COMPARATWE GROWTH QF BARLEY memos
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“nests. gov Hue Degree of M. S.
MECHIGAN STATE UNEVERSETY
Chang Won Chang

1957

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LIBRA R Y
Michigan State
University

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CONSPARLTIVE GROJTH 0F BARLEY EMBRYOS IN VITRO- AND IN VIVO

by
Chong Won (L hang

AN ABSTRACT

Submitted to the College of Science and Arts Michigan
State University of Agriculture and Applied
Science in partial fulfillment of the
requirement: for the degree of

MASTER OF SCIENCE
Department of Botany and Plant Pathology

1957

._ 47 .
Approved by w m4"mwj

Chong Won Chang
ABSTRACT 1

In an attempt to obtain a more fundamental understand-
ing of the growth of plant embryos in culture, barley embryo
development in vitro was compared to development in vivo in
the following ways: (1) by comparing their morphological
development, (2) by comparing their relative sizes and rates
of growth, and (3) by relating their growth to stages of any
bryonic development.

Most of the previous work dealing with embryo culture
has been primarily concerned with attempts at growing "pre-
mature” embryos in any way possible irregardless of the growth
patterns in culture, without determining accurately in what
morphologica1 stage of development embryos were at the time
they were placed in culture, and without comparing their de-
velopment in vitro with their normal growth in vivo. To date
only a few satisfactory results have been obtained and most
of these have been.with embryos of dicotyledonous plants, e.g.,
Van Overbeek, 33 El. (l9hl, l9h2) wherein they succeeded to
grow two proembryos of Datura. More limited success has been
obtained with immature monocotyledonous embryos: Kent and
Brink (19u7) achieving some success in the culture of immature
barley embryos; Norstog (1955) reporting rates of growth and
morphology in culture of young embryos of barley but for one
week only. In neither case, however, was a comparison made
between growth rates in culture and rate of development in

vivo. The work most similar in nature to the present study

r‘

 

 

 

Chong Won Chang
2

was that of Merry (l9h2)'who compared growth rates of embryos
in culture with those of the same aged embryos in vivo. The
only embryos, however, with which Merry had any success in
culture were in the mid-stages of differentiation at the time
of placement in culture; therefore his results have only a
very limited bearing on the present work.

In the present work, the barley variety. Hannchen
(C. I. 531, a two-rowed variety), was used for the in vivo
snd.in vitro study. Two kinds of media were used. The first
was made according to White (l95h), while the second was pre-
pared by using one part of the above basic medium and nine
parts of coconut milk. One hundred fifty embryos were cul-
tured on the second type of medium. These embryos were re-
tained on this medium.without transfer even after a two-week
period in order to see whether shoots or roots might be ini-
tiated. Eighty-eight embryos were cultured on the second type
of medium. The fact that embryos cultured on the second type
of medium.only failed to undergo differentiation, would sug-
gest that the addition of coconut milk to the basic medium
was at least one of the critical factors for differentiation
of young embryos. Orientation of the embryos on the agar
medium.had an effect in terms of the developmental morphology
in culture since it was noted that embryos placed with acutel-
lar surfaces in direct contact with the medium gave growth

patterns more nearly approaching those of embryos in vivo.

1‘

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Chong Won Chang
3

It was found from the in vivo study that the lengths
and lateral diameters of developing embryos are directly
proportional to each other, while the lengths and widths of
developing caryopses are not well correlated. ‘Hhen the sizes
of caryopses and embryos are related to the stages of embryo
development, the lengths of the caryopses show a rapid in-
crease from the time in late proembryo develOpment until
stage 2, while the lateral diameters of the caryopses and the
sizes of embryos in vivo undergo little change in measurements
during this time. After stage 2, the increase in lengths of
the caryopses gradually slow down until the middle of stage 6
is reached. Just prior to stage 5, the lateral diameters of
the caryopses undergo rapid increases in size while the embryos
increase in size at a somewhat lesser rate. By the middle of
stage 6, the lengths of the caryopses become almost constant,
while the lateral diameters are still increasing, but at a
much diminished rate. The sizes of embryos within the caryop-
ses, however, are at this time undergoing their most rapid
growth. The fresh and dry”weights of embryos show an ever-
increasing relatimnship to embryo size. In addition to this,
it was found that approximately 2/3 of the embryos' fresh
weights was due to water. Of a total of S9 spikes from.which
embryos were excised for the in vivo study, h spikes contained

embryos which averaged 0.55 x0.30 mm. initially. Their final

t.

fihong Won Chang

average size at the end of two weeks was 3.15 x 2.38 mm.
The ratio of the average final length to average final width
in vivo, therefore, was 133 z 1.

From.the in vitro study, the average initial size of
38 embryos was 0.50 x 0.30 mm., while at the end of two weeks
in culture they had attained a size of 1.20 x 0.90 mm. When
the ratio of average final.lengths to average final widths
are compared, they are seen to be identical (1.3 : l) in both
in vitro and in vivo embryos. Embryos developing in culture,
therefore, maintain length/width relationships which are iden-
tical with, and attain sizes which.approach those of in vivo
embryos. In vitro embryos differ, however, from those develop-
ing in vivo in that cultured embryos are larger at any given
morphological stage, are slower in the rate at which they pass
through the various stages, show a number of morphological
deviations from.normalembryogeny, and never attain the morpho-

logical development of stage 6.

COMPARATIVE GROWTH OF BARLEY EMBRKOS IN VITRO AND IN VIVO

by
Chong Hon Chang_

A THESIS

Submitted to the College of Science and Arts Michigan
State University of Agriculture and Applied
Science in partial fulfillment of the
requirenmnts for the degree of

NMSTER OF SCIENCE

Department of Botany and Plant Pathology

1957

‘— i // fizz/l ‘25." 7

INTRODUCTION . . .
HISTORICAL REVIEW . . .
MATERIALS AND METHODS .
In Vivo Study .
In Vitro Study .
RESULTS AND DISCUSSION
In Vivo Study

Morphological
Embryogeny

Relationship between Caryopses and Embryos
Based upon Developing Stages

Relationships between Embryo Size and Embryo
Fresh and Dry Weights

TABEE

Characteristics

0

OF CONTENTS

of Barley

O O O O O O O

Embryo In Vivo as Controls for Those In ViVO.

In Vitro Study

Development of Embryos In Vitro with those

In Vivc

Morphological Comparison

Rate of Growth, Size and Stage Comparison

iSUHMARY . . . . . . .
IIIBLIOGRAPHY . . . . .
APPENDIX C O O O O O O

Page

10
10

19
19

19

28
29
31

38
38
ho
us
1+9
52

ACKNOWLEDGMENTS

The writer wishes to express his sincere thanks to
Dr. L. W. Mericle and Dr. R. P. Mericle for their construc-
tive criticism and encouragement throughout the course of
the research and during the preparation of the thesis, and
to Dr. W. B. Drew, Dr. G. P. Steinbauer and Dr. Everett S.
Beneke for their valuable suggestions.

Thanks are also due to Mr. P. G. Coleman for his un-
reserved help in the photographic work.

To the Atomic Energy Commission for financial assist-
ance, equipment and supplies, the writer also wishes to

express his appreciation.

LIST OF FIGURES

FIGURE PAGE
1. Caryopses at different stages of deve10pment . . . 12

2. Culture chamber used during the initial two-week
pOPIOd a e a s a s a a e a a e e a e a e a a a 15

3. Plantlets formed from embryos, each 0.70 mm. in
initial length and cultured for 19 days . ._. . 16

h. Relationship between lengths and lateral diameters
Of embryos in VIVO e a e a e a a e e a a e e e 20

5. Relationshipss between lengths, and fresh and dry
weights of embryos in vivo . . . . . . . . . . . 21

6. Representative embryos at different stages of
development a e e e a a e a a e a e a a e a e e 23

7. Relationships between embryo sizes, embryo fresh
and dry weights, and caryopsis sizes in vivo, and
embryo sizes in vitro as compared to stages of
embryonic development a a a a e a e a e e e e a as

8. -Relationships between embryo lengths, and caryopsis
lengths and lateral diameters from.plants
in VIVO a a a a a a e a a e a e e e a a a e e a 27

9. Representative embryos which produced shoots and
roots, after being transferred into screw-cap
‘vials e e e a a e a a a e e e e e e a a a e 0 3h

10. Relationship between embryo lengths and lateral
diameters in Vitro e e_e e a a a e e a e e a e a 37

LIST OF TABLES

TABEE PAGE

1. Individual length, width, fresh weight, and dry
weight measurements made at two-day intervals
of immature barley embryos developing in vivo . 53

2. Individual length, width, fresh weight, and dry
weight measurements made at two-day intervals
of barley embryos and caryopses developing
in'VIVOaeeaeaeeaeeeaeeaeeea 57

3. Average size, fresh and dry weights of embryos
from (Table l and Table 2) and average size
of caryopses (from Table 2) related to
histologically determined stages of embryonic
development.................. 60

h. Individual length and width measurements made at
two-day intervals of four representative
immature barley embryos developing in vivo . . . 61

5. Culture group 1. Average growth.rate of embryos
cultured continuously on basic medium.plus
coconutmilk.................. 62

6. Culture group 2. Ratio of length/width (in mms)
of immature barley embryos developing in
culture, including total days in culture(d)
and day (d) on which shoot or root first
lppBQrOd aeeaeeeeesoeeaeeeao 63

7. Summary of three series (based upon sizes)
fromTableé.................. 68

8. Growth characteristics of a ”typical” immature
barley embryo (No. 8-6 of Table 6) in culture,
at two-day intervals, for 30 days. . . . . . . . 69

INTRODUCTION

Although the artificial culture of plant materials has
received wide attention for a number of years, embryo culture
should be distinguished from "tissue culture" in the broad
sense. The latter aims at the growth in vitro of isolated
tissues or of plant parts, while the former, embryo culture,
theoretically at least, intends to induce normal embryogeny
during embryo development (from.the time of fertilization
until embryo ”maturity" as found in the seed) like that which
is accomplished within the plant itself, that is, in vivo.

The real aim, therefore, of embryo culture should be that of
achieving continuous, normal embryonic development with the
idea in mind of the ultimate production of seedlings which

are morphologically and physiologically the same as those of
embryos which develop in vivo. In order to approach this aim,
then, it is most important to culture immature embryos as
young as possible and to trace all stages of embryonic develop-
ment until seedlings are formed. Thus, it is extremely hard
to draw any positive conclusions as to the success that is
being achieved in regard to embryonic develOpment in culture,
without first observing each stage of normal embryogenesis
within the plant itself from fertilization time until the time

when the embryo is'mature' (as found in the mature seed).

'0

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A survey of the work accomplished by researchers up to
the present time may be grouped into two categories: one con-
cerned with the production of plantlets; the other with the
continuous embryonic growth of immature embryos. In the former
case, the growth of immature embryos does not follow normal
embryonic development, but rather gives rise to small plantlets
which are the premature outgrowths of previously formed root
and shoot primordia. The latter aims at inducing the same amp
bryogenesis and ultimate formation of seedlings as would result
from.normal development in vivo. So far as the history of
embryo culture is concerned, much more research has been done
with.this latter aspect in mind, yet to date few satisfactory
results have been obtained. Among the more successful at-
tempts has been the work of Van Overbeek,.g£.gl.(l9hl, 19h2)
using materials of dicotyledonous plants in which seven pro-
embryos (0.1h mm. in diameter) of Datura were cultured, and,
for the first time, apparently normal embryonic growth.was
induced in two of them.

In monocotyledonous plants Norstog (1955), culturing
barley embryos, ranging from 0.3 to 0.8 mm. in length, reported
the rate of growth and the morphology in culture during a one-
week period only. He also succeeded in ”growing" two "pro-
embryos” which.were only 0.16 to 0.2 mm. in length. In neither
case, however, was a comparison made between growth rates of

the cultured embryos and those developing in vivo.

So far as the writer knows, the research most similar
to the present work is that of Merry (l9h2). He attempted to
culture seven-day old (post-fertilization) barley embryos
which.measured approximately 0.3 mm. in length; nine-day old
ones 0.5 - 0.6 mm. long; ten-day old one 0.7 mm. long; and
eleven-day old embryos measuring 0.8 - 0.9 mm. in length. Al-
though he did not succeed in growing any of these, he was able
to produce plantlets from twelve-day old embryos and compared
them morphologically with embryos growing in vivo.

Although the knowledge of plant embryo culture has ac-
cumulated over the years, especially research dealing with
various aspects of nutritional requirements, thus far no one
has succeeded in growing extremely immature excised monocot
embryos in culture the way they normally develop in vivo. At
this point it is quite important to compare the growth be-
haviors of embryos in vitro with those in vivo in order to
more clearly evaluate their differences. Therefore, in the
investigation undertaken here, particular attention has been
directed toward a comparison of embryo development in vitro
and in vivo in the following ways: (1) by comparing their
morphological development; (2) by comparing their relative
sizes and rates of growth; and (3) by relating their growth

to Stages of embryonic development.

HISTORICAL REVIEW

After a careful survey of the literature relating to
embryo culture as a whole, it was decided to limit the dis-
cussion to the more pertinent work: that dealing primarily
with the culture of immature embryos. Also, since the present
work was designed to study in detail immature embryos, excised
as young as possible, the culture of mature embryos has little
relationship to the aspect that the writer intended to approach.

For some unknown reason, it appears to be very hard to
grow embryos of Gymnosperms in vitro. So far as the writer
knows, no one yet has succeeded in culturing immature Gymna-
sperm.embryos, a1though.the culture of Giggko embryos was at-
tempted by Radforth (1937), that of £iggg_embryos by Leo and
Wang (l9h3), and young embryos of ngi§_were investigated by
Sterling (l9h9). The results obtained by these investigators
were more or less similar in that they failed to induce normal
embryonic growth, but did obtain undifferentiated masses of
tissue.

In the culture of embryos of dicotyledonous plants,
much greater success has been attained. Lofland (1950) was
able to culture mature embryos of Goss ium, yet failed to
grow the young, immature ones. Since certain varieties of

sweet cherries produce no viable seeds (because prior to the

time of fruit ripening, the embryo and endosperm tissue cease
development and abort) Tukey (1933) attempted to culture the
immature aborting embryos. Although he was not able to induce
normal embryonic growth, he did succeed in producing plantlets
by using Knop's complete nutrient solution and Crone's nitro-
gen free solution. He also found that immature embryos of
apple,and peach, among others, did not continue normal em,
bryonic development in culture, but rather produced plantlets.
Using a medium.containing coconut milk, Van Overbeek, gt 3;.
(l9hl, l9h2) cultured seven proembryos of Datura, 0.1h mm. in
diameter, which were 1h days old. They, for the first time,
succeeded in growing two immature embryos (of all those at-
tempted) apparently normally, without a precocious differenti-
ation into plantlets. Their success was apparently due to
certain substances within the coconut milk which was referred
to collectively as an ”embryo factor.”

As was mentioned above, it has been fairly well estab-
lished that young immature embryos of monocotyledonous plants
are much harder to grow than dicotyledonous plants. Many inp
vestigators have attempted to solve this difficult problem.

In the years following the successful growth of proembryos
of Datura with the addition of coconut milk, it has become
generally accepted that young immature embryos in vitro require
certain ”embryo factors” for their normal embryonic growth.
Therefore, a great deal of effort in recent years has been de-

voted to the determination of a mere effective ”embryo factor."

LaRue (1936) cultured young excised embryos of many
plants to determine the minimum.size of embryos which could
be grown, and also to investigate the relationship of such
culture with the developmental morphology of the embryos. He,
particularly, succeeded in culturing embryos which.were
smaller than any previously cultured. Following LaRue's
earlier work, LaRue and Avery (1938) compared the growth in
culture of immature embryos of Zisania with those growing
in vivo. By culturing,embryos, ranging from 0.2 - 0.35 mm.
they found that the size could be doubled but were unable to
develop further. In the culture of embryos 0.h to 0.7 mm.
in length, continuous cell divisions were found; any increase
in embryo size was due to cell enlargement only. Embryos in
which.more cell divisions were obtained and in which a rudi-
mentary leaf was induced, measured 0 mm. in length. Brinkigt
5;, (l9hh) grew a young hybrid embryo, which was the product
of a cross between a wild species of barley and domestic rye,
into a mature plant. Konzak gt 51. (1951) succeeded in ob-
taining seedlings by culturing young embryos of hybrids be-
tween commen barley and wild perennial barley. In the culture
of young embryos of 9352;, Lee (1952) found that the seedlings
from the cultured embryos were smaller and weaker than those
grown from.embryos developing in vivo. Interested in the
factors responsible for normal embryonic development, Curtis
(19h?) cultured orchid embryos, using a medium to which bar-
biturates had been added, and obtained instead of plantlets

o.

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only an undifferentiated mass of tissue. Kent and Brink (l9u7)
and Ziebur, gt 3;. (1950) cultured immature barley embryos
using, in addition to a basic mdneral-sucrose mixture, various
concentrations of casein hydrolysate, tomato Juice, sodium
nucleate from yeast, lactalbumin, and wheat gluten hydrolysate
to test the effect of these substances as ”embryo factors."
Theirthmmature embryos” which were most successfully cultured
were well differentiated embryos at the time of excision.
Since the primary aim of these studies was to determine the
effects of culture upon subsequent seedling growth, no com»
parisons were made with.normal embryo development in vivo.
Ziebur and Brink (1951) found that the addition of endosperm
to the culture medium would also act as an ”embryo factor.”
They reported limited success in culturing ”proembryos,” but
again without any comparison with in vivo embryogeny. Haagen-
Smit 23 5;. (l9h5) tried to grow very young embryos of maize
by the addition of coconut milk to a medium containing in
addition to Van Overbeek's basic medium, sucrose, asparagine,
and biotin, but failed t6 obtain significant results. The
first successful attempt to culture immature monocot embryos
by using an ”embryo factor" source entirely different from
any tried by previous workers was made by Pieczur (1952). He
found that young maize embryos could be effectively grown if
they were placed on a medium in which a mass of the maize endo-

sperm.tissue was already growing, yet would not grow if merely

excised endosperm (from the grain) was placed on the-medium
at the same time that the embryos were started in culture.

In a recent investigation concerned with the culture of ex-
cised embryos of cats, barley, rye and wheat, Norstog (1955)
reported the successful embryonic growth of immature barley
embryos by culturing with a modified White's nutrient medium.
to which 90 percent coconut milk had been added. or the four
smallest embryos cultured (ranging from.0.16 - 0.20 mm. in
length) two produced leaves and roots. while Norstog described
the morphology of his cultured embryos in some detail, he did
not use in vivo controls. In an earlier experiment, using a
different culture medium, Merry (19u2) compared barley embryos
in culture with those in vivo in an effort to determine their
morphological relationships. He failed, however, to induce
embryonic development and to produce seedlings when culturing
embryos younger than eleven days post-fertilization. The
youngest age at which he could produce plantlets was from
twelve-day old embryos.

After a survey of the brief history dealing with imma-
ture monocot embryo culture up to the present time, it appears
to be true that no one has yet succeeded in taking extremely
immature embryos and producing normal embryonic growth.with
subsequent plant seedlings which are morphologically and
physiologically the same as those which develop in vivo. when
previous workers have referred to ”successful" culture, they

apparently have meant the ability to keep immature monocot

It‘ll”)

embryos alive or growing for a short period of time irregard-
less of their nature of development, and without making a

critical comparison between embryo growth in vitro and in vivo.

10

MATERIALS AND METHODS

The barley variety, Hannchen (C. I. 531, a two-rowed
variety) was used for the in vivo and the in vitro study.
This variety was chosen because it has been used extensively
for radiation research here during the past four years and
is a uniformly growing plant of a long inbred line well adapted
to the Michigan climate. Plants were grown in the greenhouse
where temperatures were kept at 75° F during the day and 65° F
at night. Diurnal optimum temperature differentials for this
strain of barley should be 10° - 15° F. It is particularly
important to maintain the lower night temperature to avoid
sterility problems often encountered with higher temperatures.
Since barley is a long day plant, the day length was increased
to 20 hours by the use of artificial light in order to hasten
flowering, thus permitting extra ”crops” to be grown during

a given period.

In Vivo Study

In order to determine the growth rates of the caryopses
and embryos in vivo, samples were taken at two-day intervals
and the lengths and lateral diameters were measured. Measure-
ment of embryos and caryopses were carried out under a calibrated

dissecting microscope. There was little difficulty in

10

11

determining the exact lengths and lateral diameters of the
embryos (after excision from the fruits) and the lateral diames
ters of the caryopses. The lengths of the caryopses, however,
were much more difficult to obtain since the fusion of the two
stigmas (Fig. l) at the apical and made an exact delimitation
of that end hard to determine. This difficulty was also en-
countered by Harlan (1920). In the present study, this dif-
ficulty was resolved by carefully determining the fusion point
of the stigmas with a dissecting needle before measurements I
were made.

Formalin-acetic acid-alcohol, FAA (Johansen, l9u0),
was used for killing and fixing caryopses sampled for the
histological study. Materials were dehydrated, embedded in
paraffin by standard procedures (Johansen, l9h0) and serially
sectioned at 12-15 microns. Staining was carried out by
using saffanin.

Selection of caryopses of the same age was made pos-
sible by the fact that the caryopses in the middle of the spike
are pollinated on the same day, while in the terminal four and
the basal four, pollination occurs slightly later. Therefore,
if the caryopses of the four terminal and four basal nodes
are discarded, the remaining caryopses are, for all practical
purposes, identical. Sampling of the caryopses and embryos

was done in order, from the top of the spike toward the base.

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Figs 1.

Caryopses at different stages of development.

a.

b.

d.

3.

Caryopsis, 3.2 mm. long, containing a mid-
proembryo, size not determined.

Caryopsis, 5.6 mm. long, containing an
embryo 0.23 mm. long.

Caryopsis, 7.0 mm. long, embryo O.h mm. long.
Caryopsis 8.8 mm. long, embryo 0.67 mm. long.

Caryopsis, 9.7 mm. long, embryo 3.00 mm.
long.

 

13

Otherwise one might wonder whether the scars made on the
rachis by removing the earlier caryopses might not affect

the normal physiology of those remaining above the scars.

1h

In Vitro Study

Sterile culture chambers for this phase of the work
were of two types. The first type consisted of small plastic
cups placed in petri dishes in the bottom of which were wet
filter papers for maintaining a saturated atmosphere (Fig. 2).
Plastic cups were sterilized by soaking them in 70 percent
alcohol overnight. Chambers of this sort were used for the
initial cultures since embryo inoculation could be easily ac-
complished and measurements of embryos could be accurately
made through the petri dish lids with the aid of a calibrated
dissecting microscope. After two weeks culture, embryos were
transferred to a second type of culture chamber screw cap
vials (Fig. 3). since these had more room for upward growth
of shoots.

