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New York
State Gullege of Agriculture

At Gornell University
Ithara, N. YU.

Library

Peg. ElarkFtbolog gl

whree papers by TOalNius POLITIO, Honoriry aseis tant
in the Botanical Institute of the xoyal PCG

University of Pavia,

Trunslations by U. 6. vept. Workers

Copies received from C. J. Humphrey, september, 1923.

the titles of the papers are as follows:

On the Wlaioplasts in-ono- and vicotyledonous rlants.
lranslation of; ougli elaioplasti nelle mono- e
dicoviledoni. «Atti Ist. Bot. Unit. ruvia, 11,
14:335-361. pl. 13-15. 1914;

On special Cellular Bodies Which Form Anthocyanine.

à translation of: sopra speciali corpi cellulari
acti Ist. Bot. Univ.

che formano antocianine.
Pavia, Il, 14:363-:76. pl. 16-18, 1914;

On a special Cellular Body Found in luo Orchids.
lranslation of; sopra uno speciale corpo cellulare
trovato in due orchidee. a translation a atti
ist. Bot. Univ. Pavia, Li, Meff-Lo5, pl. 49,

1914.

i DEPT. OF PLANK paTnotoey|

73 zope fr
3 83 0 A
sate,
thout petto.

at

rey x

Cornell University

Library

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924000663470

Politis, Ioannes, Honorary Assistant in the Botanical Institute of the Royal
University of Pavia.

ON THE ELAIOPLASTS IN MONO- AND DICOTYLEDONOUS PLANTS. Translation
of: Sugli elaioplasti nelle mono- e dicotiledoni. Atti Ist. Bot»

Univ. Pavia, II, 14: 335-361. pl. xiii-xv, 1914.

In 1888 Wakker (6) ® noted for the first time within the cytoplasm of

the epidermal cells of young leaves of Vanilla planifolia and of VY. aromatica

latifolia some special bodies very strongly refractile to light, to which he
gave the name elaioplasts (formed from olio), because they consisted of a
fundamental plasmic substance in which were found included a fatty or oily
substance. About five years later Zimmermann (9), having found in the perianth

of Funkia coerulea similar bodies, took up the study of the question and ex-

amined many species belonging to a large number of families among the Mono-
cotyledoneae. Studying the elaioplasts from a morphologic point of view and
that of their diffusion, the result of this research was that he found the
elaioplasts in five other genera, of which three belonged to the Liliaceae,
one to the Amaryllidaceae, and one to the diciddscoas

The first, howéver, to study the development of the bodies in question
was Raciborski in 1893 in the genera Ornithogalum, Albuca, Funkia, and Gagea.

Raciborski (5) noted that. in these the elaioplasts appearei as little
spheres strongly refractile to light, always close to the cell nucleus; that
they did not participate in nuclear division and that they maltiplied by
neoplasia from the cytoplasm. In Ornithogalum stachyoides the elaioplasts

would multiply, according to the author, by gemmation.

a
Reference is made by number to "Literature cited
translation,

~ 2
A little later Zimmermann (10) established the presence of elaioplasts
in the stele and in the subepidermal assimilatory tissue of Psilotum, in the

internal epidermis of the lobes of the perianth of Maxillaria picta, in the

epidermis and in the parenchyma of the perianth and in the floral axis of

Beschorneria bracteata.

Finally, Kuester, comparing the elaioplasts to the oily bodies of
mosses, found that the former differed from the latter by their behavior in
their stroma and in the oily substance, but were similar to them in structure.

With the work of Kuester (3) concludes the series of publications
which, so far as I mow, have been written on elaioplasts, which, as one sees,
were encountered in only a few genera of monocctyledons, namely, five of the

Liliaceae, two of the Amaryllidaceae, and three of the Orchidaceae.

In the dicotyledonous piante nobody has Sugar a; ad

Last year I encountered in the epidermal cells of scales of function-
ally active bulbs of Hippeastrun aulicum some spherical bodies strongly re-
fractile to light, joined tegether in the form of bunches of grapes, which
in their physical properties and in their microchemical reactions were
identical to those which Wakker, Zimmermann, and Raciborski described under
the name of elaioplasts.

This fact led me to the following researches the results of which are

summed up in this contribution.

a The cellular organs observed by R. Beer (On elaioplast - Ann. Bot.
XXIII, 63-1909) in various parts of the flower of a Gaillardia can not, in
my opinion, be ascribed to elaioplasts, put to chromatophores, since in the
elaioplasts neither chlorophyll nor cther pigments are formed.

ize oe | |
. (FEST. PF Bay PATHOLOCY)

& 5 apo

I have studied many species belonging to coverià distorta families vA
of the vegetable kingdom and have established the o E io al
in 24 new genera belonging to the Monocotyledoneae, such as the Liliaceae,
Amaryllidaceae, and Orchidaceae, and also to the Iridaceae, and later also
in certain dicotyledonous plants, namely, in all species which I have
studied belonging to the family Malvaceae.

I have found elaioplasis in the following species:

\

Liliaceae: Alo& rhodacantha, Dc,, Drimia undulata Jacq., Yucca

filamentosa Linn., Ruscus racemosus Linn.

Amaryllidaceae: Clivia nobilis Lindl., Fucharis subdentata Benth.,

vHimantophyllum cyrtanthiflorum Groenl. s Himantoplyllum miniatum Groenl.,

Hippeastrum aulicum Herb., Hinpeastrum vittatum Herb., Hippeastrum reti-

culatum Herb., Haemanthus albiflos Facq., -Haemanthus coccineus Linn.,

Sternbergia Fischeriana Rupr., Sternbergia lutea Orph., Sternbergia ma-

er antbha J., Poliantnes tuberosa Linn.

Iridaceae: Watsonia humilis Mill.

Orchidaceae: Brassia brachiata Lindl., Cattleya Harrisoniae Pazt.,

Laelia anceps Lindl., Lycaste aromatica Lindi., Lycaste Skinneri Lindl.,

Miltonia Clowesii Lindl., Tetramicra bicolor Rolfe, Cymbidium aloifolium

Sw., Cymbidium Lowianum Reichb., Oncidium sphacelatum Lindl.

Malvaceae: Hibiscus Rosa-sinensis Linn., Hibiscus herbaceus Vell.,

Hibiscus liliiflorus Cav., Hibiscus tricolor Dehnh., Hibiscus syriaces

Lim., Althaea rosea Cav., Malva rotundifolia Cav., Malva sylvestris Lim.,

Gossypium arboreum Parl., Boethea cauliflora Nees.

ke

The study of the elaioplasts in these species was undertaken by me
from the microchemical standpoint and from the morrhological and biological
points of view, and I hope my observations may complete the contributions
reported in preceding papers.

Method: In the study of the plasmatic stroma of the organ in question
I have used as fixative solutions an alcoholic solution of corrosive sublimate;
alcoholic solution of picric acid, and a solution of absolute alcohol containing
© per cent of acetic acid.

In the monochromatic staining I have made use of fuchsine, eosine,
aurantine and erythrosine, and later have preferably used Zimmermann's mixture
(iodine green and fuchsine).

With these reagents I was able to demonstrate that the stroma is
eminently erythrose and nearly always presents an affinity for stains similar
to that of the nucleolus.

In the study of the distribution of the elaioplasts in diverse parts
of the single species, I availed myself almost exclusively of fresh material,
since these bodies because of their strong refractility and the characteristic
forms which they present are easily recognizable, and it would, I believe, be
unnecessary to have recourse to special stains. However, in those cases in
which the elaioplasts might be confused with the nucleus or with other bodies
included in the cytoplasm I used osmic acid, a solution of potassium iodide,
Sudan III, and Scarlet R.

With the first of these reagents, employed also by Zimmermann, the
elaioplasts in a few minutes turned brown or blackisa.

With the second of these reagents, the starch granules took on, ac-
cording to the length of time the reagent was allowed to act, a violet color

or azure blue more or less intense. The protoplasm stained a golden brown,

- 5
“the nucleus and the plastids, vrown; the elaioplasts, on the contrary, a more
or less intense brown, remained distinct from the mucleus, however, because
of the intensity of the stain and because of its [the elaioplast's] form and
diverse structure, particularly because of its mass subsequent to the con-
timed action of the reagent, and the drops cf some unstainable oil.
° With Sudan III the elaicplasts stain intensely red.

I used for this latter reagent an 80 per cent alcoholic solution and
to get better results first gently heated the sections before subjecting them
to the action of the reagent, since the oily substance of the elaioplasts,
which is easily soluble in alcohol, becomes somewhat insoluble when, as noted
by Raciborski, it is later heated. Ordinarily a few moments of inmersion in
the staining solution suffices because of the intense staining of the
elaioplasts. The sections taken from the stain are then carefully washed
with water and examined in water or in glycerine.

Finally I have obtained singularly instructive preparations using

Scarlet R (2) in the same manner as I did Sudan III.

Special Part.

Polianthes tuberosa Linn.

The elaioplasts are found in this species in the cell cytoplasm in
nearly all of the young epidermis and are of special importance because of
their notable size, which they attain in the adult stage, a fact which
facilitates greatly their study.

I observed in living celìsfa structure] strongly refractile to light

and possessing a color which varied from pale yellow to brown,

cs)

when viewed in their sections made in diverse directicns one arrives
at the conclusion that the slaioplasts are of spheroidal form. This form
changes frequently with the growth of the elaioplasts becoming irregular.
They possess a granulose or spongy structure and consist of a fundamental
protedmaceous substance in which one finds included an oily substance. These
bodies ultimately presented the following reactions:

They are easily soluble in aicohol, ether, carbon bisulphide, and
benzine; insoluble in acetic acid and chloral hydrate.

With tincture of Alkama they stain red, with Sudan III intensely
red, and with copper acetate after prolonged action they take on no particular
color.

On the other hand, the fundamental proteinaceous substance is recognized
by the brown color which it assumes with the iodine preparation, by the rose
red reactiong which is produced by Millon's reagent (especially if it is

‘applied by warming), by the yellow color which it acquires when treated with
nitric acid (or with nitric acid and ammonia), and finally by the red color
which it taxes on when stained with Raspail's reagent.

So far as concerns the development [of the bodies], it is to be noted
that in the epidermal cells of the apex cf a very young floral axis of this

species (Polianthes tuberosa) the elaioplasts apvear as very minute spherical

bodies strongly refractile to light, close to the cell nucleus. They multiply
by neoplasia from the protoplasm. Not infrequently, however, it follows that

they [the elaioplasts] participate in the process of cell division by the di.

7

partition of their plasma, a process vhich takes Place passivelyi
In the culminating point of the process of nuclear division, in the
division, that is, of the chromosones, end, in general, in the phenomena

. È an
which occur in the process of kariokinesis, the elaioplasts do not take/sactive

7?

part and, therefore, the abovewindicated division, I believe, has no
fundamental importance. On the contrary, it remains as a passive division
with the bipartition of thé plasma after the manner followed in this case,
In fact, during nuclear division the elaioplast remains inert and almost to
the moment in which bhe new membrane which begins to form comes against it.
Then it bends and takes on a semilunar form. The extremity of the thin membrane
forms and then splits into incurved points generally in two unequal parts.

With increase in size of the cell the elaioplast, grows in volume and
at complete development exceeds the dimenpions of the nucleus, becoméng often
four or five times larger than the latter. Later the elaioplast of the epi-
dermal cell, where it is found at a certain distance from the extremity of
the apex of the floral axis, begins to undergo phenomena of degeneration.
In fact, it diminishes in volume, becomes impoverished in gaily substance, and
presents some scattered vacuoles in its mass among which often there is to
be distinguished a large central vacuole. Finally in the cell, as development
advamces, nothing more than a residuum of the elaioplasts can be found, and
frequently also this comes to be completely reabsorbed in the cytoplasm in
which it is immersed.

It is to be noted that in this species the elaioplast remains almost
always throughout its life close to the cell nucleus,

In the species in question the elaiopiasts are encountered in the
epidermis and in the parenchyma of the perianth, of the floral axis and of
the peduncle, of the stamens, of the pistils, and of the young leaves; they

are absent in the ovule, in the seed, in the adult fruit, and in the roots,

Cc

Haemanthus albiflos Facq.; H. &occineus Linn.

