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This is to certify that the

thesis entitled
Cytogenetics of Haploids and Hybrids

from the Cross,

Hordeum procerum X Hordeum vulgare
presented by

Jeffrey Dean Griffin

has been accepted towards fulfillment

of the requirements for
M.S.
- degree in Botany

    

Major professor

0"] 639

 

 

 

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returned after the date
stamped below.

 

 

 

CYTOGENETICS OF HAPLOIDS AND HYBRIDS FROM THE CROSS,
HORDEUM PROCERUM X HORDEUM VULGARE

 

 

By

Jeffrey Dean Griffin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Botany and Plant Pathology
1981

Abstract

Hordeum_procerum_Nevski (2n=6x=42) was crossed with three cultivars

 

of barley, fl, vulgare L. (2n=2x=l4). Based on cytological and
morphological evidence, two classes of plants resulted. Crosses made
with 5, vulgare cv. 'Larker' resulted in polyhaploids (2n=3x=21).
while crosses made with other cultivars resulted in hybrids with variable
chromosome numbers, 2n=23=29. The presence of genetic factors in the
E, vulgare genome for resistance to chromosome elimination is proposed.
Microsporogenesis was studied in the polyhaploids and hybrids,
as well as in both parental species. fl, procerum is considered a
segmental autoallohexaploid. In addition, a mechanism for the genetic
control of meiotic pairing was inferred, which causes fl, procerum to
behave cytologically like a diploid. It is suggested that there is no
appreciable homology between the genomes of 3, procerum and fl, vulgare.

ACKNOWLEDGMENT

I wish to express my sincere gratitude to Dr. William Tai for
serving as my major professor, and providing me with the facilities,
as well as the opportunity, to undertake this research. Thanks also
to Drs. Peter S. Carlson and Stephen Stephenson for serving on my
guidance committee, to Dr. Joanne Nhallon for her encouragement
and support, to fellow graduate students Gary Bauchan and John Blackson
for their helpful suggestions, and to Liu Zhiwu for his friendship.
Finally, I wish to express my sincere thanks to my parents,

whose support made it possible for me to finish this work.

ii

Table of Contents

 

Page
List of Tables ....................... iv
List of Figures ...................... v
Introduction ....................... 1
Materials and Methods ................... 4
Results .......................... 9
Discussion ......................... 41
Appendix A ......................... 51
Appendix B ......................... 52
Bibliography ........................ 53

Table
Table

Table

Table
Table
Table
Table
Table
Table
Table

Table

10

ll

List of Tables

 

 

 

Page
Crosses Performed With Hordeum procerum as Female
Parent ....................... lO
Chromosome Numbers of Progeny of Interspecific
Crosses (fl, procerum (6X) X M, vulgare (2X) 12
Summary of Metaphase I Chromosome Associations in
Hordeum vulgare cv. 'Larker' and in Hordeum
procerum ...................... 18
Summary of Post-Metaphase I Chromosome Behavior for
All Plants Studied ................. l9
Pollen Stainability ................ 22

Summary of Metaphase I Chromosome Associations in Two
Polyhaploids from the Cross 5, procerum X M, vulgare

cv. 'Larker' 23

Summary of Metaphase I Chromosome Associations in
l-4 (fl, procerum X E, vulgare cv. 'Coho')

Summary of Metaphase I Chromosome Associations in
4-2 (5, procerum X 5, vulgare cv. 'Coho')

Summary of Metaphase I Chromosome Associations in
4-8 (5, procerum X H, vulgare cv. 'Coho')

Summary of Metaphase I Chromosome Associations in
14-2 (M, procerum X E, vulgare cv. 'Beacon')

Summary of Metaphase I Chromosome Associations in

14-7 (5, procerum X E, vulgare cv. 'Beacon') . . . . 28

iv

List of Figures
Egg;
Figure l Mitotic chromosomes of the polyhaploids and hybrids. 29
Figure 2 Spike and spikelet morphology ........... 33

Figure 3 Microsporogenesis in M, vulgare cv. 'Larker' and
in H. procerum ................... 35

Figure 4 Microsporogenesis in polyhaploids from the cross,
fl, procerum X M, vulgare cv. 'Larker' ....... 37

Figure 5 Microsporogenesis in 5, procerum X 5, vulgare
hybrids ...................... 39

INTRODUCTION

 

The study of interspecific hybrids may yield information about
taxonomic and evolutionary relationships between plants; and, when
one of the species involved is an agronomically useful one, may lead
to the transfer of genes which contribute to increased crop yields,
improved quality, resistance to environmental stress, or resistance
to pests. This generalization can be applied to most previous
interspecific hybridization work in the genus Hordeum (Schooler and
Anderson, 1979; Steidl, 1976; Starks, 1976; Morrison gt_gl,, 1959).