Two kinds of media were used. One was made according
to White (l95h). while another was prepared by using one part
of the above basic medium and nine parts of coconut milk.

The first type of medium.was used with the screw cap vials

and the second type of medium.with the petri dishes. Before
adding agar, the basic nutrient solution was adjusted to a pH
of 5.6 by the addition of 0.1 normal potassium hydroxide. The
original pH of the solution ranged from h.5 to h.6. Agar,

0.75 percent, was added to the nutrient solution just before
autoclaving at 15 lbs. pressure, 2u0° F, for 20 minutes. Coco-

:nut milk was sterilized by using a series of sintered glass

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Fig. 3, Plantlets formed from embryos, each 0.70 mm.
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filters and was added to the autoclaved culture medium asep-
tically, just prior to gelation, at the time when the medium
felt warm.to the hand. Since 9 parts of coconut milk were
added to 1 part of basic medium, it was hard to have a per-
fectly nniform.medium of these two components. If the medium
became gelatinous upon the addition of the coconut milk, it
was steamed for as short a time as necessary to obtain a more
perfectly mixed.smdium.

In obtaining embryos for culture, individual young
fruits with lemma and palea still intact were transferred
through three different rinses of 1 percent each of Kromet.1
After removing the lemma and palea, the fruits were again
sterilized with another solution of 1 percent Kromet for five
minutes, then rinsed.with three changes of sterile, triple
glass-distilled water. All of these procedures were carried
out under a glass dust shield in an inoculating room.wherein
the atmosphere had been previously water sprayed with a hand
sprayer. until the embryos were excised, sterilized fruits
were kept in sterile petri dishes which contained a small
mmount of sterile culture solution. Embryos were aseptically
removed from the young fruits with the aid of a dissecting
microscope and transferred to the agar medium.in the plastic
cups. Efforts were made to insure the placement of embryos

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18

with the nutrient agar. Because a scutellum had not yet dif-
ferentiated in the smallest embryos, 0.h - 0.6 mm. in length,
it was difficult but not impossible to orient the excised
embryos so that they would have the above position after dif-
ferentiation. The importance of the position of the young
embryos upon the agar medium.will be discussed later. It
should be pointed out, however, that very small embryos of
this type must be excised and placed in culture as quickly

as possible after the flowering spikes are obtained from the
greenhouse since it was found that with a lapse of tbme,
viability of the embryos in culture was considerably reduced.
After the excised embryos were inoculated onto the medium
containing coconut milk (the second type of medium.as mentioned
above) within the plastic cups, the petri dishes were half-
sealed lest proper aeration should be hindered and kept in a
growth control laboratory in the dark at a 60° F night temper-
ature and a 70° F temperature during the day. At the end of
two weeks embryos which were selected to be cultured for an
additional length of tbme were aseptically transferred from

the plastic cups to the screw capped vials.

19

RESULTS AND DISCUSSION
In Vivo Study

Comparisons of embryo sizes (lengths and lateral dimme-
ters) and embryo weights (fresh and dry) were made from 378
embryos dissected at two-day intervals from.59 developing
barley spikes and are shown in Tables 1 and 2, and Figures
h and 5. In order to determine changes in embryo sizes and
weights (relative to stage of embryo development and caryopsis
size) two caryopses borne oppositely on the rachis were sampled
at specific times as the spikes developed. One sample was
killed and fixed for histological determination of the stage
of embryogeny while the other sample was used for size
measurements of the caryopsis and for subsequent excision of
the embryo so that its size, weight and gross morphology could
be determdned; no pairs of such samples were made and the
average of the results obtained are shown in Table 3, be-
ginning with the earliest stage which could be dissected (late

proembryo).

Morphological Characteristics of Barley Embryogeny
Patterned after Mericle and Mericle (1957), morphological

 

and histological features of barley embryos, from.fertilization

time until “maturity” of the embryo as found in the seed, may

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Heights of embryos in

 

 

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Lengths of embryos in El.

22

be divided into two large groups: proembryos and differenti-
ating embryos. The former may be in turn arbitrarily divided
into three sub-groups: early, middle, and late proembryos.
Early proembryos include developmental stages from the one-celled
zygote to the B-celled stage; middle proembryos from.the 16-
celled stage to approximately 72 cells; while late proembryos
consist of the largest obovate-spheroidal embryos just prior
to the initial stagesof'organogenesis (organ differentiation).
Differentiating embryos may be sub-grouped into six stages.
The principal feature distinguishing stage 1 is a slight con-
vexity which.forms on the abaxial ”face” of the embryo surface.
In examining this stage threeadimensionally with a dissecting
microscope, there is a considerable overlap, however, between
this stage and that of a late proembryo, relative to size and
gross morphology (Fig. 6, a, b). Stage 2 is characterized by
the initiation of the coleoptile, at this time an incomplete
circle (collar) of tissue which makes this stage more or less
easily recognizable. Little difficulty is experienced, there-
fore, in distinguishing stage 1 and stage 2. Stage 3 (Fig.

6, b, c) also has clear morphological features: a gradual
development of a fan-shaped scutellum, a continued differenti-
ation of the coleoptile (now a complete circle of tissue) and
the initiation of the shoot primordium which appears as a
'dot' of tissue in the center of the encircling coleoptile

when viewed with a dissecting microscope. About this time the

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Fig. 6.

Representative embryos at different stages of
development.

a. Embryo, 0.23 mm. long, at late proembryo
stage.

b. Embryo, 0.h0 mm. long, at stage 1 (early
differentiating embryo).

c. Embryo, 0.67 mm. long, at stage 3 (middle
differentiating embryo).

d. Embryo, 3.00 mm. long, at stage 6c, (late
differentiating embryo).

 

root (radicle) primordium is being initiated internally.
Stage A is characterized by the formation of the first leaf
primordium.in addition to the structures developed at stage 3.
Characteristics of stage 5 and stage 6 are not as apparent
externally except for an increase in size, especially of the
scutellum. Internally, however, stage 5 shows a middle-sized
differentiating embryo with two leaf primordia, completely
enclosed by the coleoptile and a well differentiated root
primordium. In stage 6 the embryo completes differentiation
(Fig. 6, d). The scutellum.becomes full sized, additional
leaf primordia are formed (usually a total of 3 - h) within
the coleoptile, the radicle is fully formed and seminal root
primordia are differentiated (usually 3-h in this strain of
barley). As this study progressed, it was found necessary

to subdivide stage 6 into three groups of “maturing” embryos:
early (6.); middle (6b) 3 and 1.1;. (6c). These three groups
are morphologically and histologically very similar, but dif-
fer markedly in their rates of increase in size and weight,

as will be shown later.

Relationship between Caryopses and Embryos Based upon Developing
gtages (Fig. 7)

The lengths and lateral diameters of developing embryos
are directly proportional to each other (as shown in Fig. h),
therefore these two entities may be spoken of collectively as
”embryo size.” 0n the other hand, the lengths and widths of

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Fig. 7e

Relationships between embryo sizes, embryo fresh
and dry weights, and caryOpsis sizes in vivo,
and embryo sizes in vitro as compared to stages
of embryonic development.

(1)
(2)
(3)
(h)
(5)
(6)
(7)
(8)

Fresh weights of embryos (in viva)

Dry weights of embryos (in vivo)

Lengths of caryopses (in vivo)

Lateral diameters of caryopses (in vivo)
Lengths of cultured embryos

Lateral diameters of cultured embryos
Lengths of embryos (in vivo)

Lateral diameters of embryos (in vivo)

WOW in mg.

Size in m.

2dr

 

 

 

 

2.1 )-
1'e8 "
1.5 '
1.2 "
0.9)
0.6 »
e
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(O hk’flffsafitrzmgumgfffgfio-u”g ' . V
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o' -
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L “OTIS: 1‘.“.'.'-3:.::- Oo---9""‘.'.

 

“1‘11““ 1 2 5 h 5 6a 6b 6c

Preembryes Difiercntiating emu-yes

26

developing caryopses are not well correlated (Fig. 8) so that
these two entities must be considered separately. From.the-
time in late proembryo development (when measurements could
first be made) until stage 2, only the lengths of the caryopses
showed a rapid increase, with the caryopses attaining 82 per-
cent of their final length by stage 2, while the lateral diame-
ters of the embryos undergo little change in measurements
during this time. After stage 2, the increase in lengths of
the caryopses gradually slow down until the middle of stage 6
is reached. The lateral diameters of the caryopses and the
lengths and lateral diameters of the embryos, on the other
hand, gradually increase in rate of growth. Just prior to
stage 5, the lateral diameters of the caryopses undergo rapid
increases in size while the embryos increase in size at a some-
what lesser rate. By the middle of stage 6, the lengths of
the caryopses become almost constant while the lateral diameters
are still increasing, but at a much diminished rate. The amp
bryos, however, are at this time undergoing their most rapid
growth, in terms of increases in length and lateral diameter.
According to Merry (l9ul) it takes approximately 7
days after pollination for the developing embryo to reach
0.2 - 0.3 mm. in length (late proembryo stage); 8 days to
reach 0.5 mm. in length (first differentiating embryo stage);
10 days to become 0.6 mm. long (stage 3); and 12 days to reach
a length of 1.1 mm. (stage 6a). The results obtained in this

study agree essentially with those of Merry, as can be seen

003 in mm.

‘
J

7
&

~izcs of caryo

C
L...

 

 

r
, e 0. e . .
0 g . ’ . O : O .
. 0 e e .
9.0. g 0 O z .. : O . . O Q . ' O s
0 e O 0 e O O
O . g : O .
. e . 1 ‘ e
. . , .
8‘e0' : . . : .
g C
8 ° . ‘ Lengths of caryopses
O
O : .
'7.on 9 .
O O
O
O
i .
2
6e0"
. O
O
2
s 0
5.10L ;
O
O
heO' :
. 0 e
. ' . :
O
o . E O : : O . . .
O
V . : . g 0 O
E‘OL O ’ e . e . .
’ . : : : lateral diameters of
3 :o' : 0 caryopses
O O
o 3, 3;: : '
. e 0. 0e 0
2e0' . ‘2'. : . 3
no. '
(“Q
Fig.8. Relationships between 02:17er lengths, and caryopsis
1.0L __ lengths and lateral diameters-from plants in vivo
0.1; 0.8 1.2 1.6 ' 2.0 2.3: 2.3- 5.2

Lengths of embryos in in.

28

in Table 3. It should be pointed out, however, that although
the variety of barley used by Merry was Alpha rather than
Hannchen, it was a two-rowed variety and was grown under
greenhouse conditions.

The results of this study are not in agreement, how-
ever, with those reported earlier by Harlan (1926) using the
sumo variety of barley (Hannchen) but grown under field condi-
tions. Harlan found that a caryopsis 8 mm. long contained an
embryo which was 0.12 mm. in length. In the present work, a
caryopsis of this length contains an embryo 0.5 mm. long. In
addition to this, Harlan reported that a differentiating
embryo (stage 1) is found in a caryopsis which is 8.8 mm.
long, while in this study, this stage of embryonic develop-
ment was present in a caryopsis averaging 7.0 mm. in length,
whereas a caryopsis of 8.8 mm. contains an embryo, not in
stage 1, but rather in stage 5-6. In other words, Harlan's
material consisted of caryopses of much larger size, relative
to stages of embryogeny, or embryos which were much smaller

than those found in the present study.

Relationghips between Embryo Sizes and Embryo Fresh and Dry

weights
As presented in Figures 5 and 7, the fresh and dry

weights of embryos show an ever-increasing relationship to
embryo size (length and lateral diameter). At least a part

of the sudden upsurgence of weights beginning with the middle

29

of stage 6 is believed to be a reflection of an increase in
the embryos' dorsi-ventral diameters. Accurate measurements
of the dorsi-ventral diameters were not practicable because

of the errors which would probably be induced by shrinkage
during the time consumed in positioning the embryos on the
edges of their fan-shaped scutella. If embryo volume, how-
ever, could have been determined, there would probably have
been a more perfect correlation between embryo size and weight.
When fresh and dry weights of embryos are compared (Fig. 5)

it may be seen that approximately 2/3 of the embryos' fresh
‘weight is due to water. This water content is relatively con-
'stant throughout all periods of embryogeny investigated in
this study.

Embryos In Vivo as Controls for Those In Vitro
m w

 

Not only was the in vivo portion of this study under-
taken to learn more about normal embryo development 22; £3,
but also to serve as a standard of comparison for embryos
developing under culture conditions, so that it might be de-
termined to what extent embryos developing in vitro approxi-
mate normal embryogeny. In order to achieve this and, embryos
of the in vivo study must include initial stages which are as
early in development and of a size equal to or less than the
smallest ones which can be excised and placed in culture.
Furthermore, if embryos are to be maintained in culture for

a two-week period, then those developing in vivo must be

 

 

30

observed throughout a corresponding period of thme. It there-
fore becomes most important to ascertain the extent of develop-
ment, both in regard to size and stage achieved by embryos i5
gigg during this two-week period.

The smallest embryos which could be excised and placed
in culture,-with the equipment at hand, averaged 0.5 x 0.3 mm.
(ranged from.0.h - 0.6 mm. in length) initially, and were in
a late proembryo stage of development. Of a total of 59
spikes from.which embryos were excised for the in vivo study,
h spikes contained embryos which ranged from 0.50 x 0.25 to
0.60 x 0.30 mm. initially, the average of which was found to
be 0.55 x 0.30 mm. Their‘average final size at the end of
two weeks was 3.15 x 2.38 mm. which, according to Tables 3
and h, corresponds well with the average size of embryos which
are in stage 6c (3.00 x 2.30 mm.). Comparison of the initial
length and the final length results in an increase of M72 per-
cent or a final length which is 5.7 times the initial length,
attained at an average rate of 0.37 mm. per two-day intervals.
Comparison of the initial and final lateral diameters shows
an increase of 693 percent or a final diameter which is 7.9
times the initial, reached at an average rate of 0.30 mm.
per two-day interval. The development of these embryos (from
the four spikes) provides the basis for comparison of embryos

in the in vitro study with those growing in vivo.

’v

31

In Vitro Study

As shown in Table 5, Group 1 consisted of 150 embryos
cultured for two weeks in plastic cups on the medium contain-
ing 1 part of White's basic medium and 9 parts of coconut
milk. These embryos were retained on this medium without
transfer even after the two week period in order to see
whether shoots or roots might be initiated.

Series 1 of Group 1 was comprised of 30 embryos
ranging in size, initially, from 0.3 - 0.h mm. in length,
and 0.15 - 0.20 mm. in width in either a late proembryo stage,
or stage 1, of development. (Of these embryos, 7 showed growth
during the two-week period while the others did not enlarge
during this time but gradually turned brown. or the embryos
showing growth, the average initial size was 0.35 x 0.20 mm.
and the final length and lateral diameter attained during the
two weeks, was 0.7 x 0.50 mm., resulting in a doubling of
size. Two of these embryos were definitely proembryos
(initially) with no suggestion of differentiation when observed
with a dissecting microscope. In culture they each developed
into a ball-shaped mass of cells and maintained a good white
color. One was 0.35 x 0.20 mm. initially, and 0.85 x 0.65
mm. after two weeks, the length increasing by a factor of 2.u
and the lateral diameter, 3.2. The other proembryo was 0.35
x 0.25 mm. initially, and 0.90 x 0.60 mm. after two weeks,

length increasing 2 times and lateral diameter 2.h times. In

e\

32

both cases, therefore, a greater relative increase occurred
laterally than longitudinally.

In series 2 of Group 1, 6h embryos were placed in
culture, ranging in initial size from 0.h5 x 0.20 mm. to
0.60 m 0.35 mm. These embryos were in stage 1 - 2 of em-
bryogeny. Of these, 36 embryos grew, increasing in size for
two weeks and attained an average final size of 1.0 x 0.75 mm.,
after an initial average size of 0.50 x 0.30 mm. Thus, the
final average length increase was 2 times the initial, and the
final average lateral diameter was 2.5 times the initial.

Series 3 of Groupll consisted of 56 embryos in stage
3 - 5, and ranged in size from.0.70 - 0.90 mm. Of these, 26
were apparently growing at the end of two weeks. Their average
initial length was 0.80 mm. and final average was 1.55 mm.,
representing a growth increase of 1.9 times in length, while
in lateral diameter an increase of 2.3 times was obtained
(0.h5 mm., initially, and 1.05 mm., finally).

Throughout the three series of cultures described
above, none of the embryos gave rise to shoots or roots even
though they remained in culture for longer periods of time
than two weeks. The low increase in size and the failure of
shoots and roots to appear (suggesting a lack of internal
differentiation) would certainly indicate that the goal of
inducing normal embryogeny had not been attained. In other

words, satisfactory environment was not being supplied by

II

 

 

33

these culture conditions. Therefore, growth rates of these
embryos at two-day intervals are not listed individually but
are summarized as averages in Table 5. The main purpose in
mentioning these results is to call attention to the fact
that two proembryos showed increase in size in culture.

As shown in Table 6 and summarized in Table 7, Group
2 consisted of 88 embryos cultured in plastic cups on 1 part
of White's basic medium and 9 parts of coconut milk for a
period of two weeks, then transferred to vials containing the
basic medium only, where they remained for an indefinite period
of time or until roots or shoots appeared.

Series 1 of Group 2 consisted of 36 embryos, ranging
in size from.0.30 - 0.h0 mm. in length and 0.15 - 0.20 mm.
in lateral diameter. Most of these embryos were in stage 1
or were late proembryos. After two weeks, 1 embryo of this
series was growing well, and after being transferred to the
basic medium without coconut milk at the end of this time,
continued to develop and eventually (one month from.initia1
placement in culture) produced a normal appearing root (Fig.
9, a, b). This embryo (No. 8 - 6 in Table 6) initially did
not show any noticeable sign of the indentation which charac-
terizes, morphologically, the beginning of differentiation.
Therefore, this embryo was either a late proembryo or a very
early stage 1 and measured O.h0 x 0.25 mm. At the end of the

two-week period, this embryo reached a size of 0.85 x 0.65 mm.

Fig. 9.

Representative embryos which produced shoots
and roots, after being transferred into screw-
cap vials.

ae

b.

Ce

d.

6.

Embryo after 29 days in culture. Initial
size was 0.h0 x 0.20 mm. and final size Just
before root was formed was 1.80 x 1.35 mm.

Embryo No. 8-6 which was the same size as
(a) above initially and produced a normal
appearing root after 30 days in culture.

Embryo which was 0.5 mm. in initial length
and formed normal shoot root after 28 days
in culture.

Embryo with normal shoot and root which were
produced after 18 days in culture. The
original length of this embryo was 0.70 mm.

Excellent normal shoot and root which arose
from an embryo 0.55 mm. in initial length
after 21 days in culture.

 

35'

and attained a size of 1.80 x 1.35 mm. Just prior to the ap-
pearance of the root, giving an increase of 2.1 times in
length and 2.6 times in lateral diameter by the end of two
weeks, and an increase of h.5 times in length and 5.u times

in lateral diameter Just prior to root formation. Because

this embryo was the smallest one to undergo differentiation
under culture conditions, it was selected as the representative
embryo for evaluation of morphological stages of embryos in
culture and for comparison with embryos developing in vivo

(as will be discussed later).

Series 2 of Group 2 was comprised of 38 embryos which
initially measured 0.h5 - 0.60 mm. in length and 0.20 x 0.30
mm. in width, and were in stage 1 - 2 of development. At the
end of two weeks, 18 of these embryos were growing and nine
of them.gave rise to roots or shoots (Table 7). The average
initial size of embryos in this series was 0.50 x 0.30 mm. and
1.20 x 0.90 mm. at the end of two weeks; therefore, final
length showed an increase of 2.h.times the initial length and
lateral diameter increased 3 thmes. If the initial average
size of the cultured embryos is compared with the average
size of these embryos one day before the appearance of shoots
orroots (the average size attained being 1.80 x 1.h0) this
gives an increase of 3.6 times in length and h.6 times in
lateral diameter. The average two-day increment in size was

a length increase of 0.10 mm. and a lateral diameter increase

36

of 0.09 mm. The relationships between length and lateral
diameter (width) of these embryos, graphed in Figure 10,
show essentially a straight line relationship, the length be-
ing proportional to width during the two-week culture period.
Since a number of these embryos were initially rather small,
yet grew well and differentiated in culture, it is assumed
that the culture conditions used with the Group 2 embryos
were more satisfactory than those used with Group 1. There-
fore, the results obtained in series 2 of Group 2 were con-
sidered to be good enough in approaching the original aim of
the study to warrant comparison with those of the in vivo
study. I

Series 3 of Group 2 consisted of IA embryos, ranging
in size from 0.70 - 0.90 mm. in length and 0.h0 - 0.50 mm.
in lateral diameter. Since these embryo sizes were initially
too large to consider for the purposes of this work, the re-

sults obtained in this series were neglected.

2.0

1.6

1.2

0.8

0.4

 

 

a

n

ht .

mu

w 0 0

mm.

‘

mm e e e

mt e eee‘ 0

mm .x..

Pd .0. ”.~ .0.

Jul ’00s"0 “0 0‘0

a m .

E z...

.1 a e 0 dfl Is

t1 0

Mad C. “0.00“.“

e n e e o

R n.“ 000000 0

000. 0 0

e 000“?‘0000

m. flueeeeee“

0 . . O

m a a“...

00.0

P P bl P b F («lipl
8 .4 0‘ 6 2 8 h.
e .e e e e .e e
2 2 2 1 1. . O 0

Lateral diameters of embryos in m.
J

38

Development of Embryos In Vitro with Those In Vivo

Morphological Comparison

In order to compare the embryos developing in culture
with those developing in vivo it is necessary not only to comp
pare the size increases of the embryos in each case, but also
to determine the morphological stages and the phases of differ-
entiation through which the cultured embryos have passed.
while the morphological features of the various stages of em-
bryogeny are rather specific in the case of embryos developing
in vivo, the same cannot be said for those in culture. Ems
bryos deve10ping in vitro are, in general, not as consistent
in their growth patterns as those which develop in vivo, and
further, depending upon critical environmental conditions
such as nutritional and/or atmospheric factors, or perhaps as
a result of the mere mechanics of culture techniques, the growth
patterns of cultured embryos may vary slightly from one set of
cultures to another. This is particularly true of those emp
bryos which are not placed on the agar in such a way that the
developing scutellum is in contact with the medium. Thus,
orientation of the embryos on the agar medium.may be said to
have implications in terms of developmental morphology.

The following description of morphology of embryos
in culture is based upon observations of in vitro embryos
in general and embryo No. 8-6 (Table 6), in particular, which

as mentioned before, was the smallest embryo to undergo

.5

39

differentiation in culture. One of the most characteristic
features of embryos which develop in culture is that of
greater, or precocious, increase in dorsi-ventral diameter.
While in vivo embryos at stage 1 or state 2 all have a greater
length than lateral diameter and little dorsi-ventral diameter,
such embryos after being placed in culture show rapid increase
in dorsi-ventral diameter even while still in these stages.
This increase may be caused by the fact that excised embryos
are removed from whatever mechanical restrictions might be
otherwise imposed by the caryopsis coat and endosperm tissue.
The appearance of the slight indentation, which is character-
istic of the first stage (stage 1) of differentiation of

the in vivo embryos, was never observed in any of the embryos
which were placed in culture prior to the differentiation of
stage 1. Stage 2 in vitro is characterized by the initiation
of the coleoptile as an incomplete collar of tissue, as in
stage 2, in vivo. Stages 3, u and S of embryos in culture
eXhibited exceptionally poor differentiation ofthe scutellum
which resulted in the development of a ball-shaped embryo
during differentiation stages rather than the typical fan—
shaped embryo which develops in vivo. In addition, all cul-
tured embryos showed an anomalous formation of the coleop-
tile, caused by a failure of the lower "lip" (that portion

of the coleoptile circle most distant from the scutellum) to

elongate at the same rate as the rest of the structure, thereby

no

resulting in a greatly enlarged coleoptile "pore" through
which the shoot emerged prematurely. Norstog (1955) also
reported the formation of abnormal coleoptiles in cultured
barley embryos. Finally, stage 6 of differentiation, as

seen in the in vivo embryos, was never found in any of the
embryos placed in culture at any stage prior to that stage:
instead, embryos in stage 5 in vitro sooner or later directly

gave rise to shoots or roots.

(Rate of Growth,_Size and Stage Comparisons

As has already been mentioned, Van Overbeek, 33 El
(l9hl) were the first ones successful in producing seedlings
from proembryos in culture (in this case witthatura, a dicot).
Norstog (1955), has been the most successful thus far in
culturing monocot embryos of very mmall size. He induced
small embryos (one as small as 0.16 mm. in length) tosform
leaves and roots in culture. However, because the smallest
embryo also had a number of anomalies, it was suggested by
him.that this development might actually represent regenera-
tion from callus tissue. It is significant that the small
embryos cultured by Norstog are within the size range of the
proembryos of the present study. While Norstog mentioned
that his smallest embryos did not show outward signs of dif-
ferentiation, the fact that he did not compare his cultured
material to'any in vivo stages, and only recorded growth

increases of cultured embryos at the end of a one-week period,

kl

.makes it almost impossible to evaluate his results in the
light of the present study. Further, Norstog did not use the
same barley variety as was used in this investigation; neither
did he describe his growing conditions, nor did he make size-
stage relationships so that stages of cultured embryos could
be accurately determined. If they are similar to those of
Merry (l9ul, l9h2) or to the present study, then Norstog's
youngest embryos were definitely proembryos; on the other
hand, if the embryo sizes correspond more closely to those of
Harlan (1926), then it is more probable that Norstog's
youngest embryos were late proembryos or very early stage 1,
and, therefore, would be comparable to the smallest embryos
successfully cultured in the present work.

Kent and Brink (19h?) and Ziebur and Brink (1951),
using a six-rowed variety of barley, reported limited success
in the culture of a few ”proembryos" showing no outward signs
of differentiation and measuring 0.3 mm. in length initially.
No details of morphological development or growth rates were
given, and no comparisons were made with in vivo embryos of
the same age. ' I

The work mmst similar in nature to the present study
was that of Merry (l9h2) who compared growth rates of embryos
in culture with those of the same aged embryos in vivo. The
only embryos, however, with which Merry had any success in

culture were, initially in the mid-stages of differentiation

 

 

(\(Jallli ...-ill

I!) (‘3‘! ll.llll‘..

M2

at the time of placement in culture; therefore his results
have only a very limited bearing on the present work.

If the sizes and growth rates of the 38 embryos in
zitgg of Series 2, Group 2 (Table 7) are compared with the
28 embryos in vivo of the four spikes (Table A) which were
chosen as the controls for the cultured embryos, the fol-
lowing results are obtained. The in vitro embryos averaged
0.50 x 0.30 mm., initially, and attained an average size of
1.2 x 0.9 mm. at the end of two weeks, giving an average
length increase of 2.8 times and a lateral diameter increase
of 3 times at an average increment per two day interval of
0.10 and 0.09 mm., respectively. The in vivo (control)
embryos in the same stage of development averaged 0.55 x
0.30 mm., initially, and attained an average size of 3.15
x 2.38 mm. by the end of two weeks, giving an average increase
in length of 5.7 times and lateral diameter increase of 7.9
times at an average increment per two-day interval of 0.37
and 0.30 mm., respectively. When the ratios of average final
length to average final width are compared, they are seen to
be identical (1.3 : 1.0) in both in vitro and in vivo embryos.
The in vitro embryos just prior to the appearance of shoots
and roots attained an average size of 1.8 x 1.h mm. and, when
compared to the size of in vivo embryos at the end of two
weeks (by which time full differentiation has occurred), it
is seen that again the same ratio of length to width (1.3 : 1.0)

\

Jo.

I.

is obtained. Therefore, it may be said that in vitro em-
bryos maintain the same length to width relationships (for

a two-week period and through as much differentiation as
occurs in culture) as takes place in vivo (during the same
two-week period and throughout differentiation), with merely
an overall lack of size increase in the case of the cultured
embryos. These relationships are also shown graphically in
Figures h and 10.

Stages of development of cultured embryos are deter-
mined by comparing embryo sizes of the 38 in vitro embryos
(Series 2 of Group 2) to the morphological development of
embryo No. 8-6 as shown in Table 6. Justification for this
comparison may be found in the fact that the average final
size Just before the emergence of shoots or roots of those
embryos of the 38 which produced shoots and/or roots was
1.8 x l.k mm., while in the case of embryo No. 8-6, it was
1.8 x 1.35 mm. On the basis, then, of sizes and of Table 8,
the embryos of Series 2, Group 2, are assumed to have reached
stage 3 by the end of two weeks. About 30 days from initial
placement in culture, they reached stage 5 and gave rise
directly to shoots and/or roots. In vivo embryos, on the
other hand, although starting out at the same size and stage
of development as the cultured embryos, completed differenti-
ation to the end of stage 6 during the same period of time

(two weeks) in which in vitro embryos were only reaching stage 3.

.0

7‘

Embryos, therefore, differentiate more slowly in culture than
in vivo, taking a longer period of time to pass through each
stage and never attain an actual stage 6, morphologically.

Keeping in mind that cultured embryos during the two-
week period attain a size which is comparable to stage 6a
in vivo, yet a morphological stage of only stage 3, it can
be said that cultured embryos at any given stage must be
larger than the in vivo embryos of a comparable stage. Fur-
ther support for this conclusion can also be found in the
fact that cultured embryos Just prior to the appearance of
shoots and roots attain a size which is comparable to stage
6b in vivo, while only reaching stage 5, morphologically.
From.this, it then follows that the culture techniques used
in the present study must actually induce more embryonic
growth per stage than occurs in vivo. In fact, the length
of stage 3 in vitro is approximately l.k times that of the
same stage in vivo, and stage 5 in vitro is 1.8 times that of
stage 5 in vivo.

In an effort to discover whether this increased size
is due to an increase in cell size or in cell number, histo-
logical comparisons were made of embryos of the same size.ig
zitgg and in vivo. When the number of cells per microscope
field was determined for in vitro and in vivo embryos, the

cell sizes of the in vitro embryos was found, in general, to

 

Us

.\

 

hS

be 1.5 times those of the in vivo embryos. Cell size differ-
ences could, therefore, account for much of the difference
in total embryo size, yet there must also be an increase to
some extent in the number of cells of cultured embryos. It
may be concluded that the nutritive and atmospheric conditions
used in the present culture techniques promote both cell en-
largement and cell division in cultured embryos over and
above that normally occurring in vivo. While it is difficult
to pinpoint the specific factors in the culture technique
which might be responsible for these differences, the fact
that embryos cultured on the basic medium plus coconut milk,
failed to undergo differentiation, would suggest that the
addition of coconut milk to the basic medium.was at least one
of the critical factors. However, considerably more work is
needed along these lines before any more definite conclusions
can be reached as to the specific role of an I'embryo factor”
such as coconut milk. _ .
In conclusion, embryos developing in culture maintain
length/width relationships which are identical with, and at-
tain sizes which approach those of in vivo embryos. In vitro
embryos differ, however, from those developing in vivo in
that cultured embryos are larger at any given morphological
stage, are slower in the rate at which they pass through the

various stages, show a number of morphological deviations from
normal embryogeny, and never attain the morphological develop-

ment of stage 6.

1 .l Ill‘lllllllllillsl ill
Isl ’1 (v‘tilli )hll: I’.Illlu'. .Iql'l.‘.l.l.lv .

is

SUMMARY

1. The growth and development of barley embryos
in vitro were studied by comparing them to embryos in vivo
in the following ways: (a) by comparing their morphological
development, (b) by comparing their relative sizes and rates
of growth, and (c) by relating their growth to stages of
embryonic development.

2. The lengths and lateral diameters of developing
embryos in vivo and in vitro are directly proportional to
each other.

3. The lengths and lateral diameters of developing
caryopses in vivo are not well correlated.'

h. From.the time in late proembryo development until
stage 2, only the lengths of the caryopses show a rapid in-
crease, while the lateral diameters of the caryopses and the
sizes of embryos in vivo undergo little change in measure-
ments during this time. After stage 2, the increase in lengths
of the caryopses gradually slow down until the middle of stage
6 is reached. Just prior to stage 5, the lateral diameters
of the caryopses undergo rapid increases in size while the
embryos increase in size at a somewhat lesser rate. By the
middle of stage 6, the lengths of the caryopses become almost

constant, while the lateral diameters are still increasing,

h?

but at a much diminished rate. The sizes of embryos within
the caryopses, however, are at this time undergoing their
most rapid growth.

5. Fresh and dry weights of embryos in vivo show an
ever increasing relationship to embryo size. A sudden up-
surgence of weights begins with the middle of stage 6. Ap-
proximately 2/3 of the embryos' fresh weight is due to water,
this water content being relatively constant throughout all
periods of embryogeny.

6. The in vivo embryos, which averaged 0.55 x 0.30 mm.,
initially, attained an average size of 3.15 x 2.38 mm. by the
end of two weeks, giving an average increase in length of 5.7
times and lateral diameter increase of 7.9 times.‘

7. Cultured embryos, which initially measured O.k5 -
0.60 mm. in length and 0.20 - 0.30 mm. in width.were still
growing at the end of two weeks and some of them gave rise to
roots er shoots. The average final size of these embryos was
1.20 x 0.90 mm. at the end of two weeks; therefore, final
length showed an increase of 2.h times the initial length and
the lateral diameter increased 3 times. If the initial average
size of these cultured embryos is compared with their average
size one day before the appearance of shoots or roots, this
gives an increase of 3.6 times in length and h.6 times in

lateral diameter.

h8

8. When the ratios of average final length to average
final width are compared, they are seen to be identical (1.3 :
1.0) in both in vitro and in vivo embryos at the end of two
weeks. In addition to this, if the sizes of the in vitro
embryos Just prior to the appearance of shoots and roots are
compared to those of in vivo embryos at the end of two weeks,
it is seen that again the same ratio of length to width (1.3 :
1.0) is obtained. Therefore, it may be said that in vitro
embryos maintain the same length to width relationships as take
place in vivo, with merely an overall lack of size increase in
the case of the cultured embryos.

9. While the morphological features of the various
stages of embryogeny are rather specific in the case of em-
bryos developing in vivo, embryos growing in vitro, ara,in
general, not as consistent in their growth patterns.

10. Embryos developing in culture maintain length/
width relationships which are identical with, and attain sizes
which approach those of in vivo embryos. In vitro embryos
differ, however, from those developing in vivo in that cul-
tured embryos are larger at any given morphological stage,
are slower in the rate at whiCh they pass through the various
stages, show a number of morphological deviations from.normal
embryogeny, and never attain the morphological development of

stage 6.

 

a.

.1...) .‘II. .I

4|!" III;\(A.

 

.h

1+9

BIBLIOGRAPHY

Brink, R. A., D. 0. COOper and L. E. Ausherman. 19hh. A hy-
brid between Hordeum.jubatum and Secale cereale reared
from an artificially cultured embryo. Jour. Breed.

35: 67’7Se

Cross, G. L. 1937. An improved method of staining with
fast green. Proc. Oklahoma Acad. Sci. 17: 69-70.

Curtis, J. T. l9h7. Undifferentiated growth of orchid emp
brgos on media containing barbiturates. Science 105:
12 .

Haagen-Smit, A. J., R. Sui, and G. Wilson. 19h5. A method
for the culturing of excised, immature corn embryos
in vitro. Science 101: 23k.

Harlan, H. V. 1920. Daily development of kernels of Hannchen
barley from flowering to maturity at Aberdeen, Idaho.
Jour. Agri. Res. 19: 393-k29.

Johansen, D. A. l9k0. Plant Microtechnique. McGraw-Hill
Book Company, Inc., New York.

Kent, N., and R. A. Brink. l9h7. Growth in vitro of immature
Hordeum embryos. Science 106: 5h7- .

Konzak, C. F., L. F. Randolph, and N. F. Jensen. 1951. Embryo
culture of barley species hybrids. Cytological studies
of Hordeum sativum.x Hordeum bulbosum. Jour. Heredity
(4.2: 12;-13uo .

IaRue, C. D. 1936. The growth of plant embryos in culture.
Bull. Torrey Bot. Club 63: 365-382.

, and G. S. Avery. 1938. The development of the
embryo of Zizania aquatics in the seed and in artificial
culture. Bull. Torrey Bot. Club 65: 11-21.

Lee, A. E. 1952. The growth of excised immature sedge embryos
in culture. Bull. Torrey Bot. Club 79: 59-62.

So

Loo, S. W., and F. H. Wang. l9h3. The culture of young
conifer embryos in vitro. Science 98: Shh.

Lofland, H. B. 1950. In vitro culture of the cotton embryo.
Bot. Gaz. 111: 307-311.

Mericle, L. W. and R. P. Mericle. 1957. Irradiation of
developing plant embryos. I. Effects of external ir-
radiation (x-rays) on barley embryogeny, germination,
and subse uent seedling development. Amer. Jour. Bot.
(In press?

' Merry, J. l9hl. Studies on the embryo of Hordeum sativgg.
I. The development of the embryo. Bull. Torrey Bot.
Club 68: 585-598.

. 19h2. Studies on the embryo of Hordeum sativum.
II. The growth of the embryo in culture. Bull. Torrey
Bot. Club 69: 360-372.

Norstog, K. J. 1955. The growth of barley embryos on coconut
milk media. Bull. Torrey Bot. Club 83: 27-29.

Pieczur, C. 1952. Effect of tissue cultures of maize endo-
sperm.on the growth of excised maize embryos. Nature

170 3 21.1.1-2}.lz e

Radforth, W. N. 1937. The development in vitro of proemp
bryos of Ginkgo. Royal Can. Inst. Trans. 21: 87-9h.

Sterling, C. l9h9. Preliminary attempts in larch embryo
culture. Bot. Gaz. lll: 90-9h.

Tukey, H. B. 1933. Artificial culture of sweet cherry
embryos. Jour. Heredity 2h: 1-12.

Van Overbeek, J., M. E. Conklin, and A. F. Blakeslee. 19k1.
Factors in coconut milk essential for growth and de-
velopment of very young Datura embryos. Science 9h:

350-351.

. l9k2. Cultivation in vitro of small Datura em?
bryos. Amer. Jour. Bot. 29: H72-h77.

White, P. R. l95h. The cultivation of animal and plant cells.
The Ronald Press, New York.

 

O

Q Q
0 I 0
e e
o
_
0.. o
C
_
.
e
o
o
e

Ox

‘\

 

51

Ziebur, N. K., and R. A. Brink. 1951. The stimulative ef-
fect of Hordeum endosperm on the growth of immature
plant embryos in vitro. Amer. Jour. Bot. 38: 253-

256.

, L. H. Graf, and M. A. Stahmann. 1950. The effect
of casein hydrolysate on the growth in vitro of im-
mature Hordeum embryos. Amer. Jour. Bot. 37: lab-1&8.

 

APPENDIX

52

53

Table 1. Individual length, width, fresh weight, and dry weight measurements
made at two-day intervals of immature barley embryos developing in vivo.
(Four basal and four terminal grains of each head were not included.

 

Length Width Fresh Wt. Dry Wt. Length Width Fresh Wt. Dry Wt.

 

in mmI in mm. in ms. in mg. in mm. in mg. in_mg. in E21.

Head No. 1 Head No. 8
0.90 0.65 0.110 0.025 0.90 0.75 0.055 0.020
1.75 1.20 0.270 0.055 1.50 1.10 0.310 0.045
2.70 1.90 1.515 0.340 2.95 2.15 1.675 0.430
3.20 2.30 1.980 0.760
3.35 2.40 2.275 0.880 Head No. 9

0.35 0.15 0.028 ----

Head No. 2 0.90 0.70 0.050 0.015
0.55 0.30 0.015 --- 1.70 1.20 0.350 0.075
1.15 0.65 0.120 0.040 2.40 1.55 0.610 0.319
1.70 1.15 0.230 0.070 3.00 2.05 1.560 0.470
2.60 1.85 1.370 0.250 3.30 2.25 2.305 0.625
3.00 2.15 1.890 a 0.390 3.65 2.45 3.195 1.080
3.05 2.40 2.260 0.400

Head No. 10

Head No. 3 0.35 0.20 0.030 --—-
0e85 0045 0.040 Co 015 0.90 0.60 -..- ...-
l.20 0.90 0.120 0.045 1.70 1.15 0.380 0.080
2.10 1.60 0.550 0.155 2.35 1.55 0.730 0.190
3.10 2.10 1.805 0.460 3.05 2,00 1.375 0.475

3.35 2.15 2.320 0.715

Head No. 4 3.40 2.35 2.990 0.910
0.20 0.15 ---- ----

Head No.

Head NC. 5 0.35 0.15 0.009 ---
0.95 0.55 0.045 0.015 0.80 0.50 0.055 0.015
1.00 0.90 0.135 0.025 1.55 1.05 0.305 0.050

2.25 1.50 0.480 0.120

Head No. 6 2.65 2.00 1.305 0.270
0.25 0.15 00005 ...-'- 3030 2030 2. 260 0.700
0.75 0.40 0.050 0.020 3.40 2.55 3.030 0.870
1.05 0.60 0.080 0.020
1.85 1.40 0.550 0.160 Head No.

2045 10 55 00700 0.255 0095 0. 65 M -"""""‘
1.90 1.25 0.350 0.090

Head NO. 7 2.25 1065 Co 595 001100
0020 0.15 0.009 -."— 3020 2.05 1.760 O. 610
0.95 0.40 0.049 0.025 3.30 2.05 2.190 0.800
1.10 0.75 0.150 0.040
1.65 1.20 0.445 0.095 Head No.

3.05 2.00 1.735 0.665 1.20 0.85 0.120 0.035
3.20 2.10 2.395 0.765 1.90 1.40 0.550 0.090
2.10 1.60 0.830 0.140

Sh

 

 

Length 'Width Fresh Wt. Dry Wt. .Length 'Width Fresh.Wt. Dry Wt.
in mm, in mm, in mg. in gg. in 2g. in mg. in gg. in mgL_
Head No. 14 Head No. 22
0.55 0.30 --- --- 0.20 0.15 0.005 ---
1.15 0.65 0.135 0.025 0.60 0.30 0.025 0.005
2.35 1.55 0.735 0.265 1.60 1.15 0.340 ——--
2.25 1.55 0.760 0.195
Head No. 15 2.55 1.90 1.190 0.285
0.25 0.15 0.005 ---
0.40 0.25 --- --- Head No. 23
-——- --- ---- --- 0.20 0.15 0.005 --—-
1.70 1.30 0.360 0.060 0.65 0.35 0.020 0.010
2.85 2.00 1.280 0.450 1.35 0.85 0.185 0.035
1.85 1.40 0.490 0.122
Head No. 16 -... .... -.... ...__
0.50 0.25 --- --- 3.25 2.15 2.170 0.870
1080 1025 00370 00145 3055 2035 30070 .....-
2.45 1.80 0.800 0.290
Head No. 24
Head No. 17 0.85 0.45 0.050 0.020
0.20 0.15 0.002 --- -- --— --- ---
0.70 0.35 0.040 0.015 2.15 1.60 0.580 0.125
g 2.70 1.85 1.320 0.380
Head No. 18 ‘ 3.05 2.15 2.010 0.495
0.85 0.55 0.110 0.015 3.35 2.20 3.110 0.895
3.40 2.25 3.160 0.900
Head No. 19
0.45 0.25 0.010 --- Head No. 25
--— --- --- --- 1.05 0.70 0.075 0.025
2.25 1.75 0.700 0.255 1.60 1.45 0.500 0.090
3.50 2.45 2.760 0.880 Head No. 26
2.35 1.65 0.910 0.170
Head Noe 20 2075 20 05 10 430 00295
0.45 0.25 0.010 ..-..
.... --—. ...-- -—- Head No. 27
2.25 1.75 0.700 0.255 0.65 0.30 0.050 ---
O..- ”. “.-- -..-'- 0075 00 45 00075 00 015
4.25 2.50 3.710 --- 2.55 1.75 0.810 0.215
2.90 1.85 1.545 0.375
Head No. 21 3.05 2.05 2.055 0.605
1.85 1.30 0.335 0.060 A
2.50 1.70 0.795 0.210 Head No. 28
3.30 2.10 1.990 0.770 0.55 0.35 0.035 ---
3.45 2.35 2.950 0.840 0.95 0.55 0.055 0.010
3.70 2.60 3.695 0.940 1.95 1.35 0.420 0.135
2.45 1.55 0.910 0.195
3.05 2.00 1.575 0.350
3.20 2.10 1.995 0.550
3.30 2.35 2.840 0.750

55

 

 

 

 