In all the species of this genus which I have studied the elaioplasts
appear, on microscopic examination, in the adult stage, under various forms,
which may be considered as variations of a fundamental type of globose form.
They frequently appear continuous, although in reality their form results
from the union of many minute spheres which may be separated from one another
by pressure on the cover glass of the preparation. Fach of these spheres
consists of a plasmatic stroma in which is found included an oily substance.
With osmic acid (solution 1 per cent) the elaioplasts become almost instan-
taneously black (Pl. XIV, fig. 4). After treatment for a few seconds with
Sudan III the single spherical elements which constitute the elaicplasts
coalesce and from their mass oily drops appear, while the mass and the drops
assume an intense red color. The oily substance resists the action of
acetic acid and of chloral hydrate, but dissolves easily in absolute alcohol,
ether, chlorpform, and benzineg It, however, resists the action of these
latter solvents following heating or following the action of osmic acid,

The elaioplasts attain greater dimensions than those of the cell nucleus.
They have a light yellow color and are found in the adult stage close to the
cell nucleus or a little distance therefrom. Their manner of development is

comparable to that described for Polianthes tubefosa.

The elaioplasts of this species are found in the epidermis of ali the
scales of the bulb, in both epidermises of the leaf, and finally in the

epidermis of the floral axis and of the perianth.

Himantophyllum miniatum Groenl., H. Cyrtanthiflorum Groenl.

=

In these species I had occasion to study, in addition to the vegetative
organs, also floral organs. With the aid of methods indicated by Wakker, that
is, plasmolyzation of the cell, I was easily able to demonstrate that the
elaioplast could be found in the cytoplasm. In this case a better reagent is
a 15 per cent aqueous solution of potassium nitrate, with the addition of a
small quantity of eosine. with such a solution, which has a marked power of
disintegration, I brought ab-ut the plasmolysis and the contraction of the
interior protoplasm from which the elaioplasts separated, which remained for
a long time visible and maintained their prinitive form. In the cells of
the ovary, in process of development, the elaioplasts in the younger stage
appeared as littie aggregates of rotund or oval bodies endowed with strong
refractility and frecuertly were cicse to the cell nucleus. Fach group did
not exceed the size cf the ceil nucleus.

Later, however, when the flowers nave reacned almost their ultimate
size and begin to open in the epidermal csils of the ovary, wnich are
preferably chosen for the stuay cf the development of the elaioplasts, these
will wax in volume and in number and each grows conse:ventiy becomes greater
but never attains to the dimensions of tne cell nucieus. n this stage,
which may be the culminating point of their development, the elaioplasts
begin to lose theér original form anc primitive structure ari show phenomena
which, I believe, are tc de resardel as dezenerative.

On the conirary, all the smallest oval or rotunî oedies appear at
first to flow together, forming a single mass. The fusion takes place

gradually in a manner which may follov all tne mimits phases of the total
disappearance of the charact@Pisvic structure of tre elaicplast. This mass,

7

at first homogeneous, then aggregated or grumose, is finally granular,
having a very irregular form frequently with tree-like ramifications

(Pl. XV, fig. 1). It is rofractile and presents yellow reflections.

In its interior it presents in turn one or more central globules, which
are at the starting point of the tree-like ramifica:ion. Such forms show
all the reactions of the proteinaceous substance and are encountered in
epidermal cells of all parts of the open flower but absent in the epidermis
of the leaves and of the floral axis, in the tissue of the lobes of the
anther as well as in the stem and in the pollen grains. It is also useless
to seek for them in the seed, in the ovule, in the root tips, and in the

deeper tissues of all the other organs.

Hippeastrum vittatum Herb., H. reticulatum Herb.

The bulbs of tais species were examined both in the dormant and
functionally active stages. In the external epidermis of the scales of the
bulb during this period of dormancy the elaioplasts assume varied forms
(Pl. XII, figs. 4, 5), whica may be considsred as representing the ultimate
or final phases of their development; in fact, they appear as single masses
without any structure, often very large, strongly refractile to light,
colorless or light yellow.

Such a mass is of most irregular form and may appear now grumose and
lobate, now gramlose with treeelike ramifications, and finally as a col-
lection of very minute rounded bodies. Such masses are immersed in the
cytoplasm. They are generally found close to the cell nucleus and show the
following reactions:

With a solution of iodide of potassium they stain intégely brown,
treated with nitric acid and then with ammonia they take on a yellow color,

The color reaction to Millon's reagent when warmed is brick red.

Treated with sulphate of copper and then with potassium hydrate, they
take on a violet tint.

The substance, therefore, of the mass under examination is of a protein»
aceous nature, Moreover, as is shown, it yet remains to be established,
with the aid of the misrochemical reactions indicated by Zacharias (8) (in
his last work on the chemical constitution of protoplasm and of the mucleus),
toy which of the proteinaceous substances which enter into the constitution
of the cell protoplasm can be ascribed that of the following bodies:

(1) In a 10 per cent solution of sodium chloride they are insoluble
and do not expand.

(3 They swell and are insoluble in hydrochloric acid diluted according

to the following proportions: Four parts of hydrachloric acid to 3 parts of

watere

1

tu

(3) They remain unchangeà in a 20 per cent solution of magnesium sul-
phate.

(4) In methyl greon (used on sections made from fresh material as
well as on those fixed by immersion in absolute alcohol) they show no color.

(6) With the mixture methylene blue plus fuchsine S [ 1 volume
(250 con. H26 plus 0.25 g. fuchsine S) plus 1 volume (250 ccm. H20 plus
0.25 g. methylene blue) ] used on sections treated for 12 hours in 5 per
cent hydrochloric acid and then in absolute alcohol, the bodies and. the
nucleoli stain intensely red; the chromatin of the nucleus blue, the plasma
of the nucleus brilliant red.

(6) In sections treated with carmine acid the body and the nucleoli
remain colorless and the chromosones stain red,

(7) Submitted to th¥ action of glycerinated pepsin for a period
of 20 hours, the bodies and the nucleoli swell up and are not dissolved.

Such reacticns lead cone to tne conclusion ine, the fundamental svd-
siance of the eluiople.ts denaves towaras the atcve-iumed reixzents like
the substance of nucleoli.

As already said, the elaioplasts encountered are found under the
forms above iescribed in the cells of the epidermis cf the scales of dormart
bulbs; in actively functioning bulbs; in-the avygrary; they iseparate or
avpezr in various stages of a dissolutional process (Pi. XIIL, figa 3; È)
while in long cells they are accompanied by new elaioplasts (PI, SLL, fis,
Fu el These last are-of notazle size, colorless, and refractile, and

ike grapes. Each of these

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little spheres consists of a plasmetic stroma ani of an oily substance. The
stroma shows all the reactions indic.ted as characteristic of protetimaceous

substance. And, where the secticnus are fixed in en alconclic soluticn of

corrosive sublimate, they mav ve stained «nith fuchsine, er: throsine, and eosine.

. 13

The oily sudstance is resistant to the action of concentrated hydro-
chloric acid, of acetic acid, of a 50 per cent caustic potash solution, and of
chloral hydrate. It is easily soluble in alcohol, ether, chloroform, xylol,
and benzine. It becomes black with osmic acid and ultimately stains with Sudan
III, Scarlet R, alkanine, and cyanine.

Similar elaioplasts in the adult stage are found in non-determined
positions, which have no relation to tne cell nucleus. They afe ercowed,
in silivion te +. passive movement impressed. upon them ov the cytoplasmic
streaming, with a movement of their own, They appear, in fact, to cross the
cell cavity, to arrive a: ine wall, nesivaste for a fer moments, then recross
tue cell Dy returning to tre point from weich trev departed. Such a movement
is sometimes slow, sometimes rapid, and often interrupted by a rest of greater
cr less length. Contemporaneously with the movemext of translation, they

show a rotatory mcvemert.

Shee:

Besides those which appear in the bulbs, elaioplasts are found in the
epidermal tissues of the ovary.

They, in open flowérs, are of spheroidal and irregular form, resulting

and dimensions

from the union of colorless or pale yellow spherical bodies, the number/ of
which are variable. Their size is often very considerable, since in complete
development they often approach and even surpass, the dimensions of the
nucleus. They have a development similar to that of the elaioplasts of the

Liliaceae, described by Raciborski. In their senile stage their greatest

degree of refractility diminishes and they appear sluggish.

- 14
With their intorpidity appear mumerous vacuoles which give them a
granular or spongy aspect and which, on becoming greater, often form a single
vacuole which, increasing in size, terminates by bringing about the disap-

pearance of the claioplasts.

.

Hippeastrum auticum Herb.

In the cells of the external epidermis of the scales of some dormant
bulbs of Hippeastrum aulicum was found immersed. in the cytoplasm a body which,
so far as I could determine, had not hitherto been observed; and I present
here a summary of the results of a study undertaken «by me as to the chemical
and structural constitution of the same.

The body is endowed with a refractile elevation; it is found situated
in the cell in a non-determinate position and has dimensions which vary from
10 to 20 microns, and in certain cases even more. It possesses a spherical
form and comprises a central part, c, and a peripheral part, b (PI, XIII,
fig. 9).

The central part is of variable form and dimensions, homogenous
structure, and vacuolated. This latter character is that which predominates
and presents bimodality in the diverse phases of the development of the body,
following which the vacuoles are unequal but of uniform type, sparse through»

s

out the mass of the central part, or superior in size to a single central

vacuole surrounded by others of smaller dimensions. Tn not infrequent cases

in which they show marked vacuolization (Pl. XIII, fig. 6) it may be noted

that the central vacucle, increasing and dissolving under other circumstances,

terminates in a manner comparable with the central part. In such a case the

body presents nothing othsr than a very large vacuole limited by the peripheral

portion.

,

15

This is less refractile in the central part, presents a finely
granulose structure, and envelops the central part like an involucre.

Finally, so far as concerns the relation between the central part
and the peripheral part, I observed that in the fixed and stained
preparations the first is almost always separated from the second by
means of a colorless halo (Pl. XIII, fig. 9, a)»

Stuining Methods. In the examinution of the body under consideration

I find fresh material desirable or fixed material treated with different
reagents. As fixatives I have employed absolute alcohol contuining 5 per
cent of acetic acid » platinum chlorideplus acetic acid (Merkel), satur~
ated solution of corrosive sublimate, acetic acid plus osmic acid plus
platinum chloride (Hermann) , Flemming!s solution, Rabl!s solution, Keiser's
solution, chromic acid, alcoholic or ayueous solutions of picric acid, osmic
acid.

Among these I have obtained the best results with the first four.

With regard to the stains, without emomerating «ll the reagents which
I used, I note here only those which have given good results.

With Flemming's triple stain (safranine plus gentian violet plus
Orange g.) the nucleoli and the bodies stained red und the chromatin of the
macleus violet.

With the triple stain Enrlich-Biondi-Heidenhain, used on material fixed
with absolute alcohol plus 5 per cent acetic acid, the nucleus and the body
stained intensely rei.

With acid fuchsine, cosine, erythrosine, and surantia, I obtained a
beautiful stain of the body and of the nucleus. With such stains it is, bopbe

noted that the central part of the body stains more intensely than the

peripheral part,

- 16.

Microchemical Studies. The bodies present the following reactions:

In water, aside from even at high temperature, the body does not
disappear.

In absolute alcohol, even after prolonged immersion, it is resistant.

In sulphuric ether, it is insoluble.

In a solution of iodide of potassium, the central part stains an in-

tense brown and the peripheral part golden busin,
us

With Millon's reagent,if agitated and heated, the central part takes
on a marked brick red color and the periphoral part stains a light rose
yellow.

With Raspail?s solution, the central vart takes on an intense rose
wohLany.while the periphery is stained less intenselya.

Treated with a solution of Trommer's reagent (copper-alkaline solution),
the body assumes a violet color, and this color becomes more distinct in
the central part of the body.

With nitric acid and ammonia, the body assumes a yellow color; with
Sudan III, it stains weakly; with osmic acid it becomes light brown; with
acetate and chloride of iron and with bichromate of potassium, the body
presents no particular color eee: with copyer acetate in aqueous solution
(after some: days) it shows no coloration.

Among the reagents thus listed from the reactions obtained through the
use of Millon, Raspail, and Trommer's soluticns, there remains no doubt as
to the proteinaceous nature of the suostance which forms the central part
of the body, and these reactions indic:te the particization of a substmate

Similar in nature to the formation of the peripheral part of the body.

17

The reactions with ferric salts, with potassium bichromate, and with copper
acetate, exclude the presence of tannic or resinous substances. The stain
reactions obtained with Sudan III and osmic acid do not make known the pres -
fence of the smallest quantity of oleose substance or the protcinaceous na-
ture of the body, considering the fuct that even crystals of protein react
to these stuins (1).
Conclusions. The body in question, occupying the same position as the

elaioplasts, of.4 composition analogous to them, ani being found, moreover,
in the genus Hippeastrum, where these latter {the elaioplasts] were found,
I believe to be nothing more than a form of their [the elaioplasts'] develop
ment.

Eucharis subdentataBenth.

So far as reg.rds the study of the history of the develowment of the
elaioplasts in this genus, I have been unable to examine the floral axis
in process of develcpment, or the bulbs in germinative activity, and an
unable to make any pronouncement.

I examined solely the bulbs following the stage of blooming ani enccun
tered the elaioplasts in the external epidermis of their scales.

They occur immersed in the cytoplasm and are generally of notable di-
mensions. The color varies from yellow to brown and in form the elnioplasts
are roundish or irregular.

Often arops of cil may develop from their mass. Examining one by one of
the scules, I noted that in all cases they [the elaioplasts] are not of ir-

regular forn and variable size and thet in no case are they wholly lacking.

ig.

Such elaioplasts, when treated with Millon, Raspail, Trommer’s reagents,
cola à proteinaceous reaction, while when treated with osmic acid they turn
black; they stain intensely red with Sudan III und Scarlet R.

Sternbergia Fischeriana Rupr ., S. lutea Orph., S. macrantha J.

If one examines the bulbs of these species in the stage of their
functional activity one will find in the cells. of the external epidermis of
their scules colorless elaioplasts which are strongly refractile to light and
which in the adult stage attain much larger dimensions (Pl. XIII, fig. ll).
Such elaioplasts are of a more irregular form and result from the union of

numerous little spheres which, together, comprise fundamental proteinaceous

and oily substances. .

Ornithogalum caudatum Fac.

All that I have noted hers for the elaioplasts of this species may be
found in the work cited from Raciborski and is as follows:

"The elaioplasts are found isolated or bound together in the large
epidermal cells of the walls.of the ovary and lying near the nuclei."

Having had 4&t my disposition Sidi sei I undertook also in

this species the study of the elaioplasts and will speak briefly of the method
e KE

of their development in the storage organs.

In the epidermal cells of the scales of the bulb, whether they be adult
of young, provided it is in the period of vegetutive activity, one finds
DI elaioplasts, which at complete development present themselves as little
spheres seueneiy refractile to light, united in one or more groups, immersed
‘in the cytoplasm, and situated in a position having no relation to the

nucleus. They are colorless or light yellow and move actively in a manner

already indicated. .

19

If one examines the same bulb during its dormant period, it is easy
to discover in the cytoplasm of all or of some of the epidermal cells a
body of homogeneous and vacuolated structure, sometimes grumose, very
voluminous, and which fuchsine, cosine, and aurantia staan actively. It
shows all the characteristic reatitions of proteinaceous substance, Such
bodies present a form which is extremely variable (Pl. XIV, figs. 3, 6, 8);
they are of a color which varies from yellowish to brown and often present
a central A granulose part which stains more intensely at the periphery when
treated eri a solution of iodide of potassium. They do not behave other=

wise than do the elaioplasts which, after a short period of functional ac

“Ae fe
*

tivity, degenerate and take on later the aspect and form alluded to. In
fact, by the end of the flowering period the elaioplasts which one finds

in the external epidermis of the scales of the bulbs begin to show phenomena
of degeneration. Before the little spheres which constitute the body dis-
pose themselves somewhat disorderly they lose their form and coalesce, thus
contributing to the formation of a single unified mass.

Theffusion occurs at the points of contact and occurs contemporaneously
throughout the elaioplast, or gradually, beginning from fixed points. In
this period of its evolution it is possible to find sometimes in the plasma,
and more especially in the neighborhood of the mass of the elaioplast, one
or more spherical refractile bodies, which become black when treated with
osmic acid, react to Sudan III, to cyanine, and to Alkanna, and at the same
time show all the reactions of proteinaceous substance, Such bodies probably
represent the elements of the elaioplast separated from its mass, first

having been derived through fusion, or probably formed by neoplasia.

20

The forms under which the mass appears following the fusion of the
spherical bodies are, as has been said, extremely variable. In fact, such
a mass may appear as a large sphere (from which are sent cut one or more
treelike ramifications) (Pl. XIV, fig. 9, fy g), spheroid, ellipsoid, and even
grumose, thus presenting prominent lobes,

Thus does one see how in this degenerating elaioplast there develops
a unified mass from the normal form of an aggregate of spherical bodies
which represent the perfect development by diverse successive stages, in
which the single spherical elements which constitute it come little by
little to finally join together. Probably the fundamental substance of the
elaioplast during this process undergoes some chemical modification, con=
sidering that it, being at first colorless, begins to become yellowish,
and ends frequently by becoming intensely brown, Moreover, the oleose
substance in the advanced stages of degeneration of the elaioplast appears
in the form of minute drops, then diminishes in size and finally disappears.

Following the disappearance of the oleose substance there remains the
proteinaceous substance, which contimes for some time to represent the
elaioplast, reduce to lumps or granules which later dissolve and disappear.

When the bulb resumes its functional activity there form anew in the
è
epidermal cells of the scale elziorlasts, which multiply through a process
of true gemmation as well as by neoplasia’(Pl, XIV, fig. 9). In fact, I
have observed that they appear as a sphere at the surface of which in com
plete development there forms a tiny spherical embossment. This growth
ends on reaching a certain size (which is generally much inferior to that
of the individual which produces it) and afterwards there develops another

Similar one which behaves nearly in the same manner.

al
This repeating process accurring again produces a considerable increase in
the number of individuals, which occasionally become isolated one from
another by continuing to live isolated and independent. In general, they.
remain united until the. end of their life, forming a chain in which they
attain the size of the degenerative elaioplast.

Such a chain may also break in two or more parts. There may be observed,
moreover, some cases in which the gemmae form contemporaneously at two or
more points of the primitive elaioplast (Pl. XIV, fig. 9, d), and then, ate
taining the size of the mother sphere, one may find attached to it at dif-

ferent points two or more chains of daughter individuals.
Drimia undulata Jacq,

If one examines the bulbs of. this species during the period of their
active growth one will find the elaioplast in the cytoplasm of all the
epidermal cells of the scales. The elaioplasts appear as a group of little
Spherical bodies endowed with strong refractility offrpale ochraceous yellow,
and variable in number. In examining one by one the scales of various bulbs
in diverse condition, depending on the stage of development and age, one
would be able to note in the epidermal cells the diverse forms which the
elaioplasts assume and which correspond to the diverse stages of their de-
velopment. Among these will be noted the more important.

x The completely developed elaioplast appears in the form of a cluster
[grape-like ]. Later, in successive stages, the spherical bodies bécome

confluent but form a homogeneous, roundish mass, with, ramifications of a

granulose substance,

22

The fusion occurs at points of contact of the spherical bodies and
may take place contemporaneously throughout the entire mass of the elai-
oplast or, gradually, commencing at definite points. ‘Treating this forma-
tion with absolute alcohol, the oleose substance dissolves out rapidly and,
if we later submit it to a solution of potassium iodide, one will observe
a residuum of fundamental proteinaceous substance.

In this species, besides the bulbs, there were examined the young and
adult leaves in which, in the epidermal tissues as well as in the assimila-

tory parenchyma, the elaioplasts were lacking.

Alo# rhodacantha DC,
The elaioplasts occur in this species in the epidermis of the axis and
of the floral organs; they are lacking in the deeper tissues of the organs
above indicated, in the epidermis and in the parenchyma of the leaves, in

the young and-adult rootse

Yucca filamentosa Linn.

"There is present, in addition to the cell nucleus, also a peculiar
body which stains brown on treatment with osmium." (li).

In the lobes of the perianth of this species one finds an elaioplast
in the cytoplasm of each cell of the epidermal tissue. It is of spherical
form or nearly so, colorless, or slightly yellowish, of. smaller dimensions
than fe a and is markedly refractile.

In the youngest stage dt is found always near or in the neighborhood of
the nucleus; in the adult stage some of them are slightly remote from the
mucleus, while still others maintain a median position. With osmic acid they
become black; with Sudan III they stain intensely red (Pl, XIII, figa 10)

and show all the reactions of proteinaceous substance, in which respect their

structure is more similar to that of the elaiovlasts of Polianthes tuberosa,

23

Their distribution is characteristically irregular; in fact, one does
not find them in the young epidermis. In the leaves, both young and adult,
with the aid of osmic acid one can find no trace of the elaioplastse They
are found, however, always in the epidermis of the pedicels) ot the floral

bractse

Ruscus racemosus Lim,

If one examines the epidermal tissue of the still young scaley leaves
of this species, one will find in the cytoplasm of some or all of the cells
elaioplasts having, in the adult stage, dimensions which frequently exceed
those of the nucleus and a variable form which assumes a form characteristic
of the elaioplasts of Ornithogalum umbellatum. Such elaioplasts ‘are color-
less and refracti]}e and do not assume a fixed position in the cell.

Watsonia humilis Mill.

From studies made by me it appears that in the Iridaceae the elaioplasts
are not diffused things, as in the case of the Orchidaceae, the Amaryllidaceae,
and the Liliaceae.

In fact, wherever I have examined many representatives of such families

f

I have found them only in Watsonia humilis.

1

They appear in this species in the epidermis of tubers. When completely
developed they are large (voluminous), of dimensions more or less similar to
those of the nuclaus, of rotund form, oval or irregular, and pale ochre in
colore

In many respects their structure is notably that of a constitution of
small and very numerous spherical elements and in the further stages of their

development they show vacuoles which render them granulose or spongy.

ail
Sudan IIT, Scarlet Rs Alkanna, and cyanine stain them and osmic acid
blackens them.

Miltonia Clowesii Lindl.

In the floral axis and in the perianth of this species, in each of the
.epidermal cells, and in those of the parenchymay there is to be found an
elaioplast immersed in the cytoplasm situated generally near the cell nucleus.
These elaioplasts resemble in physical properties and in microchemical
reactions those described above.

Laelia anceps Lindl.

If one examines the epidermis of the floral axis of this species one
will find in each cell an elaioplast in the form of a single sphere extremely
refractile and presenting in its complete development a reaction identical

to the reacfions of the preceding species. -

Cattleva Harrisoniae Paxt.

The elaioplasts are found in this species in the epidermis of the leaves
"during process of growth, In the adult stage they appear in groups of tiny
spheres extremely refractile to light. Such groups have the form of bunches
of grapes and are relatively smaller,

Lycaste aromatica Lindl,, L. Skinneri Lindl,

If one examines the leaves of the perigonium of this species one will
find in each cell of the epidermis an elaioplast immersed in the cytoplasm
or close to the cell nucleus or a little distance from the latter. Such
. elaioplasts in the adult stage are of irregular form, granular structure, and
of a size slightly inferior to that of the nucleus. They show all the

reactions peculiar to proteinaceous and oleose substances.

25
Cymbidium aloifolium Sw., Cy Lowianum Reichb.

In this species one finds elaioplasts in the floral axis, in the ovary,

in the leaves of the perigonium, in the lip, and the gynostemium.

They are found immersed in the cytoplasm of the epidermal cells and
result from the aggregation of very numerous little sphersswhich are strongly
refractile to light (Pl. XV, fig. 6). Their size upon completion of develop»
ment is most remarkable, since they come to occupy half, or even more, of
the cell cavity. Such elaioplasts are of irregular or spherical form and
roll around or cover nearly perfectly the cell mucleus (Pl. XV, fig. 5),
or they can rarely be found any distance away from the cell nucleus. In the
most advanced stage they los# their characteristic structure and become
granulose and vacuolated,

Brassia brachiata Lindl.

The elaioplasts are found in this species in the epidermis of the ovary
of the floral organs and are wanting in the leaves and in the adult roots.
They are cui or irregular in form and where they have separated in the
first stage of their'evolution they show in their inferior some vacuoles
which increase in number, giving to the elaioplasts a granulose appearance
(Pls XV, figs 4).

Oncidium sphacelatum Lindl.

The elaioplasts of Oncidium efhacelatum resembl¢.more nearly those of

the preceding species, They are found in this species in the epidermis of
the floral organs. In their final stage they break up and thus multiply
themselves (Pl. XV, fig» 3). Thus it follows that in one and the same
cell may be often found more than one elaioplast. Such a method of multi-

plication is described also by Raciborski in Ornithogalum comosum and in

De Eckloni.

26

Tetramicra bicolor Rolfe.

The elaioplasts of this species occur in the epidermis of the floral
axis and of the leaves of the perigonium. They are found in the adult stage
generally some distance from the cell nucleus. They are of spheroidal form
and of a pale ochre color and present reactions which perfectly parallel
those characterizing the elaioplasts of the preceding species.

Malvaceae.

The elaioplasts are found in all species of Malvaceae alluded to, which
I have examined in the epidermal cells of young sprouts, of the petiole, of
the leaf blade, the floral peduncle, the calyx, and the corolla, They are
wanting in the ovule, in the seed, in the adult fruit, and in the rootse
They are of grape-like or irregular form and result always from the union of
very minute spheres the size and number of which are variable (Pl. XIV, fig. 7;
Pl, XV, fis. 2), Each spheze consists of a plasmatic stroma, including an
eleose substance. The plasmatic stroma treated with an aqueous solution of
iodide of potassium assumes an intensely brown color. Treated with nitric
acid and then with ammonia it turns yellow. Treated successively with sul»
phate of copper and potassium hydrate it takes on a violet color.

The reaction to Millon's reagent is red, especially where the material is

heated.

a7

The oleose substance is soluble in 5 per cent alcoho},in ether, and
in chloroform. It is insoluble in acetic acid, in chloralchydrafs; it stains
with Sudan III and with Scarlet R and turns black when treated with osmic
acid,

The development of these bodies is comparable to that of the elaioplasts
of the preceding species. In fact, in epidermal cells in process of division
of the growing point they do not participate in the phenomena of kariokinesis.
They appear at first to turn around the nucleus like little refractile spheres
which, moreover , increase in number as they progress in their development,
increasing slightly in volume. In the advanced stages of their development
they are of a granulose structure (Pl. XIV, figs. 2, 5) and present in their
intetipr. numerous vacuoles. When the epidermal cells aye passed beyond the
stage of their complete development it is difficult to find in them any
elaioplasts because they, degenerating, have disappeared,

From the results here given regarding the elaioplasts found in the
ie and those hitherto found in the Monocotyledoneae, they resemble
these latter perfectly in position, in form, in ce in structure,

and in chemical composition,

28

The examination of the following species failed to show any elaioplasts:

Acalypha hispida Burm,

u obovata Benth.
Alpinia nutans Roxb.

Amyris balsamifera Linn.
Andropogon Schoenanthus Linn.
Anthurium magnificum Lind.

n lucidum Kunth,
Ardisa japonica Blum.
Aristolochia Gigas Lindl.

Pistolochia Linn.
TS Dracunculus Li Linn.
Artocarpus rigida Blum.
Asparagus plumosus Baker,
Bambusa mitis Poir.

-, " nigra Lodd.
Bégnonia foliosa H. B,
imperialis Lem.
maculata Raddi,
Bixa Orellana Linn,
Boehmeria biloba Mia.
Bulbine frutescens Willd,
Caesalpinia tinctoria Domb.
Caladium amabile Versch.

n esculentum Vent.

n

Caladium violaceum Hort.
Cassia Fistula Linn.
Contradenia grandiflora Endl,
Ceratozamia robusta Mig.
Cineraria amelloides Linn.
Cinnamomum aromaticum I, Grahe |
Cistus ladaniferus Linn.
Clerodendron Balfouri Hort.
li fallax Lindl,

Clusia flava Jacq.
Cneorum tricocum Linn,
Coccoloba excuriata Lim.
Coffea arabica Linn,
Coriaria myrtifolia Linn.
Crinum ensifolium Roxb.

" giganteum Andr.
Croton cascarilla Benn,

" cornutus Andr'.
Cycas circinalis Linn.

" revoluta Thumb.
Cyperus alternifolius Linn.
" minimum’ Forsk,

" Papyrus Linn.
Cyrthanthera magnifica Nees.

29

Datura arborea Linn,
Dianthus Caryophyllus Linn.
Dieffembachia picta Schott.
Echeveria cocinea DC.
Eranthemum aspersum Hook.
Eupatorium Morisii Vis.
Fatsia japonica Decne.
Ficus elastica Roxb.
Fittonia gigantea Linden.
Galipea ovata St. Hill.
Gardenia florida Linn.

It tubiflora Andr,.
Goldfussia glomera glomerata Nees,
Grevillea robusta A. Cume
Gynura aurantiaca DC.
Halesia hispida Mast.

Ilex paraguayensis Hook.

Illicium floridanum Ellis.
Ipomea Leari Paxt.

Ixora javanica DC,
Jasminium grandiflorum Linn.
Justicia carnea Hook,
Laurus glandulifera Wall,
Leontopodium alpinum Cass.
Linnsea borealis Linn,
Magnolia grandiflora Linn.
Maranta argyrophyla Linden.

" arundinacea Linn.

" splendida Hort.
Mesembryantheman barbatum Linn,

" curvifloram Haw,
Myrcia acris DC.
Musa textilis Née.

Opuntia glaucophylla Wendl.
Origanum Majorana Linn.
Pandanus javanicus Hort,
Pellionia pulchra P. E. Br.
Philodendron giganteum Schott.
Phyllis Nobla Linn.

_Pieraena excelsa Lindl.

Piper Cubeba Lim.

" longum Linn.
Plumbago grandiflora Tenor.

Plumeria alba Lim.
Polygala myrtiflora Linn.
aa umbellata Thumb.
“Quercus Suber Linn.
Rivina laevis Linn.
Ruellia Devosiana Hort.
Salvia involucrata Cav.

" officinalis Linné
Sansevieria guienensis Willd.
Sassafras officinale Nees,
Sempervivum californicum Hort.
Strobilanthes Deyeriana Hort.
Stromanthe sanguinea Lond,
Tamarindus indica Linn.

Thea viridis Linn.
Tournefortia laurifolia Vent.
Tradescantia zebrina Morte
Zingiber officinale Rosc.
Xanthochimas pictorius Roxbg.
Xylophylla Arbuscula Sw.

" falcata.

‘4_flceata.

n macrophylla Horte

dò

ui

Relation bétween Crystals of Calcium
Oxalate and Elaioplasts.
The afthors who have occupied themselves with a study of the relation
between crystals of calcium oxalate and elaioplasts are the following:
Wakker observed that in the epidermal cells of Vanilla planifolia,
contemporaneously with the disappearance seta elaioplasts, there appeared
erystals of calcium oxalate, He, however, noted that in V. aromatica
‘latifolia the epidermal cells, Although in the young stage being supplied with
elaioplasts, lacked the production of crystals in the successive stages of
their development and forced i. the conclusion that the crystals of the
A
epidermis of V, planifolia could have no direct relation to the elaioplasts.
Warlich (7) subsaquently observed that the elaioplasts in Vanilla
planifolia in the advanced stages of their development became birefractile and
that in their interior there is formed a crystal of calcium oxalate which was
later disclosed, Besides this, there is always in the adult epidermal cell
accompanying the crystal a birefractile rotund body which shows a black cross.
It might probably represent the ultimate residuum of the elaioplast. The
conclusion at which this author arrives is opposed to that of Wakker, since
he believes that there exists in this case a special relationship between
the elaioplasts and the crystals of calcium oxalate, Relative to such an
opinion as the above, Zimmermann expresses himself as follows:
These facts, however, appear to me to require confirmation. In any

event, it can not be said to be true of the occurrence of such phenomena.

Thus Wakker asserts that a cultivated species under the name of Vanilla

31

planifolia latifolia clearly shows elaioplasts in the epidermis of the
leaves, although they exhibit no relationship to calcium oxalate within the
cells concerned, Even though I have made observations on the aforementicned

Vanilla sp. neither in this one nor in Ornithogealum umbellatum and 0. nutans

could I demonstrate a single trace of diffraction in the elaioplasts, although
I examined them in all possible stages of their development,

With the opinion of this last author I am in agreement also because I
have never encointered crystals of calcium oxalate in relation to the
elaioplasts in the anaeous species in which I followed, to the last stages
of their {the elaioplasts!] development.

The Origin and Morphological and Biological Significance of the
Elaioplasts.

Raciborski admits that the elaioplasts cwe their origin to a secretion
of cytoplasm. On the contrary, I contend that they are of nuclear origin.
Such an hypothesis is supported both by the fact that the elaioplasts appear
during their first stage close to the nucleus and by the fact that the
chemical nature of their substance redcts toward solvents and staining reagents
like the substance of the nucleus, as I have established.

So far as concerns the morphological significance of the elaioplasts we
have the following opinions: According to Wakker, it [the elaioplast] is
probably derived from me taporphos is of the chlcroplasts. This opinion, as

Raciborski observes, is opposed by the fact that they have diverse methods of

multiplication,

34

Zimmermann found in many cases that the elaioplasts showed a manifest
Similarity to the fungous organisms and as a consequence they do not exclude
the question of possible parasitism or symbiosis, Such an opinion was later
combated by Raciborski, whos from studies which he made on the development
of the elaioplasts in the vegetative organs of the Liliaceae, reached the
conclusion that they [the elaioplasts] may be considered as normal organs
of the respective cells. I, never having been able to observe the formation
of spores in numerous cases in which I observed the Glaioplasts, do not
believe it is possible to support the opinion of Zimmermann.

On the other hand, if it is a question of parasitism, the spherical
bodies which constitute the elaioplasts ought to present the characteristic
structure of the cell, Such a structure I have not been able to distinguish
in the many preparations which I have made, not even in cases in which they
exhibit notable size, even when I used the most powerful immersion objective
and the strongest compensating oculars.

If, again, it may be added that it [the elaioplast] may be treated as
.a parasitic organism, it ought not to have the characteristic chemical
constitution which is presented in the elaioplast and should appear anywhere
in the cytoplasm and not constantly next to the nuclens,

I have sought also to cultivate the elaioplasts obtained from very thin
sections of the external epidermis of scales of the bulbs of Hippeastrum

aulicum and Haemanthus albiflos in suitable nutrient media, sugar solutions

of varying concentration, Knop?s solution, and gelatine. In the attempts
made they have not shown the least tendency to grow, and after a few days

begin to degenerate.

33

The tesearches of Stahl on mosses. were done to show that the cleose
bodies of these plants serve as organs of defense against the ravages of
Snailse Raciborski maintains that one may attribute even to elaioplasts
such a function, based on observations that the floral axes of such
Species of Gagea and Ornithogalum are not eaten by animals even when these
are found in dry soil, although the leaves are not respected. Such hypotheses
do not to me seem probable since it is to be noted that the elaioplasts
develop and function solely during a given period of the evolution of the
organs in which they are found, then disappear; henne they would be incapable
of protecting any organ whatever during its life against any herbivorous
animal. Therefore, even the direct observations exclude completely the
possibility that the elaioplasts have any defensive function to perform since;
~moreover, I have found in the leaves of Malvaceae (in which elaioplasts were
.present) little pits caused by snails. These [snails] ate also the floral

axes of Funkia coerulea and Polianthes tuberosa which I fed them,

Conclusions.
‘In the species which I have examined the elaioplasta are found in the
adult stage within the cytoplasm, now close to the nuclaus, now distant from
it, They occur generally in the form of sma 11 spheres attached in gràupsy
rarely in the form of single spheres, During their development they are
able to assume diverse forms but for the most part in a given species there

predominates a single form dependent on the number and size of the little

.

spheres which compose them,

34

Zimmermann observed that in the perianth of Funkia coerulea the

elaioplasts possess an active movement of displacement and of rotation which,
also, I, too, was able to demonstrate and which I observed to be characteristic
of elaioplasts in the form of: grape-like clusters.

In regard to the position of the elaioplasts it is to be noted that
with rare exceptions they are found in the epidermis of the floral organs.
Their presence in the other organs is inconstant.

Their multiplication may obtain after three months, by neoplasia of the
protoplasm; by gemmation; or by division, which takes place passively. The
first constitutes the normal method of multiplication, the second is rareyy
the third and last represents an exception and accompanies .a division of the
plant cell. A typical example of the miltiplication by gemmation may be

observed in the epidermis of the scales of new bulbs of Ornithogalum caudatum;

while the passive division èccurs during the division of the épidermal cells
of the floral axis, of the ovary, and of the perianth lobes of Polianthes
tuberosa.

So far as concerns the biological study of the elaioplasts, from the
scope of the surprising and varied stages of their development, and the as-
signment to them, in so far as possible, of functional significance, my ob-
servations were repeated, not coi anda vegetative organs. of mapy Liliaceae
and Amaryllidaccae, but also on their bulbs examined in diverse conditions
with regard to the age of the bulb as well as stage of vegetative activity or

dormancy. The authors who preceded me in the study of the elaioplasts were

not able to study all the phases of their development since they {the

elaioplasts] do not occupy such reserve organse

36

From the studies I have made on bulbous species in which I have found
elaioplasts the following may be deducted:

(1) That the elaioplasts, besides occurring in vegetative organs, may
be found in the external epidermis of all the scales of the bulb.

(2) That the elaioplasts in bulbs, after a short existence, degenerate,
then disappear,

(3) That of the two substances of which elaioplasts consist the first
to disappear is the oleose substance, while the proteinaceous substance
persists for varying lengths of time»

(4) That in dormant bulbs the elaioplasts appear without definite
structure and assume varied forms which are to be considered as representing
the ultimate phases of their development.

(5) That in each renewal of functional activity of the bulb elaioplasts

form anew.

The form of the elaioplasts is singularly and particularly elevated

in seme bulbs of Hippeastrum aulicup and of Ornithogaium caudatun.

The principal results of my researches, then, may. be thus summarized:

(1). I have found elaioplasts in 27? new species referable -to 19 new
genera of Monocotyledoneac.

(2) I have found that there are elaioplasts even in some Dicotyledoneae
(where none were ever found before), namely, in the Malvaceae, the one family
from the many examined by me which possess thema

(3) The elaioplasts are not to be considered as parasites (as thought
‘54 Zimmermann) nor as organs of defense (as maintained by Raciborski), but
as specific organs of the cell in which they form and whose function is that

of elaborating oleose nutritious substance.

38

(4) The fundamental substance of tho elaioplast is similar to the

substance of the nucleolus,

(5) In bulbs there are formed new elaioplasts with each renewal] of

their [the bulbs: Jvegetative activity.

Botanical Institute of Pavia, August, 1911.

(1)

(2)

(3)

(4)

(5)

: (6)

(7)

(8)

37
LITERATURE CITED,

Buscaglioni, Ly,

Un nuovo reattivo pet l'istolozigi vegetale, Malpighia,

Anno xii, v. xii, p. 5.
Ehrlich, P., Krause, R., etc.
1910. Enzyklopéidie des Mikroskopischen Technik. Wien, p. 450.
Kuster, W.
1894. Die Oclkirper der Lebermoose und ihr Verhiltnis zu den
Flaiop}astenyj Inaugural-Dissertation, Basel.
Miller, L.
1893. Grundzige einer vergleichenden Anat. d. Blimenblitter.
Nova Acta d. K. Leop, Carol, Wyetsch. Ak. d. Naturforscher,
Bd. sen pe 64, |
Raciborski, M.
1893. Ueber die Entwicklungsgeschichte der Elaioplasten der
‘ Liliaceen. Anzeiger der Academie der Wissenschaften
in Krakau, p. 259.
Wakker, J. Ho
Studien Uber die Inhaltskòrper der Pflanzenzellen.
Pringsheim's Jahrbùcher, Bd. xix, pe 423.
Warlich, H.
1892. Ueber Calciumoxalat in den Pflanzen, Marburger-Inaug.-
Diss.
Zacharias, E.
1909. Die chemische Beschaffenheit von Protoplasma und Zellkern,

Progressus rei botanicae, Bd. 3%, p. 67.

| 38
(9) Zimmermann; As

Ueber die Elaioplasten. Beitruge zur Morphologie und

Physiologie der Pflanzenzelle, Bd. i. p. 185.
(10)

1894. Sammel-Referate aus dem Gesammtgebiete der Zellenlehre.

Beihefte zum Botanischen Centralblatt, p. 165.

£ =

Fig. 1-2.
n aes
n Di
Tt Fia
n L,5,8.

n o

n 6,9.

" 10.

elaioplast; a = nucleus; la

39

PLATE LEGENDS.

leucoplast; ¢ = cytoplasm; g = oii drop.
PLATE XIII.

Epidermal cells of the perianth of Polianthes tuberosa Linn.

Elaioplast in adult stage stained with Sudan III.
Elaioplast passively divided,
An epidermal cell of a scale of the bulb of Hippeastrum

vittatum Herb, e = elaioplast in adult stage stained with

Sudan III,

e ~ old elaioplast of which the oily
substance has disappeared.

e = the proteinaceous substance begins to
dissolve.

Hippeastrum vittatum Herb. 5, Elaioplast in adult stage.

4, &, Elaioplasts in degeneration colored with acid

fuchsine (the material was fixed in absolute alcohol con-
taining 5 per cent of acetic acid).

Hippeastrmm reticulatum Herb. Elatoplast in the adult stage

treated with absolute alcohol and then stained with acid

fuchsine.

Special body found in dormant bulbs of Hippeastrum aulicum

Herb. central part; b = peripheral part; a = colorless

cz
halo; a + central part with large-vacuole.

Epidermal cells of the perianth of Yucca filamentosa Linn.

Elaioplasts colored with Sudan III,

uo

Fig. ll. Sternbergia lutea orph. Eldioplast in adult stage.

" 12. Ansgpidermal cell of the bulb of Drimia undulata Jacq. with an

adult elaioplast.
PLATE XIV.

Fig. 1. Haemanthus albiflos Facg. The little spheres which accompany

the alaioplast separate on:pressure from the cover glass on the
preparation.

da 2, Hibiscus Rosa-sinensis Linn. An epidermal cell of the ovary with

an elaioplast in adult stage.

w 4, Epidermal calls of a scale of the bulb of Haemanthus albiflos Facde
each one containing an elaioplast in the adult stage, treated
with osmic acid. |

di 5. Hibiscus tricolor Dehnh. An epidermal cell of the ovary with an

elaioplast in the adult stage.

di 7. Hibiscus herbaseus Vell. An epidermal cell of the perianth with

an.elaioplast in the form of a bunch of grapese
n 3,6,8. Odd form of elaioplast occurring in the epidermal cells of the
scales of the bulbs of Orhithogalum caudatum.

tt 9. Various stages of gemmation of the elaioplasts of the bulbs of

Ornithogalum caudatum.

Fige

Lar

Co

de

5,6.

hi

PLATE XV.

Himantophyllum miniatum Groenl. Cells of the epidermis of tha

ovary with elaioplasts in process of degeneration..

Hibiscus liliiflorus Cav. Epidermal cells of the ovary each of

which contains an elaioplast close to the cell nucleus.
Oncidium sphacelatum Lindl. Epidermal cells of the ovary con

taining elaioplasts in advanced development. Each of them is

fragmented, and thus we have several elaiòplasts in each cell.

Epidermal cells of the ovary of Brassia brachiata Lindl. with

elaioplasts of granulose appearances

Cymbidium Lowianum Reichb, Epidermal cells of the ovary (fig. 5)
and of the perianth (fig. 6). In the first the nucleus remains
completely covered by the elaioplast which is stained.black,

after brief treatment of the sections with osmic acid 1 per cent.

# Note: -- Figures 4, 6, 7, 8, 9 of Plate XIII were drawn with No. 3

ocular and oil immersion objective 1/2 Zeiss; all the others were drawn with

No» 9 objective, and No. 4 Koristka oculare

a

March, 1923. Translated by H, B, Humphrey.

Ath dell'Ist Botanico Univ. di Pavia Vol. XIV Tav: XIII
. XIV av

Luv Tacchinardi e Ferrari - Pavia J Politis - Sugh Elaoplast

Ati dell'Ist Botanico Univ. di Pavia Vol. XIV ‘Tan: XIV

oo 9% 3° 4 LI

| Politis - Sugli Elaioplasti

l'Autore del. Lit Tacchinardi e Ferrari Favia

Ath dell'Ist Botanico Univ. di Pavia Vol. XIV Tav. KV
av.

J. 9

I Politis - Sugli Elaioplasti

l'Autore del. Lit Tacchinardi e Ferrari Favia

Politis, Ioannes, Honorary Assistant in the Botanical Institute of the Royal
University of Pavia. eis

ON SPECIAL CELLULAR BODIES WHICH FORM ANTHOCYANINE.®/ Translation
of: Sopra speciali corpi cellulari che formano antocianine. Atti

Ist. Bot. Univ. Pavia, II, 14: 363-376. pl. xvi-xviii. 1914.

Following the complete abandonment of the hypothesis as to the chloro-
phyllian origin of anthocyanine, many authors turned their attention to the
substances which they found in plant cells, especially to sugars and tannins.
They sought to establish the existence of some genetic relation between them
and the anthocyanine pigments.

Wigand (19, 20)>/ at first admits that the tannic substance should be
considered as a chromogen (generator of color), because there seems constantly
to appear such a substance in the cell in which later anthocyanine pigments
come to be organized and also because he notes that the presence of these
latter coincides with that of the tannin in floral leaves and in red autumn
foliage.

The conclusions reached by this author we will find more amply confirmed
in the works of Wiesner (18), Tschirch, Aufrecht, Kutscher, Detmer, Reinke,
Pick, Molisch, Dennert, Bauer (1), etc.; in recent times in those of Overton
(12) Buscalioni and Pollacci (1), Mirande (8) and Laborde (7).

Buscalioni and Pollacci (1) supposed, however, that to the oxidases be-
longed the task of transforming certain substances in the anthocyanine pig-

ment, while to the reductases probably belonged the function of determining

their dissolution.

2/ See also: Atti Acc. dei Lincei, xx, giugno 1911.
b/ Reference is made by number to "Literature cited," near end of

translation.

aS

The hypothesis that the formation of the anthocyanine compound is re-
lated to the phenomena of axidation found much support in the works of Mirande,
Molliard (11), Miss theldale (17), V. Grafe (4, 5), Combes (2), etc., and was
applied latterly in a particular manner by Palladin (14). According to this
author, there exists in all plants chromogens and oxidases. The oxidases
settled upon the chromogens end may give crigin to anthocyanine pigments, and
these may break up by the dimirution of the process of fxidation and by the
acceleration of the procasses of reduction.

More recent research led Palladin (13) to admit the existence of com-

pounds to which he gave the name prochromogens and from which, through the

intervention of an enzyme, there might be derived a chromogen. These latter
may ve produced in large numbers when the functional activity is more intense,
namely, in the spring, while during a great part of the growth period they
may form in small quantities by completing the process of cxidation.

With respect to the relation which exists between the anthocyanine and
sugars, the research of Overton (12), which was fully confirmed by that of
Molliard (10) and Palladin, revealed the existence in some plants of a direct
relation between the production of anthocyanine and the accumulation in their
tissues of sugars resulting from culture.

Finally, Combes(3), following on his research on gaseous changes during
the formation and disappearance of anthocyanie ‘ pigments, was led to consider
these substances as glucoside compounds, which originated in a more oxidizable
ambient or fluid than that normally present and is not differentiated from
that which forms under orddnary conditions according to their stage of more
advanced oxidation. This point of view conforms with the opinion maintained
for a long time by many authors, namely, that the anthocyanic compounds ate
to be considered as glucosides, an opinion which found new confirmation in the

recent researches of Portheim and Scholl (16), and in those of V. Grafe(4, 5).

-~j3-

These are in a few words the principal results of the numerous and im-
portant studies that have been undertaken relative to the mechanism of the
formation and chemical nature of anthocyanic compounds.

For a long time the fact has been observed that anthocyanine is not
found in solution, solely, in the cell sap, but in rare instances also it may
be found in the form of little spheres. (6, 9).

As to the origin, chemical constitution, structure, development,
morphologic and biologic significance of such colored corpuscles, accurate
research, so far as I am able to say, is lacking.

In the flowers of Billbergia nutans Wendl., I have encountered the fact

which relates to the formation of anthocyanine and which, I believe, also is
important in making one acquainted with the complicated structure of the
protoplast.

In this plant in the part of the petals colored azure blue, in each
cell of the epidermis and of the parenchyma, is to be noted a granulose proto-
plasm, and immersed in it are abundant round or oval chloroplasts (sometimes
active and full of starch, sometimes a little changed, preferably close to
the cell wall), a relatively small nucleus, and, finally, a characteristic
body, to which may be attributed a particular biologic significance. I have
given it the name of cyanoplast or generator of anthocyanine.

The cyanoplasts, nicely bounded by the cytoplasm which surrounds them,
are distinguished principally by their intense azure blue color. They are of
variable dimensions and attain, in complete development, considerable di-
mensions, while at first they are hardly visible. In general, it is to be
noted that they are to be found toward the margin of the petals(Pl. X¥I, fig.2),
and major dimensions approach those of the cyanoplasts which are found in the
i iaia discus: They are generally spherical, show an involucre the cavity
of which is occupied by an azure blue pigment, not resembling in any respect

the nucleus or the chromatophores; in each cell may be found constantly from

the £inct n ainnta nuamnanlacet

4
è

Sa
Microchemical examination. The agure blue pigment which is found in

the involucre of the cyanoplasts presents the following reactions;

With acids it stains red, With alkali it takes on a greenish yellow
color. With nicotine (1, p. 356) in dilute solutions it becomes green. It
decolorizes easily with oxygenated water and with sulphurous anhydride. It
is slightly soluble in cold water and dissolves quickly in warm water, and
Slightly acidulated water.

Through the microchemical examination of the pigment of the cyanoplast
one finds present a substance which behaves toward acid, alkali, alkalcids,
solvents, and oxidizing substances like anthocyanine. Therefore, we may con-
clude that the pigment referred to belongs to anthocyanine and should not be
confused with cther pigments,

While it is easy to determine the principal chemical properties of the
pigment of the cyanoplast, it is difficult to determine the chemical nature of
its involucre.

On the whole I have tried to find out if the cyanoplast possdsses a
plasmatic stroma as do the chromatophores and the elaioplast. Such a stroma
I was unable to find, although, using many methods of fixation, such as picric
acid, chromic acid, acetic acid, absolute alcohol in an aqueous or alcoholic
solution of corrosive sublimate, I have not been able to fix and stain it,

In all attempts made with the above-named liquids the cyanoplasm dissolves

and disappears.

Verifying the absence of a plasmatic stroma, I tried to determine the
nature of the involucfe of the cyanoplast.

The mode Tmneans] of resistance of the cyanoplast to attack by the
several solvents, such as water, alcohol, neutral salt soluticns, acids and
dilute alkalis, and potassium iodide solution, shows that in them it is pos-
sible to distinguish a peripheral region which differentiates from the color

subsfance by a greater resistance to the action of the determinative solvent,

- 5-

If one uses a sclution of potassium iodide, one will note that it
acts so slowly on the cyanoplast that it is possible to follow with the
microscope the various phases of its dissolution. The action of the reagent
becomes manifest with the yellowing of the mass of the cyanoplast within
which there appears one or more cavities. With progress of the action of
the reagent, these latter will increase in ia and terminate in a manner
which results in the development of a great vacuole bounded by a dense wall
which later dissolves and disappears.

If one uses the same reagent containing some drops of hydrochloric
acid, the cyanoplasts stain red and shine on the yellow background of the
protoplasm in a manner which enables one to follow, even better, the above-
alluded-to phases of their dissolution.

These reactions and others which I have been able to make with dilute
alcohol, with very dilute alkeli, and especially with neutral salt solutions
serve to show the independence of the two substances which constitute the
cyanoplast.

Nevertheless, as will subsequently be show, there are those instances
in which the cvanoplasts appear to be colorless, namely, deprivelof said
pigment, and, in such cases, we are able to investigate microchemically as
to the chemical nature of their involucre and of the substances contained
within it. *

Such colorless cyanoplasts are of homogeneous structure, oily appear-
ance, and possess high refractility. They show the following reactions:

In water, at ordinary temperature, they vacuolize and end by dissolv-
ing completely. ihe passeive quickly in absolute alcohol or in alcohol
diluted to 50 or 75 per cent, and likewise in ether, in chloroform, in 1 per
cent

hydrochloric acid, in very dilute alkali, in neutral salt solutions,

and, finally, in glycerine. With a solution of potassium iodide they stain

- Ga
yellof ana then disappear, dissolving slowly. With Millon's reagent, at
first cold, then gently heated, no definite reaction takes place. With
sodium coralline, aniline blue.tincture of Alkanna, Sudan III, and cyanine,
they do not stain. With 1 per cent osmic acid, they turn black almost in-
stantly, and, following this treatment, they show an evident resistance to
the solvent action of water and dissolve less readily in alcohol. With
ammonium, potassium, and sodium carbonate, and with potash and ammonia, they
become yellow. With ferric acetate and with ferric chloride, they become
blackish blue. With bichromate of potash in concentrated solution, they
stain reddish brown. Methylene blue in aqueous solution they absorb in a
short time. (15).

The complexity of the reactions above brought out is not such as to
lead one to any safe conclusions as to the nature of the substances which
constitute the involucre of the cyanoplast. On the other hand, one may
conclude that they do not belong to proteinaceous substances. Moreover,
the reactions obtained through the use of ferric salts, potassium bichromate,
alkali, alkaline carbonate, and methylene blue show that a chromogen of
tannic nature is present in this involucre. But, since, following the
transformation of the tannin in the anthocyanine, the involucre does not
disappear but rather is renderéd the more evident, there follows the deduction
that in its formation it may contribute to the substances which contribute to
its still unknowa constitution.

Development. At the stage in which the corolla begins to emerge from
the calyx cup, if one examines the epidermis (ventral and dorsal) and the
parenchyma at the margins of the azure blue petals, one will be able to find
all the stages of the development of the cyanoplasts. They appear at first

as very minute spherical bodies scarcely visible, colorless or pale blue,

(PI. XVII, fig. 1)) immersed in the cytoplasm, without showing any proper

mo}

TA

Later, retaining their spherical form, they increase in size and soon
assume a more intense color, attaining in complete development dimensions
relatively greater(Pl. XVII, fig. 2).

In this period of their evolution the cyanoplasts indicate marked
modifications, in so mach that they lose their globose form, assuming odd,
extremely variable shapes (Pl. XVII, fig. 3), and reveal at the same time a
very delicate membranous involucre, within which there is found included the
colored substance. This occurs in the form of a central globule of con-
siderable size or many spherical or oval corpuscles of variable size, which
may be found indefinitely distributed withingthe involucre or else borne
toward the extremity of the preloaee ion which occure in this stage. The
number of such corpuscles varies according to the dimensions of the cyanoplast.
This latter, moreover, assumes a form and dimensions which are extremely vari-
able, and permits the supposition that they become fragmented. In fact, many
cyanoplasts are more or less thin in the median part and the two or three
swollen extremities are somewhat far apart. It is not even snob that
there sometimes follows a miltidivision, since one finds in the cytoplasm some
ting rotund colored bodies which are also azure blue and which probably repre-
sent the daughter POR cut off Fo the mass of the adult cyanoplast,
but in this regard my observations are incomplete. I could not follow this
multiplication. Rather would I believe that the extremely variable form which
the cyanoplasts take in this stage of their evolution is due to phenomena of
degeneration and at the same time that the other bodies (corpicciuoli) to
which I have referred represent young cyanoplasts formed by neoformation from
the protoplast.

The fact remains that from the beginning in each cell one f inds con-

stantly a single cyanoplast which on becoming adult is accompanied by other

Sp@irical bodies of light color and of smaller dimensions.

ee te
n Following the metamorphosis above referred to » We come to the color-
ing substance of the cyanoplasts, diffusing it into the cell cavity subse-
quent to the shrinkage and rupture of their involucre. This [the involucre ]
finally disappears completely and the cell sap, which at first was perfectly
colorless, little by little, as the cyanoplast degenerates, takes dn at first
a pale blue color, then becomes intensely blue,

Origin and biologic significance. In the protoplast, as has been noted,
one finds organs diverse as to constitution, structure, and functional value
or worth, some of which arise from preexisting organs; others, on the contrary,
originate directly from the protoplast by neoformation. Among these latter,
as one may deduct from the history of their development above described, is
the cyanoplast.

; As to the appearance of the pigment, it may be said that it coincides
generally with that of the cyancplast. There are cases, however, in which
the latter shows complet¥ development independent of its pigment. Thus, for
example, examining the flowers of the little plant which I found in an ob-
scure corner of the greenhouse (under conditions of humidity and temperature
differing from those of the majority of speciments examined by me) and in
which only the apical parts of the pale petals showed azure blue color, I
observed that in the margins, and especially toward the apex, some, among the
mumerous cyanoplasts, were completely developed and colorless (Pi. WI, fig.
1); others, on the contrary, were developed but intensely azure blue in color;
and, finally, still others showed diverse intensity of color.

In this case, then,the gradual transformation in the anthocyanine may

be assisted by a substance already extant within the cyanoplast through

metabolic precesses which take place within the same.

-9+

Such a substance may be a chromogen of tannic nature the presence of
which may be inherent within the young, colorless cyanoplast, since it may
be colored in a particular manner by the use of salts of iron, osmic acid,
and potassium bichromate.

Finally, so far as concérns the biologic significance of the bodies
in question, I believe that they shovld be considered as specific organs on
which depends the production of anthocyanine.

Facts analogous to those described for Billbergia nutans I have found

in my study of many other plants, such, for example, as the following:

Liliaceae: Convallaria japonica Linn. (frutti).
Iridaceae: Iris fimbriata, Vent. (fiori).

Orchidaceae: Laelia anceps Lindl. (fiori).
Ranunculaceae: Aquilegia glandulosa Fisch. (fiori).

Ericaceae: Erica carnea Linn. (fiori).

Labiatae: Nepeta glechoma Benth. (fiori)&

Verbenaceae: Clerodendron Balfouri Hort. (fiori).

Caprifoliaceae: Weigela rosea Lindl., Weigela japonica Thunb. var.

rosea (fiori).

i LO
Laelia anceps Lindl.
The petals and sepals in this species are of a vivid lilac rose color.

The elongated lip has in front a reddish purple spot and is ornamented in the
interior and in the throat with dark red stripes.

If one examines all these colored parts of the flower, one will find
almost always that the pigment to which they owe their color is diffused in
the cell sap ;in inquiring into its origin one should examine the sparse
red spots of the gynostemium, which are of late formation.

Such spots are due to a group of epidermal cells each one of which con-
tains a single cyanoplast (Pl. XVI, fig. 4). This cyanoplast is found im-
mersed in the cytoplasm. In its adult stage it is of dimensions comparable
to those of the nucleus, is reddish purple in color, has no definite position,
and presents an involucre in which is included a colored substance which
presents all the characteristic reactions of anthocyanine. In fact, this
pigment is soluble in alcohol and especially in acidulated water, becomes
green with nicotine and greenish yellow with alkali. It decolorizes easily
with oxidizing substances.

On the other hand, the presence of an involucre in the cyanoplast
may be demonstrated through the use of 50 per cent alcohol, 3 per cent
hydrochloric acid solution, 1 per cent solution of acetic acid, 1 per cent
solution of chromic acid, and, above all, by oxygenated water or by osmic
acid, By treating the sections with oxygenated water, one may set free from
the cyanoplast its pigment and thus place easily in evidence its involucre
which appears colorless as a vesicle which remains immersed in the cytoplasm.
Treated with osmic acid, on the contrary, the involucre becomes blackish and
vacuolized (Pl. XVII, fig. 9).

It is to be noted, moreover, that the above-named reagents do not act

for a few minutes, but, if the action is prolonged, the involucre dissolves

and disappears.

Pee ae

So far as concerns the development of the bodies in question, it is
to be noted also that in this plant, as in the preceding one, they are not
derived by division from preexisting bodies and are not transmitted from the
mother cell to daughter cells but arise through neoplasia of the protoplast.
In fact, they appear at first in the heart of the cytoplasm as very minute
colorless spheres (Pl. XVI, fig. 3 ¢), in the interior of which hater begins
to form the red pigment (Pl. XVI, fig. 3 c).

Such spheres grow and in complete development may sometimes attain the
dimensions of the nucleus.