The most ambitious program of Hordeum interspecific hybridization
to date was that initiated by D. G. Hamilton and carried out by
J. N. Morrison and others at Ottawa, Canada. They attempted 111
interspecific cross combinations, about half of which involved
cultivated barley, fl, vulgare. They were able to produce fourteen
viable hybrids by using embryo culture techniques (Morrison gt_gl,,
1959). All hybrids between 5, vulgare and species outside section
Cerealia were sterile, and studies of meiotic pairing indicated no
homology between the parental genomes (Morrison and Rajhathy, 1959).

Nilan (1964) and Price (1968) reviewed the interspecific crosses
attempted in Hordeum. They also pointed out that hybrids between
barley and distantly-related species are sterile and exhibit no

homology between genomes.

Schooler and Anderson (1979) have taken a somewhat different
tack. Utilizing a fertile amphiploid from the cross (fl, brachyantherum
X H, bogdanii)(2n = 6x = 42) as the female parent and H, vulgare cv.
'Traill' (2n = 4x = 28) as the male parent, they were able to produce
F1 hybrids with a chromosome number which varied from 21 to 35.
After backcrossing to diploid H, vulgare (2n = 2x = 14) and recovering
diploid progeny, they reported some preliminary observations of gene
transfer.

This same general approach, that is, crossing an amphiploid of
a hybrid between two wild species to barley, followed by a backcrossing
program, has been attempted by previous workers at this university
(Huang, 1975; Steidl, 1976) without any appearance of startling
success. Genome relationships have also been studied between Hordeum
species, and between Hordeum and Agropyron species, by previous
workers in this laboratory (Murry, 1975; Huang, 1975; Starks, 1976).

In the fall of 1978, I began a crossing program in an attempt
to generate hybrids between H, vulgare and diploid wild species.
This program was later expanded to include tetraploid and hexaploid
species or cytotypes of species. 0f the 74 crosses attempted, only
those between cultivars or species from section Cerealia produced
fully developed, viable seeds. Some other cross combinations did
set seed, but although they included a reasonably well developed
embryo, endosperm development was poor and the seeds did not germinate.

Attempts to save some of these embryos via embryo culture, using

Norstog's Barley Medium II (Norstog, 1973) were wholly unsuccessful.

In the early winter of 1980, an encouraging development appeared
which was to lead to further research. A hybrid seedling was obtained
from the cross, H, procerum (2n = 6x = 42) X H, bulbosum (2n = 2x = 14)
using a modified version of Norstog's medium.

This led to the consideration of the possibility of producing
and studying hybrids between H, procerum (2n= 6x = 42) and H, vulgare
(2n = 2x = 14). Subrahmanyam (1977) reported that this cross resulted
in tetraploid hybrids, as well as triploid polyhaploids of H, procerum.

The questions which it was hoped the study would answer were:

1. Would the cross, H, prOcerum (6x) X H, vulgare (2x) yield
hybrids and polyhaploids, as other workers have reported?

2. Would there be differences between barley cultivars in the
degree and frequency with which their chromosomes were
eliminated by the H, procerum genome?

3. What would cytogenetic studies of H, procerum hexaploids and
polyhaploids reveal about the nature of its three genomes?

4. What would cytogenetic studies of the hybrids reveal about

the degree of homology, if any, between the genomes of

H, procerum and H, vulgare?

MATERIALS AND METHODS

 

PLANT MATERIALS

 

Two Hordeum species were used in this study. The first was a

wild species collected in Argentina, Hordeum procerum (PI No. 266196),

 

which was obtained as seed from the USDA Cereal Crops Research
Branch, Beltsville, Maryland. Previous reports (Covas, 1950; