant  that  the 
quantity  of  mandelonitrile  present  was  much  greater  than  that  of  benz- 
aldehyde.  This  would  indicate  that  the  hydrolysis  of  amygdalin  in  in- 
tact seeds  was  not  the  same  as  in  seed  homogenates.   In  the  latter  case 
the  products  formed  are  benzaldehyde  and  cyanide  with  the  mandelonitrile 
considered  an  unstable  intermediate  (104). 

Interest  was  also  directed  at  the  presence  of  phenolic  acids  in 
peach  seeds.   Using  gas  chromatographic  techniques,  benzoic,  o-hydroxy- 
cinnamic,  2, 6-dihydroxybenzoic,  o-hydroxybenzoic,  p-hydroxybcnzoic, 
2,4-dimethoxybenzoic  and  mandelic  acids  were  isolated  and  tentatively 
identified.   Of  primary  interest  was  p-hydroxybenzoic  acid  since  it  had 
been  reported  in  the  literature  as  having  growth  regulator  actions  (105). 
With  this  in  mind,  it  was  necessary  to  determine  if  p-hydroxybenzoic  acid 


69 

existed  in  peach  seeds.   Using  extraction  procedure  for  phenolic  acids 
(113),  gas  chromatographic  analysis  of  various  derivatives  were  made  of 
the  extracted  components.   Positive  identification  was  made  for  p-hydrox- 
ybenzoic  and  a  strong  indication  was  noted  that  L-mandelic  acid  was 
present  in  the  seeds.   The  latter  has  been  reported  to  occur  in  peach 
seeds  (R.  H.  Biggs,  Unpublished  data),  and  found  to  inhibit  germination 
of  alfalfa  seeds  at  10~°  M  concentrations.   Jones  and  Enzie  (46)  identi- 
fied a  growth-inhibiting  substance  from  peach  flower  buds  as  being 
mandelonitrile. 

Since  degradation  products  of  amygdalin  were  found  to  occur  in 
seeds,  attempts  were  made  to  assess  the  influence  of  cyanide,  benzalde- 
hyde  and  mandelonitrile  on  germination.   Cyanide  treatments  indicated 
that  only  at  the  highest  concentration  tested,  1.0  M,  was  an  inhibitory 
influence  shown.   Recently,  it  has  been  reported  that  some  plants,  par- 
ticularly Vicia  sp.,  have  the  capability  of  metabolizing  cyanide  and 
converting  it  into  non-toxic  compounds  (34,  69).   It  was  observed  that 
hydrogen  cyanide  (■'■'^C)  was  incorporated  into  asparagine  in  a  number  of 
plant  species.   This  was  thought  to  be  accomplished  by  cyanide  coupling 
with  serine  directly  to  form  the  4-carbon  chain  of  beta-cyanoalanine. 
The  beta-cyanoalanine  could  then  form  asparagine,  or  by  addition  of  a 
gamma-glutamyl  group,  form  gamma-glutamyl-beta-cyanoalanine.   It  was 
concluded  that  cyanide  had  little  influence  on  germination,  except  at 
concentrations  considered  quite  high.   Interestingly,  this  indicates 
that  peach  seed  do  contain  a  cyanide-resistant  mechanism  for  respiration. 
Furthermore,  the  subsequent  seedlings  were  much  greener  and  exhibited 
other  characteristics  that  accompany  nitrogen  fertilization.   Thus,  it 
was  concluded  that  the  tissues  were  incorporating  cyanide. 


70 

In  contrast  to  the  results  of  cyanide  treatments,  concentrations  of 
4.2  mg/g  mandelonitrile  and  11.0  mg/g  benzaldehyde  completely  inhibited 
germination  of  excised  embryos.   It  was  noted  that  mandelonitrile  in- 
hibited at  a  weaker  concentration  and  that  the  intermediate  concentra- 
tion of  both  compounds  had  an  action  that  was  reversible.   Thus,  if  high 
enough  concentrations  of  benzaldehyde  or  mandelonitrile  did  occur  in 
seeds  they  could  be  inhibitory  and  the  action  could  be  transitory  if  the 
compounds  were  subsequently  degraded  (104). 

The  possibility  that  subsequent  derivatives  of  benzaldehyde  could 
be  involved  in  seed  germination  was  investigated.   Thus,  the  influence 
of  benzoic  and  p-hydroxybenzoic  acids  on  dormant  peach  seeds  was  tested. 
Benzoic  acid  had  little  influence,  but  concentrations  of  p-hydroxybenzoic 
acid  at  3  X  10"^  and  10~  M  significantly  increased  the  degree  of  germi- 
nation as  compared  to  the  control. 

The  fact  that  p-hydroxybenzoic  acid  has  been  found  to  have  growth 
regulatory  properties  (105)  and  its  presence  in  peach  seeds  suggested 
that  it  may  play  a  role  in  dormancy.   The  growth  regulatory  activity  of 
p-hydroxybenzoic  acid  has  been  established  for  woody  cuttings  of  Ribes 
rubrum  (105).   Pilet  (78)  has  reported  that  p-hydroxybenzoic  acid  at 
low  concentrations  causes  a  stimulation  of  the  growth  of  stem  sections, 
while  at  high  concentrations  it  inhibits  growth.   The  inhibition  was 
apparently  due  to  the  stimulation  of  lAA-oxidase  and  subsequent  decrease 
in  auxin  level  (116).   Also,  the  activation  observed  for  lower  concentra- 
tions of  p-hydroxybenzoic  acid  indicated  that  it  acted  on  several  other 
biochemical  processes  which  were  connected  with  growth  (78). 

In  Ribes  rubrum,  p-hydroxybenzoic  acid  was  present  in  the  range  of 
0.2  -  1.0  ug/g  of  fresh  tissue  (105).   The  quantity  found  in  peach  seeds 
was  approximately  0.12  ug/g  of  fresh  tissue.   Thus  it  appears  that  the 


71 

tissue-levels  of  p-hydroxybenzoic  acid  in  both  dormant  Ribes  woody  stems 
and  dormant  peach  seeds  were  similar.   In  the  case  of  Lens  stems,  inter- 
node  sections  were  stimulated  to  elongate  at  10~  M  concentration  (78). 
At  higher  concentrations,  the  growth  of  stem  sections  was  inhibited. 
The  quantity  isolated  from  peach  seeds  was  in  the  range  that  was  inhibi- 
tory in  the  Lens  bioassay.   Thus,  it  could  be  inhibitory  to  the  seeds. 
However  the  data  was  such  with  peach  seeds  that  this  point  can  be  con- 
sidered a  matter  of  conjectual.   Yet,  it  was  shown  that  p-hydroxybenzoic 

-2      -1 
acid  would  stimulate  peach  seed  germination  at  3  x  10   to  10   M  con- 
centration.  This  stimulation  was  from  adding  p-hydroxybenzoic  acid  to 
the  external  media  for  120  hours.   Thus,  the  internal  concentration  could 
have  been  much  lower.   The  bioassay  would  not  demonstrate  an  inhibitory 
effect. 

The  ethanolic  extracted  inhibitory-complex  obtained  from  peach 
seeds  was  studied  with  the  use  of  paper  chromatography  and  other  physical 
treatments  in  order  to  obtain  some  clues  as  to  its  identity.   The  R^ 
values  obtained  on  paper  chromatography  correspond  with  the  inhibitory 
area  obtained  by  Bennet-Clark  and  Kefford  (8)  using  the  same  solvent 
with  an  alcoholic  extract  from  Ribes  sp.   This  inhibitory  area  was  termed 
the  beta-inhibitor  complex.   Recently,  the  beta-inhibitor  concentrated 
from  an  acidic  fraction  from  extracts  of  dormant  maple  buds  was  thought 
to  be  a  complex  of  phenolic  substances  (86) .   However,  the  phenolic  com- 
pounds described  were  found  not  to  be  identical  with  any  of  the  phenolic 
compound  previously  proposed  as  being  members  of  the  beta-inhibitor 
complex.   Recently,  many  of  the  phenolic  compounds  associated  with  the 
beta-inhibitor  complex  have  been  identified.   Koves  and  Varga  (53)  re- 
ported the  identification  of  many  phenolic  compounds,  among  which  were 
several  of  the  hydroxybenzoic  acids. 


72 

The  data  on  the  level  of  the  inhibitor-complex  during  the  chilling 
period  showed  that  it  did  not  change  drastically.   In  fact,  at  the  end 
of  chilling  period,  the  level  seemed  to  be  greater  than  anytime  during 
chilling.   This  finding  was  in  line  with  that  found  by  Villiers  and 
Wareing  (106,  107)  for  dormant  organs  of  Fraxinus  excelsior.   Briefly, 
chilling  has  no  effect  on  the  level  of  inhibitors  in  the  tissue  but  ter- 
mination of  dormancy  was  accompanied  by  a  buildup  in  growth  promotors  in 
the  seed.   This  may  be  the  case  for  peach  seeds  since  the  capacity  to 
germinate  increased  with  increases  in  the  duration  of  the  chilling  period. 

The  products  of  amygdalin  degradation,  mandelonitrile,  benzaldehyde 
and  cyanide  do  not  appear  to  directly  influence  the  breaking  of  peach 
seed  dormancy.   However,  it  would  seem  that  a  hydroxylated  derivative, 
p-hydroxybenzoic  acid,  of  the  benzaldehyde  oxidation  product,  benzoic 
acid,  exhibits  some  stimulatory  influence  upon  dormant  peach  seeds. 
Furthermore,  L-mandelic  acid,  as  well  as  other  phenolic  compounds,  may 
be  involved  in  peach  seed  dormancy. 

The  induction  of  germination  of  peach  seed  by  thiourea  substantiated 
previous  reports  that  this  chemical  will  terminate  seed  dormancy  (76, 
97).   Furthermore,  it  supported  earlier  observations  (30,  80)  that  the 
growth  of  the  subsequent  seedlings  was  not  abnormal  if  the  proper  envi- 
ronmental condition  were  maintained  during  germination. 


SUMMARY  AND  CONCLUSIONS 

Investigations  were  initiated  to  determine  the  relation  of  certain 
phenolic  compounds  to  peach  seed  germination.   The  phenolic  compounds 
of  primary  interest  were  those  which  are  degradation  products  of  the 
glucoside,  amygdalin,  namely,  mandelonitrile  and  benzaldehyde,  and  their 
immediate  by-products.   The  following  conclusions  were  made  based  on  the 
research  conducted: 

1.  Mandelonitrile  and  benzaldehyde  at  1.4  to  11.0  mg/g  of  perlite 
inhibited  germination  of  excised  embryos,  but  did  not  stimulate  dormant 
seeds  to  germinate.   Quantitative  determinations  of  these  2  compounds 
from  peach  seeds  by  gas  chromatography  indicated  that  the  majority  of  the 
mandelonitrile  and  benzaldehyde  was  released  between  72  and  96  hours 
after  the  start  of  imbibition  and  thereafter  only  trace  amounts  could  be 
observed.   Only  at  this  time  was  the  tissue-level  high  enough  to  be  con- 
sidered inhibitory  to  germination,  yet  it  showed  no  correlation  with 
germination.   Furthermore,  determinations  made  at  weekly  intervals  during 
chilling  indicated  that  only  trace  amounts  were  present  at  any  of  the 
sampling  times  during  chilling.   Therefore,  it  was  concluded  that  mandelo- 
nitrile and  benzaldehyde  have  no  direct,  inhibitory  or  promotive  influ- 
ence on  the  germination  of  peach  seeds. 

Cyanide  had  little  effect  on  reducing  the  per  cent  germination  at 
concentrations  less  than  1.0  M.   From  observations  on  the  increased  size 
of  seedlings  in  several  cyanide  treatments,  it  was  postulated  that  the 
tissues  were  incorporating  cyanide,  however  no  measurements  of  the  in- 
crease in  glucoside  content  was  made. 

73 


74 

2.  The  fact  that  phenolic  acids  could  be  produced  in  dormant  peach 
seeds  as  a  result  of  the  metabolism  of  mandelonitrile  and  benzaldehyde, 
led  to  an  investigation  of  phenolic  acids  in  dormant  peach  seeds,  and 
several  phenolic  acids,  including  the  hydroxy  and  methoxy  derivatives  of 
benzoic  acid,  were  found.   Of  prime  interest  was  the  finding  that  p-hydrox- 
ybenzoic  acid  at  concentrations  of  3  x  10"^  to  lO"-""  M  would  slightly  stim- 
ulate the  germination  of  dormant  peach  seeds..  However,  quantitative  de- 
terminations showed  that  approximately  0.12  ug  of  p-hydroxybenzoic  acid 
was  present  per  1.0  g  of  tissue  on  a  wet  weight  basis  which  was  below 
that  found  to  be  necessary  in  the  external  media  for  germination.   Deter- 
minations of  the  tissue-levels  of  p-hydroxybenzoic  acid  in  germinated 
seed  was  not  conducted.   L-mandelic  acid,  a  product  of  mandelonitrile 
hydrolysis,  was  also  shown  to  be  present  in  amounts  in  the  range  of  0.005 
to  0.05  ug/g  of  fresh  tissue.   The  influence  of  L-mandelic  acid  on  peach 
seed  germination  was  not  studied. 

3.  The  inhibitor-complex  level  of  peach  seeds  which  appeared  on 
paper  chromatograms  at  an  R^  of  0.6  -  0.8  was  found  to  be  essentially  the 
same  after  chilling  as  prior  to  chilling.   Thus  this  complex  does  not 
appear  to  be  involved  in  the  maintenance  of  dormancy  of  peach  seeds. 

The  inhibitory-complex  had  similar  characteristics  to  the  beta-inhibitor 
complex  reported  to  be  found  in  other  plant  tissues. 

4.  Experiments  with  thiourea  supported  previous  research  and  showed 
that  the  time  of  embryo  removal  from  the  seed  coat  and  associative  tissue 
after  seed  imbibition  had  little  influence  on  the  amount  of  abnormal 
seedling  production. 


APPENDIX:      GAS   CHROMATOGRAMS   OF   STANDARDS 


/ 


76 


0) 

en 

c 

O 
Q. 

to 

q: 


0       4 


0       4       8 


Time 


Fig.  13.   Gas  chromatograms  of  the  propyl  esters  of  benzoic 
acicl(a)  and  mandelic  acid(b).   Retention  time  in 
minutes.   (See  Table  6  for  parameters  of  gas 
chromatograph . ) 


77 


u 


4 


!! 

i  ! 


I     « 


iFii 


i-i'l 


I 


--J  \j 


U 


%^ 


10 

I   &9 


14. 


Gas   Ghroinatogi*ai.is   ei   the   propyl   ggtoi'g   of   o-liydroxybenseie 
acid(a)   and  p-hydroxyboHKoio  acid(b).     Retention  time  in 
minutes.      (See  Table  6  for  parameters  of  gats  ehromatogi-aph. ) 


78 


(D 

c 

o 

CO 

q: 


0 


4       8 

Time 


12     16 


Fig.  15.   Gas  chromatograms  of  the  propyl  ester  of 

2, 6-dihydroxybenzoic  acid.   Retention  time 
in  minutes.   (See  Table  6  for  parameters 
of  gas  chromatograph . ) 


79 


0) 
(J) 

C 

o 

Q. 
if) 
(D 

Q:: 


0       4       8      12     16 

Time 


Fig,  16.   Gas  chromatograms  of  the  propyl  ester  of 

2,4-dimethoxybenzoic  acid.   Retention  time 
in  minutes.   (See  Table  6  for  parameters  of 
gas  chromatograph, ) 


80 


0) 
fj) 

c 

o 

Q. 

CO 

0 

q: 


0       4       8       12 

Time 


Fig.  17,   Gas  chromatograms  of  the  propyl  ester  of 
o-hydroxycinnamic  acid.   Retention  time 
in  minutes.   (See  Table  6  for  parameters 
of  gas  chromatograph. ) 


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BIOGRAPHICAL  SKETCH 

The  author,  James  Bruce  Aitken,  was  born  August  1,  1938,  in  Orlando, 
Florida.   He  received  his  secondary  education  at  the  Lakeview  High  School 
in  Winter  Garden,  Florida  between  the  years  of  1953  and  1956.   He  at- 
tended Clemson  University  in  Clerason,  South  Carolina  and  was  granted  the 
degree  of  Bachelor  of  Science  with  major  in  Agriculture  in  January,  1962 
and  was  also  granted  the  degree  of  Master  of  Science  with  major  in 
Horticulture  from  Clemson  University  in  January,  1964. 

In  1964,  he  was  granted  an  assistantship  from  the  Department  of 
Fruit  Crops,  University  of  Florida  to  study  toward  the  degree  of  Doctor 
of  Philosophy.   He  entered  the  University  of  Florida  in  April,  1964,  and 
completed  his  work  towards  the  degree  of  Doctor  of  Philosophy  in  August 
1967. 

He  is  a  member  of  Alpha  Zeta,  Phi  Sigma  and  Gamma  Sigma  Delta  hon- 
orary fraternities.   He  is  also  a  member  of  the  American  Association  for 
the  Advancement  of  Science,  and  The  American  Society  for  Horticultural 
Science. 

He  is  married  to  the  former  Patricia  Ann  Dillard  and  they  have  one 
daughter,  Amy. 


90 


This  dissertation  was  prepared  under  the  direction  of  the  chairman 
of  the  candidate's  supervisory  conunittee  and  has  been  approved  by  all 
niembers  of  that  conuiiittee.   It  was  submitted  to  the  Dean  of  the  College 
of  Agriculture  and  to  the  Graduate  Council,  and  was  approved  as  partial 
fulfillment  of  the  requirements  for  the  degree  of  Doctor  of  Philosophy. 


August,  1967 


Supervisory  Committee; 


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Dean,  College  of  Agriculture 


Dean.  Graduate  School 


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