Later there begins to appear on them phenomena of degeneration. The
cyanoplasts, in fact, diminish in volume, while they present sparse vacuoles
in their mass, among which one may often distinguish a single larger central
one. Finally, the cyanoplast disappears and its pigment expands into the
cavity of the cell, coloring the cell sap, which was at first perfectly
colorless,

It is to be noted, finally, that in this species when the cyanoplast
at first appears it is colorless and its pigment begins to appear in the
interior later.

In such colorless cyanoplasts, treated with iron acetate, osmic acid,
potassium bichromate, and with methylene blue, one is able to establish

easily the presence of tannin.

Aguilegia glandulosa Fisch.

Examining the flowers in this species, one often is able to find in
the violet-coloréd petals some cells in which the color is not diffused but
is bounded instead, in one or more united spherical bodies. This pregents
a development and structure of the cyanoplast similar to those described
above. Often in one or more points of their surface they show protuberances

(P1, XVII, fig. 7 €); their pigment shows all the reactions of anthocyanine.

Ser
Similar cellular bodies containing anthocyanine are found in the

flowèrs of Nepeta glechoma.

Weigela rosea Lindl., N. japonica Thunb.

With an idea of getting an exact conception of the method by which the
réd color forms in the flowers of this species, I have studied many specimens
and have found that they are not derived from preexisting substances soluble
in the cell sap but have organized themselves into special spherical bodies
immersed in the cytoplasm. These, being at first hardly visible, increase in
size little by little and later, having reached a definite size (Pl. XVII, fig.
5}; degenoreffe and dissolve completely, coloring the cell sap, which was at
first colorless.

According to the data obtained from reactions which I got, the pigment
of such body belongs to anthocyanine. It is contained with the membranous in-
volucre the presence of which may be demonstrated by application of the usual

methods described above, and particularly by using oxygenated water.

Convallaria japonica Lim.

In this species the cyanoplasts are found in process of formation in
the epidermal and subepidermal cells of the adult fruit and, above all, in
those of the ultimate strata of the seed coat in contact with the seed. Here
the cyanoplasts in the adult stage are spherical, relatively small, immersed
in the cytoplasm, and situated in an indeterminate position (Pl. XVIII, fig.2).

They are at first present as very minute spheres azure blue in color
(Pl. XVIII, fig. 1), or as colorless and refractile bodies which give all the
reactions peculiar to tannin.

Such spheres grow, assume an inténse coloration, in the end dissolve

and color the cell sap, which f fin the first to this time has been perfectly

colorless (P1.XVIII, fig. 3).

sia
Their azure blue pigment shows all the reacticns cf anthocyanine,
since with acids it colors viclet, with alkali it beccmes greenish yellcw,
and with nicotine green; it dissolves, finally, in alechcl and in slightly
acidulated water. Thus in this species one may demonstrate easily, treating
the sections with oxygenated water, that the cyancplasts pessess an involucre

which encloses the pigment elluded te.

Iris fimbriata Vent.

In Iris fimbriata cne finds the cyanoplasts in the epidermal cells

of the ovary and at the basal part of the petals in cpen fl-wers.

The cyancplasts are here larger (Pl. XVII, fig. 6), in exact relation
to the develcpment cf the cell, and they present an invelucre sgh beccmes
evident whenever cne uses meth:ds ab-ve indicated.

It is a nctable fact that the cyancplasts in this species may appear
in complete development, colorless, ctlered in varying degrees cf intensity.
In these eclerless bedies, by the use cf salts of iren, bichrcmate cf pctash,
osmic acid, and methylene blue, the presence cf tannin may be easily establish:

ed, and, in others, applying suitable reagents, cne may demcnstrate the fact

that their pigment belcngs to anthocyanine.

Clerodendron Balfouri Hert.

The pigment to which is due the colcraticn cf the red ccrolla of this
species is, generally, in open flowers, dissolved in the cell sap.

However, he who examines many flowers will ccnclude that the origin
of this pigment is like that of anthocyanine hitherto alluded tc.

In fact, in not infrequent cases is to be fcund, immersed in the cyto-
plasm, a red spherical bedy (Pl. XVIII, fig. 7), in the cell in which the cell

sap is perfectly cclcrless. This body represents the cyancplast.

= Ia
oo It originates by neoformation from the protoplasm, like the cyanoplasts
of the preceding species, and toward the end of its evolution, like these,
dissolving, the intense red color spreading through the cell sap (Pl. XVIII,
figs 9).

In each cell one generally finds a single cyanoplast. They show an
involucre the cavity of which is occupied by the coloring substance. The
involucre is made evident by treating “Fhe sections with oxygenated water or
with osmic acid and the colcring substance presents all the reacticns
characteristic of anthocyanine, since with water and alechrl it dissolves
easily, with alkali it assumes a greenish yellow color, with nicotine in dilute
aqueous solution it becomes green, and, finally, it decolorizes easily with

sulphurous anhydride or with oxygenated water.

Erica carnea Linn.

In order to study the cyanoplasts of this species, it is necessary
to examine specially the calyx which, like the corolla, is colored rose
violet. Such coloration is due to a pigment which behaves like anthocyanine
and which forms in the cyanoplasts. They are relatively very large and are
generally found in numbers greater than one per cell (Pl. XVIII, fig. 4).
Their structure and their development resemble perfectly those of the
cyanoplasts of the preceding species. In the ultimate stages of their
evolution they show vacuolization; finally, they dissolve completely and
cause coloration of the cell sap, which at first was colorless (Pl. XVIII,
fig. dl,

Besides the cyanoplasts, one should note in this species the presence
of little spheres which are refractile to light (Pl. XVIII, fig. 4), which
one finds singly(rarely more than one)per cell. They are in the epidermis

as well as in the parenchyma of the leaves, of the sepals, and of the petals,

-~ 15 -
These little spheres turn brown on treatment with osmic acid, stain with
Sudan III, with Scarlet R, and with tincture of Alkanna. They do not present
the reactions of proteinaceous substances. As a consequence, they should be
referred to as elaiospheres.
Likewise in this species, in rare instances, one may find some colorless

cyanoplasts, which show all the reactions of tannin.

Conclusions.
ke

From the assfmbly of facts above presented I believe that one may draw
the following conclusions, limited naturally to those cases which ;I studied:

(1) The anthocyanine develops autonymously.

(2) Anthocyanine does not form in ordinary vacuoles, nor is it derived
from a preexisting substance in solution in the cell sap, but is formed, on
the contrary, within a special organ which I have designated cyanoplast.

(3) The cyanoplast originates directly from the protoplast by neofor-
mation .

(4) The cyanoplast is void of proteinaceous substance and showsan in-
volucre the chemical nature of which is unlmown and in which may be found tannin
substances.

(5) Anthocyanine may be derived from substances belonging to the group
of tannins, since the presence of such substances is found in cyanopiasts in
which anthocyanine develops.

(6) Certain substances extant within the involucre of the cyanoplast
may become transformed in the anthocyanine by means of special metabolic
processes.

(7) External agents may suspend the transformation in the anthocyanine
of the substances resident in the cyanoplast and then it remains colorless.

(8) The cyanoplast presents a definite development terminating in de-

generation and the spreading of its pigment through the cell cavity.

- 16-

(9) The color of the anthocyanine varies (red, violet, or azure blue),
first depending on the influence of the cell sap. One ought not here to con-
sider all these red, violet, and azure blue pigments as constituting a single
compound which colors according to the degree of acidity of the cell sap, but,
according to the results of the chemical research of other authors, it may be

asserted that anthocyanines are different in their properties.

Botanical Institute of Pavia, July, 1911.

(2)

(2)

(3)

(4)

(5)

(6)

(7)

La
LITERATURE CITED.

Buscalioni and Pollacci
1903. Le antocianine ed il loro significato biologico.
Atti dell'Ist. Bot. dell'Università di Pavia, v.
viii, p. 189.
Combes, R.
1909. Rapports entre les composés hydrocarbonés et la formation
de l'anthocyane. Annales des sciences naturelles, 9. ser.
1910. Les echanges gazeux des feuilles pendant la formation et
la destruction des pigments anthocyaniques, Rev. gén.
de Bot., t. xxii.
Grafe, V.
1906. Studien Uber das Anthokyan. Sitzungsberichten der Kaiserl.
Akademie der Wissenchaften in Wien. Mathem. naturw.

Klasse, Bd. cxv, Abt. i.

1909. Studien Uber das Anthokyan. Sitzungsberichten der Kaiserl.

Akademie der Wissenchaften in Wien. Mathem, naturw.
Klasse, Bd. crviii, Abt. i, Juli.

Hildebrand, F.
1863. Anatomische Untersuchungen tber die Farben der Blithen.

In Pringsch. Jahrb. iii, p. 59.

Laborde, J.

1908. Sur le mecanisme physiologique de la coloration des

raisins rouges et de la coloration automale des

feuilles. C. R. A. S,

DAB
(8) Mirande, M.
2907. Sur l'origine de l'anthocyanine deduite de l'observation

de quelques insectes parasites des feuilles. C.R.A.S.,t.cx1lv:

(9) Moll, H. v. D.

Vegetabilische Zelle, p. 47.

(10) Molliard, M.

1907 Action morphogenique de quelques substances organiques sur

d_ 23
les vegéteaux superieurs. Rev. gén. de Bot., t. xix.

(12)

1909. Production expérimentale de tubercules blancs et de tubercules
noirs & partir de graines de Radis rose. C. R. A. S.
(12) Overton
1899. Beobacht. und Vers. uber das Aufreten von rothem Zell-
saft bei Pfl. Jahr. f. wiss, Bot., t. xxxiii.

(13) Palladin, W.

1908. Ueber die Bildung der Atmungschromogene in den Pflanzen.
Ber. der deut. Bot, Ges., H. 6.

(14)

1909. Ueber Prochromogene der pflanzichen Atmungschromogene.
Ber. der deut. Dot. Ges., Bd. xxvii.

(15) Pfeffer, W.

Untersuchungen lbef die Aufnahme von Anilinfarben in lebende
Zellen, Untersuch. a. d. botan. Instit. zu Tubingen, Bd.
ii, pa Li

(16) Portheim, L., and Scholl, E.

pos Untersuchungen tiber die Bildung und den Chemismus von

Anthokyanen: . Berichte der Deut. Bot. Ges., H. 7.

= 19 +
(17) Wheldale, M,
1909. The colours and pigments of flowers, with special reference
to genetics. Proceedings of the Royal Society, B., v. 81.
(18) Wiesner, I.
1862. Einige Beobacht. ub. Gerb. u, Farbstoffe d. Blumenbdtter.
In Bot. Zeit.
(19) Wigand
1862. Einige Sitze Uber physiologische Bedeutung des Gerbstoffes
und der Pflanzenfardbe. In Bot. Ztg.
(20)
1837. Die Rothe u. Blaue Farbung v. Laub. u. Fruchte. In Bot. Heft.

Forsch. dà. Bot. Garten z. Marburg.

Tav: XVI.

I Politis - Cianopiasti

Lit Tacchinardi e Ferran’ Favia

l' Autore del.

AHi sa Botanico Univ. di Pavia VoL. XIV.

Ati dell Isk Botanico Univ. di Pavia Vol. XIV.

l'Autore del.

Lit Tacchinardi e Ferrari Favia

I Politis - Cianoplasti

Ath dell'Ist Botanico Univ. di Pavia Vol. XIV ‘Ten: AVIM
r detti ii ile i

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i Tacchinara e rer?
[ Autore del. ¢ Lit tacenih

Politis, Ioannes. H v
les, Honorary Assistant in the Botanical I
nst e of
Royal University o of Pavia. ee

ON A SPECIAL CELLULAR BODY FOUND IN TO ORCHIDS, Translation of:
Sopra uno speciale corpo cellulare trovato in due orchidee. a/

Atti Ist. Bot. Univ. Pavia, iL. 14: 377-383. pl. xix. 1914,

In two species of orchids, Coeloswe Heels Lindl., and Fris

stellafa Lindl., a fact was noted which confirms the object of the present

Coelogyne £ristata Lindl.

In the leaves of the perigonium and in the gynostemium of this

study. ha

species there is to be noted in each cell of the epidermal tissue and of
that immediately adjacent thereto a granulose protoplasm in which may be
found immersed a very large spherical mcleus, abundant leucoplasts,
Which are round or oval, and generally active(with amilacei in rclusions),
situated preferably roundabout the mucleus, and, finally, a body which,
from its chemical constitution, is different from that as yet found in
the plant cell.

Such a body is distinguished by its physical property and varticularly
by the special and characteristic refractility with which it is endowed, In
the living cell it appears spherical, colorless, of homogenecus aspect, and
of considerable dimensions, so much so that in its complete development it
may attain to dimensions slightly inferior to those of the micleus.

It is found in the cytoplasm now in the midst of the ceil, now near

the cell wall, withoùt relation either to the nucleus or to the leucoplasts.

There is ordinarily one per cell, rarely, however, two or more, Such bodies

are found even in the epidermis of the floral axis.

a/ See also: Rendiconti dell! Acc, dei Lincei, anno 1911.

- De

Microchemical examination, The studies wé#e made én fresh material.

Thin “le . a = 2
pices of epidermal tissue were immersed in different reagents and then
examined under the microscope,

The bodies presented the following reactions:

In absolute alcohol, in ether, in chloroform, after a few moments of

immersion, they dissolved.

In water at ordinary temperature they vacuolized slowly but did not
disappear, ,

They dissolved in 10 per cent sodium chloride, in 10 per cent solution
of potassium nitrate, in alkali, and in very dilute acids.

With solutions of potassium iodide they stain intensely brown, while
at the same time they vacuolize and, after the appearance of numerous vacuoles,
disappear.

With the use of Millon's reagent (cold),.the body assumes a rose color.

Applying contemporaneously to the sections concentrated solutions of
sugar and sulphuric acid (Raspail's reagent), it dissolves instantly. As a
consequence, one does not succeed in obtaining a clear reaction,

Immersing the sections in a drop of nitric acid, they take on a
yellowish color.

Sudan III, Scarlet R, and tincture of Alkanna, no color results.

With iron chloride and iron acetate PE aqueous solution, they take
on a blackfeh blue color after a short immersion.

Tith bichromate of potash in concentrated solution, they acquire a
distinctly reddish brown color.

With osmic acid, they turn black after a short time.

With ammonium carbonate, potassium carbonate, and sodium carvonate,

they stain yellow, and, with the progress of the action of the reagent, there

- 3- H

is often ods oe i 3
crved in its interior some grsnules or numerous little spheres due

t re A
o a precipitation. These granules dissolve in water and absorb many aniline

dyes.

With alkaloids (caffeine, chinine, nicotine, morphine, cocaine, atropine,
strychnine, and codeine), in 2 per cent solutions, one obtains sometimes a
scant precipitation similar to that obtained with the carbonates,

Methylene blue in aqueous solution is absorbed with avidity and commu-
nicates to the body, after a short time, a beautiful color.

Examining the reactions alluded to, we reach the following conclusions:

In Sudan III, Scarlet R, and tincture of Alkanna, we did not get the
characteristic color for oleose substances.

The reactions obtained with a solution of iodide of potassium, with
Millon's reagent, and with Trommer's reagent are the same as those which in
the microchemical examination serve to show proteinaceous su stances,

Osmic acid, ferric salts, bichromate of potash, alkaline carbonates,
alkaloids, and methylene “vlue behave precisely as in the presence of tannin.

From this complex of reactions one may conclude that within the body
under examination there exist proteinaceous and tannic substances, while oleose

substances are lacking.

Colorization. In some works by Pfeffer (1, 2) 2/ may ve found refer-

ences to the absorption of aniline dyes by the living cell. It is there to
be noted also that a color substance incapable of osmosis may be contained in

the cell sap because of the presence of diverse bodies. From these are noted

to date fluoroglucin and tannin. This latter, according to Pfeffer, may be

determined by any of all of the absorbable aniline dyes except rosolic acid.
Having established by means of many reagents the presence of tannin in

the body under examination, I believed it worth while to check the observations

of Pfeffer, using, in addition to the colors used by him, some others.

a/ Reference is made by number to "Literature cited," near end of translation.

= Me

Tae stains which I used are the following: Aniline red, gentian violet,
dahlia violet, aniline blue, malachite green, iodine sreen, methyl green,
thionine, methylene blue, safranine, neutral red, nisrosine, chrysoidine,
vesuvine, Bismarck brown, Congo red, and lisht green.

Of these staining materials, aniline red, gentian violet, dahlia violet,
malachite green, iodine zreen, methyl ¢reen, thionine, methylene blue, safraning
neutral red, vesuvine, Bismarck brown, and light green were able to determine
in a very short time, by use of the most dilute solvtions and without any
previous fixation, the presence of tannin in the body in question.

Nisrosine, on the contrary, Congo rei, and aniline blue were not ab-
sorbed even in concentrated solutions.

Development. The body in question does not participate in the phenomer
of kariokinesis in the cells of the floral axis during their course of develop-
ment. It appears first in some and then in all the cells as a very minute,
little sphere, extremely refractile to light, immersed in the cytoplasm and
enveloped frequently by a fine, granulose substance of tamnic character.

Such spheres grow with the growth of the cells in which they occur and,
arriving at completed development, reach dimensions siizhtly inferior to those
of the nucleus.

Later, namely, when the fowers begin to wither, one often observes in
these bodies noticeable degenerative phenomena; in fact, they lose their rotund
form, their homogeneous aspect, and assume irregular, varied forms, while there
appears in them numerous vacuoles which give them a spongy appearance.

Among these vacuoles, which are situated at the periphery, “there often
appear in the form of a ball those which expand as if an internal pressure

; A - vu i
were to durst them. Not infrequently one finds two vacuoles nsarly touching

-5-
‘each 1 ; 3
ch other and, where there is a fracture of the partition separating them,
it di ri :
t dissolves, thus resulting in the formation of a single vacuole. Finally,

Cd

following the fusion of many of these vacuoles, there forms a single large
one bounded by a thick wali.

Often, aside from the formation of the vacuole, one notices appearing
from one or more points of the surface of the body a refractile substance
of homoseneous appearance. If, now, one uses a solution of iodide of potas-
sium, one may be able to distinguish two substances which behave diversely;
one which colors intensely brown within and another which assumes a yellow
color peripherally. In the central part there sometimes appear granules of
variable dimensions.

Summarizing, we observe the followine: That the body in question pos-
sesses a round form, homogeneous structure, and is colored orowm in a solution.
of potassium iodide. On degenerating, it vacuolizes, its form becoming ir-
regular. ‘ith reagents herein above referred tc one can observe two distinct

substances.

Eria stellata Lindl.

In the leaves of this species, in each cell of the epidermis and of
the parenchyma, is to be noted in the cytoplasm as well as in the nucleus
and in the leucoplasts, which are roundabout it, a colorless spnerical body,
strongly refractile to light, and presenting the following resections:

Treated with a solution of potassium iodide, it stains yellow or brow-
ish, according to the degree of the concentration of the solution.

ith concentrated or dilute alkali, it dissolves and disappears rapidly.

With sulphuric acid and with other mineral acids in concentration, it

becomes invisible after a short immersion.

a
In water at ordinary temperature, it vacuolizes but does not disappear.
In saline solutions of sodium chloride, potassium sulphate, and am-

mon iur i .
onium chloride, after 24 hours, it appears as a vacuole bounded by a thick

wall,

This is not soluble in alcbhè),nor in acetic acid, sulphuric acid,
nor dilute hydrochloric acid.

With Millon'ts reagent heated ever so little, one o>taings a more de-
cided reaction; in fact, immersing the tangential sections taken from the
ventral or dorsal surfaces of the leaf blades in a drop of the reagent, the
body, retaining its original form, assumes:.arcddish color which, after slow
warming of the preparation, becomes intensely brick red.

With especially rich colorinz substance(quinoline, Sulan III, Scarlet
Rh, and sulphate Nilblau), one obtains no color reaction. One obtains, on
the contrary, an important reaction by using 1 per cent osmic acid, Treating
sections similar to the precedins with this reagent, a rest number of the
bodies in question turned entirely black, while some showed a black interior
part Gue to tannic substance and an external peripheral colorless part which
is refractile. This, with solutions of potassium iodide, stains brownish
yellow, shows all the other special reactions of proteinaceous substance,
and is, therefore, to be considered as forming a plasmatic involucre in which
tannic substance is included. Such an involucre, in preparations fixed in
alcohol and then treated with a mixture of fuchsine and methyl green, appears
granulose and distinctly red in color.

The body under examination, moreover, because of the. tannin which it

contains ,turns reddish browntwith bichromate of potash in concentrated so-
lution, bluish black in iron acetate and iron chloride, brown in 1 per cent
chromic acid, yellow when treated with alkali and with alkaline carbonate.

It in fact, absorbs many of the aniline colors without any previous fixation.
!

ue

From the sum of these observations it is evident that this body con-
Sists of two substances and that one of these is proteinaceous and the other
tannic in character,

Finally, it is to be noted that in the neishborhood of this body may
frequently be found numerous Spherical corpuscles, isolated or in groups,
endowed with Brownian movement. They are almost invisible in indifferent
solutions, and may be observed in sections treated with proper reacents,.

With osmic acid, in fact, they turn black; with bichromate of potash
and chromic acid they become intensely brown; with iron acetate and iron
chloride they take on an obscure blue color; they show, moreover, all the
reactions of tannin, and it is here, probably, that they will be found re-
lated to the body under examination and which represents the products of

its activity.

Origin and Morphologic and Biologic Significance
of the Cellular Bodies under Examination.

The, protoplast in the course of its functional activity, ag has been
noted, produces often by neoplasia the more important bodies to which belong,
according to the results of the history of their development above described,
also the body in question.

As far as concerns its morphologic significance, its method of
multiplication, chemical composition, and the morphologic characters which
this body presents, the idea that it may have some connection with chroma-
tophores is to be excluded; for, I believe that especially because of the
presence of tannin, it should be considered related to cyanoplasts (3) and
because of the presence of the vesicle of tannin ?y which it is to be
distinguished by its reaction from proteinaceous substances which show more
evidently, and because of the special biologic function which it may perform,

it should be considered related to cyanoplasts.

Vigia
So far as concerns this latter, I have sought to study if-this body,
having for its location the epidermal cell and containing tannin, has not
the function of protecting the organs in which it is found, from the ravages
of snails (as supposed by Stahl regardinz some plants containing tannic sub-
stances), but the results which I obtained were completely negative. Moreover,

the snails would eat without damasze to themselves the leaves of Eria stellata

and the floral axes of Coelozyne cristata which were given them.

Conclusions.

In Eria stellata and on Coelokxyme cristata there was revealed the

presence of a body which up to that time had not been described by any pre-
vious author. It forms by neoformation from the protoplasm and shows re-
actions of the proteinaceous substances and also those of tannic nature.

It does nct disappear durinz the life of the cell in which it is found. It
is not usei as an organ of defense against the ravages of snails.

What biologic action it may have I do not now lmow.

Botanical Institute of Pavia, Octcber, 1911.

Ù
oO
I

LITERATURE CITED.

(1) Pfeffer, W.
Ueber Aufnahme von Anilinfarben in lebende Zellen.
Untersuchungen aus dem botan. Institut zu Tubingen,
Bony day De LEI
(2) sic
1897. Pflanzenphysiologie, Bd. 1.

(3) Politig, LL

1911. Sopra speciali corpi cellulari che formano antocianine.
Rendiconti della R, Accademia dei Lincei, v. xx, ser.

5° p. 828.

PLATE LEGEND,

PLATE XIX.
& = special cellular body; n = nucleus; 1 = leucoplast; ci = cytoplasm.

Fig. 1. Epidermal cells of the perianth of Coclocme £fistata Lindl.
: . 2. Cells of tho lower epidermis of an adult leaf of Eria stellata Linil.
s 3, 4, 5. Three epidermal cells of the perianth of Coelo“yne Cristata.
The special cellular body is represented stained (without

preventive fixation) with lisht zreen (fig. 3), with Methylene

blue (ric. DD, with saframine (fic. 5).

Note:- The ficures were draw with No. 4 ocular and No. 9 Koristka

odjective.

March, 1923 Translated by H. B. Humphrey.

AR dell’ Ist’ Botanico U

ro Ue di Pavia Val Jay.

l'Autore del. [ Politis - Speciale corpo cellulare

Lit Tacchinardi e Ferran Favia

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