.Length Width Fresh.Nt. Dry Wt. ILength Width Fresh Wt. Dry Wt.
in mm in mm. in mg._ in gg. in mm. in mm. in mg, in mgI
Head No. 29 Head No. 35
0.60 0.30 0.060 ---— 0.75 0.40 0.025 ---
1.00 0.65 --—-- --—- --- ---- --- ----
1.65 1.25 0.375 0.050 2.55 1.80 0.930 0.225
2.05 1.55 0.590 0.110 2.70 1.95 1.395 0.465
2.80 1.95 1.605 0.445 3.40 2.05 2.355 0.735
3.30 2.35 2.740 0.725
3.35 2.40 3.180 0.850 Head No. 36
0.35 0.20 0.020 .....
Head No. 30 1.00 0.55 0.085 0.025
0.35 0.20 0.040 --- 1.45 1.00* 0.180 0.050
0.75 0.40 0.055 0.015 2.25 1.65 0.800 0.210
1.35 0.85 0.200 0.040 2.65 1.90 1.295 0.320
2.30 1.55 0.660 0.155 2.75 2.05 1.565 0.485
2.95 1.95 1.340 0.350
3.20 2.20 2.055 0.445 Head No. 37
0027 00 15 00 005 fl
Head No. 31 0.60 0.30 0.020 0.005
0035 00 20 “-.. ...- 1025 0085 O0 105 00035'
0.95 0.55 0.070 0.030 1.90 1.35 0.400 0.085
1.50 1.10 0.280 0.090 2.55 1.80 0.985 0.230
2.25 1.60 0.810 0.155 3.05 2.05 1.710 0.365
3.05 2.00 1.650 0.380
30 20 20 20 20 585 O0 730 Head N00 38
3.45 2.50 2.850 0.950 0.50 . 0.25 0.020 ----
3.70 2.50 3.905 0.970 0.90 0.60 0.050 0.015
1.70 1.20 0.315 0.085
Head No. 32 2.25 1.75 0.865 0.205
00 12 00 06 ...“- “—- 2070 1090 10355 00320
O. 50 00 25 “..- "'"""'- 2075 20 05 10755 00415
0.85 0.55 0.045 0.020 2.85 2.20 2.195 0.615
1.85 1.30 0.400 0.085
2.25 1.75 0.875 0.175 Head No. 39 4
3.00 2.05 1.505 0.320 0.35 0.20 0.045 —--
1.05 0.70 0.085 0.045
Head No. 33 1.35 1.10 0.195 0.065
0020 0011 -..— ...“ 2025 10 65 00 690 00155
0.65 0.30 0.040 0.010 2.50 1.85 1.080 0.260
1.20 0.85 0.155 0.030 3.10 2.05 2.000 0.530
1.60 1.10 0.300 0.055
2.05 1.40 0.895 0.140 Head No. 40
0.50 0.25 0.040 0.010
Head No. 34 0.90 0.70 0.065 0.020
00 35 O0 20 ...-'- ...—- 1080 1025 0.400 00 W5
0.55 0.30 0.065 0.010 2.30 1.75 0.720 0.230
1.20 0.85 0.125 0.025 2.80 2.05 1.615 0.400
2.20 1.70 0.705 0.170
3.10 2.10 1.625 0.355

‘56

 

 

 

Length ‘Width Freah.Wt. Dry Wt. Length Width Fresh.lt. Dry Wt.
1g mm. in gg._ in_gg. 1n gg. 1n_mm. in mm. ig_gg, in mg.
Head N00 [.1 Head N00 43
1.95 1.25 0.225 0.080 0.25 0.15 ---— ----
3.15 1.85 1.135 0.480 0.65 0.35 0.010 0.015
3.30 2.15 1.920 0.910 1.30 0.85 0.070 0.050
1.90 1.35 0.395 0.085
Head No. 42 2.20 1.65 0.635 0.145
1.15 0.75 0.165 0.030 2.80 2.05 1.455 0.345
2.15 1.40 0.505 0.110
2.65 1.75 1.150 0.320 Head No.
3.00 2.00 1.900 0.480 1.00 0.65 0.050 --—-
3.10 2.20 2.395 0.595 1.85 1.45 0.475 0.120
3.20 2.35 2.565 0.660

57

Table 2. Individual length, width, fresh weight, and dry weight measure-
ments made at two-day intervals of barley embryos and caryOpses develop—

ing in vivo. (Four basal and four terminal grains of each head were not
included.5

 

 

Embryos Caryonses
Length Width Fresh Wt. Dry'Wt. Length Width
in gm. in gg. in mg. in 2g. in gm. in_ggL
Head No. 1
0.16 0.08 -—--- --—- 5.92 1.76
{0.1. 0.08 ...-- ..--- 5.60
0.16 0.08 ---- —---— 5.60 1.76
0.56 0.32 0.025 --- 8.60 2.08
{0.48 0.32 0.020 —-—-- 8.00 2.08
0.56 0.32 0.020 --- 8.16 2.24
1.12 0.80 0.140 0.025 8.96 2.56
{1.04 0.80 0.125 0.020 8.96 2.56
1.12 0.80 0.150 0.030 8.96 2.72
2.00 1.28 0.620 0.110 9.76 4.16
Head N00 2
“-.. ‘"'-" ----- ---- 40 64 10 68
---- ---- ----- ----- 4.64 1.68
---- --- --- ---- 4.64 1.60
0.35 0.18 0.010 --- 7.36 2.08
‘0.35 0.16 0.010 ---- 7.36 2.08
0.40 0.18 0.015 --- 7.20 2.08
0.32 0.16 0.010 --- 7.36 2.08
1.12 0.72 0.110 0.025 8.48 2.72
‘1.12 0.72 0.100 0.030 8.48 2.72
0096 0064 0.095 00 020 8048 20 56
1.60 1.28 0.400 0.075 9.60 3.20
1.92 1.28 0.625 0.090 9.60 3.36
2.88 1.92 1.635 0.335 9.60 3.84
‘3.04 1.92 1.910 0.370 9.60 4.00
2.88 1.92 1.350 0.310 9.60 3.84
Head No. 3
0.27 0.18 --- --—- 6.40 1.92
{0.27 0.18 --- --- 6.40 1.92
0027 00 18 W“ -"""" 6. 56 1.92
006‘ 0032 00035 ...-~- 8016 2008
{0072 0.40 00 035 """"" 8096 2024
0.64 0.32 0.040 --- 8.64 2.24
1.44 1.12 0.220 0.035 9.12 3.00
‘1.44 0.96 0.210 0.030 9.28 3.00
1.44 0.96 0.250 0.035 9.28 3.20
2.40 1.60 1.015 0.165 9.60 3.50
3.04 2.08 1.875 0.495 9.60 3.84

58

 

 

 

 

Embryos Caryopsoa
.Length Width ‘Fresh.Wt. Dry Wt. LLength. ‘Width
in gg. in ya. in;gg. in gg. in_gg. in mm.
Head No. 4
(0.56 0.32 0.030 -—-- 8.00 2.24
0.65 0.32 0.035 ---- 8.48 2.24
*0.48 0.24 0.015 ---- 7.84 2.08
0.64 0.32 0.030 ---- 7.84 2.08
0.48 0.32 0.015 --- 8.00 2.08
{1.28 0.80 0.185 0.035 8.64 2.56
1.28 0.80 0.175 0.030 8.64 2.56
x1.12 0.80 0.145 0.025 8.64 2.56
2.08 1.28 0.625 0.090 9.60 3.52
2.72 1.76 1.285 0.245 9.76 3.68
Head N00 5
0.80 0.48 0.095 ---- 8.80 2.24
0080 00100 00075 ...-0.- 8064 2024
1.44 0.96 0.210 0.030 9.12 2.88
1.44 0.96 0.240 0.035 8.96 2.72
2.24 1.28 0.610 0.110 9.44 3.00
2.23 1.29 0.620 0.125 9.44 3.00
2.72 1.76 1.385 0.270 9.44 3.36
2.89 2.08 2.170 0.500 9.44 3.84
3.20 2.40 3.260 0.970 9.44 4.00
Head No. 6
0.32 0.16 0.010 --- 6.88 1.92
‘0.32 0.16 0.015 —-- 6.88 1.92
0032 0016 00015 "'"""'- 700‘ 1092
0.88 0.64 0.095 0.020 8.96 2.43
‘0.96 0.64 0.100 0.020 8.96 2.40
0.96 0.64 0.100 0.030 8.96 2.45
1.76 1.12 0.445 0.070 9.44 3.00
Head No. 7 '
1.28 0.88 0.155 0.025 8.64 2.72
1.12 0.80 0.145 0.030 8.80 2.72
1.92 1.28 0.450 0.090 8.64 3.00
2.08 1.44 0.580 0.100 8.80 3.20
2.56 1.12 1.275 0.305 8.80 3.36
2.88 1.12 1.800 0.350 8.80 3.52
3.20 2.08 2.798 0.828 8.96 3.84
3.20 2.24 3.350 1.075 9.12 4.00
Head No. 8
0006 0003 """""" -"-"'" 3068 1060
[0.08 0.05 ---- ----- 4.48 1.12
0.08 0.05 ---- ---- 4.48 1.12
0.48 0.24 0.020 0.010 7.68 2.08
{0.48 0.24 0.030 0.010 7.68 2.24
0.64 0.32 0.030 0.015 8.16 2.21
1.20 0.80 0.155 0.030 8.96 2.57

 

 

 

Embryos Caryonsea
Length Width Fresh Wt. Dry Wt. ALength Width
in an. in mg. 'in mg, in m . in gg. inggg
Head No. 8 (continued)
0.96 0.66 0.100 0.025 8.64 2.40
2.23 - 1.44 0.630 0.100 9.28 3.20
Head NC. 9
0064 0032 00035 ..“" 8032 2008
0.64 0.32 0.030 ---- 8.32 2.08
1.04 0.72 0.115 -—--- 8.64 2.56
‘1.04 0.64 0.115 ----- 8.64 2.40
1.44 0.96 0.245 0.050 9.12 2.56
1.60 1.12 0.290 0.075 9.28 2.88
2.56 1.60 1.100 0.255 9.44 3.52
2.88 1.92 1.920 0.460 9.44 3.84
Head No. 10
0.11 0.01 ----- --- 4.00 1.60
‘0.13 0.01 -—--- --- 4.48 1.60
0.13 0.01 ----- ---- 4.96 1.12
0.56 0.24 0.015 --- 7.52 2.08
{0.64 0.32 0.035 --—-- 7.52 2.24
0.56 0.32 0.020 --- 7.68 2.08
1.04 0.64 0.115 0.035 8.80 2.24
1.04 0.64 0.130 0.030 8.80 2.24
Head No. 11
{0.64 0.32 0.040 —--- 7.84 2.23
0.72 0.40 0.045 --- 8.16 2.23
1.04 0.64 0.115 0.020 8.96 2.56
1.12 0.72 0.110 0.020 8.96 2.56
1.76 1.12 0.345 0.080 9.12 3.20
1.76 1.28 0.365 0.085 9.12 3.20
2.56 1.60 0.990 0.240 9.28 3.36
2.88 2.23 2.410 0.450 9.28 3.50
Head No. 12
-—-- ---- ---—— ---- 3.52 1.60
--- --- —-- ---- 3.52 1.60
---- --- ---- ----- 3.84 1.60
0.48 1.60 0.025 --—-- 7.36 2.24
‘0.36 1.60 0.020 --- 7.20 2.08
0.40 1.60 0.025 ---- 7.20 1.92
0.96 0.64 0.100 --- 8.48 2.56
1.04 0.64 0.110 --- 8.48 2.72
2.08 1.44 0.655 0.100 9.76 3.68
1.92 1.28 0.495 0.080 9.76 3.68

60

Table 3. Average size, fresh and dry weights of embryos (from Table 1
and Table 2) and average size of caryOpses (from Table 2) related to

histologically determined stages of embryonic development.

Stage of embryo Size of embryo Fresh weight
of embryo

A -

B -

c -

D _

E -

F 0.25 x 0.15

0 0.30 x 0.15

G-l 0.35 x 0.20

1 0.40 x 0.20
(0.45 x o. 20)

2 0.50 x 0.25
(0.60 x 0.30)

3 0.70 x 0.35

4 0.80 x 0.40

5 0.90 x 0.50

68 1.20 x 0.80

6b - 1.80 x 1.20

6c 3.00 x 2.30

0.005
0.005
0.015

0.025

0.035

0.045
0.055
0.075
0.150
0.450

2.500

Dry weight
of embryo

0.005

00 010

0.010
0.015
0.020
0.045
0.075
0.850

Size of
caryopsis

505 x 109
6.0 x 1.9
6.0 x 1.9

7.0 x 2.0

8.0 x 2.0

8.3 x 2.1
8.5 x 2.2
8.6 x 2.3
9.2 x 2.7
9.6 x 3.7
9.7 x 3.9

Table 9.

61

Individual length and width measurements of barley embryos

developing in vivo made at two-day intervals from four representative

spikes.

Embryos
of Spikes

Average

 

 

Days in vivo
4 6 8
1.20 2.60 .00
1.15 1.85 2.15
1.95 2.45 3.05
1.35 1.55 2.00
1.65 2.0 2.80
1.25 1.55 1.95
1.20 2.25 2. 0
1.20 1.75 1.90
1.25 2.33 2.88
1.23 1.67 2.00

10

PE
to
0

Mr
0 0
HM
00

E

E

N
0

O
Kn

E
E

N
0

N
N

N
00 H
at N
00

A) A)
C O O
c— kg
c> \nca

N
I

N
\n

N
0
K.)
(I)

62

Table 5. Culture group 1. Average growth rate of embryos cultured contin-
plus coconut milk (no culture transfers).

uously on basic medium

No. of embryos
cultured

No. of embryos
growing at the
end of two weeks

Average initial
size ' in mm.

Average final
size in mm.

Percent increase
in size

Series 1

30

0.35 x 0.20

0.70 x 0.50

100 x 150

Series 2

64

36

0.50 x 0.30

1. 00 x 0075

100 x 150

Series 3

56

26

0.80 x 0.45

1.55 x 1.05

94 x 133

63

Table 6. Culture group 2. Ratio of length/width (in mm.) of immature barley
embryos developing in culture, including total days in culture (d) and day

(d) on which shoot or root first appeared.

One part basic medium (White,

1954) plus nine parts coconut milk was used as the culture medium for two
weeks, after which embryos were transferred to basic medium only (without
coconut milk).

Embryo

0

Culture No. 1

l

\D" 00 NI 0\ Vi 3‘ U.) M

E

0.35

E

0.40

0.25

0.70

Culture N o. 2

1

\OQQO‘UIl‘U)

0.30

Days in culture

 

 

 

 

 

 

 

 

2 4 6 8 10 12
9.95 9.95 0.90 1.00 1.15 1.29
0045 0060 0065 0075 0090 0095
9.25 9.29 1.95 1.15 1.25 1.49
0.45 0.65 0.70 0.80 0.90 1.00
0,20 9,95 9,99 1.09 1.10 1,99
0.40 0.55 0.60 0.65 0.75 0.85
9.15 9.29 9.25 1.99 1.95 1.15
0.45 0.55 0.65 0.70 0.75 0.85
9.39 ....

0.15

0.60 0.70 0.80 0,90 9,95 ____
0.40 0.50 0.55 0.60 0.65

1,10 1,25 1,30 1,45 1,55 1,25
0.70 0.95 1.10 1.25 1.35 1.50
1,39 1,60 1,80 1,95 2,05 2,40
0.90 1.65 1.30 1.40 1.50 1.60
9.55 9.95 9.29 9.99 9.95 1.99
0.35 0.50 0.55 0.60 0.65 0.75
9.99 ..250 1.19 ...51 2 1.49 1.45
0.45 0.60 0.65 0.75 0.85 0.95
0,25 ____

0.20 "“
1,99 1,40 1,55 1,15 1 0 2,00
0.75 0.95 1.10 1.25 1.40 1.50
9.95 9.99 9.29 1.99 1.19 ....
0.40 0.50 0.55 0.65 0.70

9.99 9.29 9.25 1.99 1.95 1.15
0.45 0.55 0.65 0.75 0.80 0.90
9.49

0.20

0,30

 

Total

NO‘

FJFJFJFJ

E09

.3

.Hlsr
.88

F‘F‘
O
c>c>

Hg
0

0‘
\fl

53F“
.014
uxC)

{HE
NO‘
\h

tJF°
43a:
c>c>

E

1.20

Remarks

(26d) Shoot
(28d)
(26d) Shoot
(28d)
(20:1)

(266) Shoot
(28d)

(20d) Root
(22d)
(23d)

(18d)
(21d) Shoot
(26d)

(21d) Shoot
(23d)

(23d) Shoot
(26d)

611

Days in culture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Embryo O 2 4 6 8 10 12 Total Remarks
Culture No. 3
1 9.25 ____
0.15
2 0,55 9,99 0,65 0,65 0,10 0.15 0.90
0.35 0.35 0.40 0.45 0.50 0.55 0.60
3 0,65 0,85 1,00 1,10 1,15 1,25 1,40
0.35 0.50 0.70 0.75 0.80 0.85 0.85
4 0.60 0.80 0,90 0 o 1.15 1,25 1,50
0.40 0.50 0.60 0.65 0.75 0.80 0.85
5 0.90 1.10 1,50 1,60 1.85 2,00 2,19
0.55 0.70 0.90 1.05 1.15 1.20 1.30
6 0,60 0,75 0.90 1,00 1,15 1,55 1,50
0.35 0.35 0.60 0.65 0.75 0.90 1.00
7 9.55 9.95 9.99 9.99 9.95 1.95 1.15 1.60 (27d)
0.25 0.40 0.50 0.55 0.60 0.65 0.75 1.10
8 9.99 9.19 9.95 9.29 1.99 1.95 1.59 ....
0.25 0.45 0.60 0.70 0.80 0.90 1.00
9 0,90 0,12 1,49 1,50 1,95 1,85 9,99 9,29 (18) Root
0.55 0.70 0.85 0.95 1.00 1.10 1.40 2.00 ' (200)
Culture No. 4
1 0,60 0.80 0.90 9,95 1,05 1,15 1,25 1,55 (30d) Shoot
0.40 0.45 0.55 0.60 0.65 0.75 0.85 1.65 (32d)
2 9.95
0.15
3 9,50
0.25
4 9.55 9.45 9.45 9.59 ____
0.25 0.40 0.45 0.45
5 9.25 1.99 1.55 1.59 ____
0.55 0.75 0.85 0.90
6 9.99 9.29 9.99 9.95 ....
0.30 0.40 0.55 0.60
7 9,49 0,55 0,65 0.70 ____
0.25 0.40 0.50 0.55
8 9.55 9.29 9.29 1.99 1.15 .... .... ....
0.35 0.40 0.55 .0.65 0.75
Culture No. 5 '
1 , 9,99 1.20 ____
0.60 0.75
2 9.55 9.55 .... ____
0025 0030
3 0,95 1,20 1,45 1,60 ____
0.65 0.80 1.05 1.20
A 9.99 9.95 ____
0.20 0.40
5 9.69 9.15 9.29 _ .... __
0.35 0.45 0.50
6 9.59 9.55 9.95 ....

 

Days in culture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Embryo 0 2 4 6 8 10 12i
Culture No. 6
1 0,45
0.25
2 9.59
0.30
3 9.55 9.15 9.29 9.25 1.9.5 1.99 1.29
0.30 0.40 0.55 0.65 0.70 0.75 0.85
4 9.3.9 ....
0.20
5 9.49
0.25
6 9.29
' 0.20
Culture No. 7
1 ‘ 9.99 1.95 1.2.9 1.25 1.45 1.55 1.99
0.35 0.65 0.80 1.00 1.00 1.05 1.10
2 1.00 1.20 1,49 1,60
0.60 0.75 0.90 1.05 .
3 9.29 9.29 1.99. 1.92 1.9.5 1.95 ....110
0.35 0.50 0.60 0.65 0.65 0.70 0.70
4 9.39
0.15 '
5 9....56 9._80 _.2_0 0 9.25 1.95 1.15 1.2.9
0035 0045 0055 0065 0065 0070 0080
Culture No. 8
1 9,55 0,55 ,Q,45 0,50 0,50
0.20 0.20 0.25 0.35 0.45
2 9.3.5 ...350 9.25 _.4_0 0
0.20 0.25 0.25 0.25
3 9.29 9.25
0.15 0.15
4 9.15
0.10
5 ____ _._. ....
6 9.49 9..9 9.99 9.95 9.15 9.99 9.95
0.25 0.35 0.40 0.50 0.55 0.60 0.65
7 9.49 9.99 9.19
0.25 0.35 0.45
8 9.49 9.59 9.55 9.99 9.95
0020 0025 0035 0045 0050
9 9.39 9.99 9.19 9.15
0.20 0.35 0.40 0.40

Total

1,80
1.35

65

Remarks

(30d) Root

(32d)

66

Days in culture

Embryo . 0 2 4 6 8 10 12 Total Remarks

Culture No. 9

1

2
3
A
5
6
7
8

Culture No. 10

l
2

QGWFW

9.59
0.25
9.45
0.25
9.45
0.20
9.49
0025
9.59
0.30
9.25
0.20
9.55
0.35

Culture No. 11

l

O‘Utbw

0,30
0.20

9.49
0.20
9.45
0.20
9.55
0.20
9.45
0.30
9.55

0.25-

 

PP
88

9.90
0.65

9.25
0.65

 

 

 

0,85
0.55

0.65

0.75

 

 

 

 

 

SDFJ
said
uzc>

turd
Sica

 

 

 

 

 

 

2.45
1.25

1.25
1.35

(31d) Shoot

(33d)

(28d) Root
5 (30d)

(21d) Root

(23d)

Embryo

0

Culture No. 12

1

CnQO‘Ut3‘WN

9.45
0025
9.45
0.26
9.3.5
0.20
9.49
0.25
0,50
0.30

9.55
0.30

Days in culture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2 4 6 8 10 12
0,55 0,65 0,20 0.80
0.35 0.35 0 45 0.55
0,15 9,55 9,95 0,95 1,00 1,05
0.35 0.45 0 60 0.65 0.75 0.85

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Total

\J'CO‘
O

6'?

Remarks

(31d) Shoot

(33d)

68

Table 7. Summary of three series (based upon sizes) from table 6.

Series 1 Series 2 Series 3
No. of embryos 36 38 14
cultured
No. of embryos
grown for two 1 18 10
weeks
No. of embryos
formed shoots l 9 6

or roots

Avera e initial

size length x 0.35 x 0.20 0.50 x 0.30 0.80 x 0.45
width) in mm.

Average final

size (length x 0.85 x 0.65 1.20 x 0.90 1.82 x 1.12
width) in mm. (for two weeks)(for two weeks)(f0r two weeks)
1.80 x 1.35 1.80 x 1.40 2.20 x 1.85
(for total (for total (for total
days) days) days)
112 x 225 140 x 300 ' 127 x 148

(for two weeks)(for two weeks)(for two weeks)
Percent increase
in size 350 x 575 260 x 366 175 x 311
(for total (for total (for total
days) days) days)

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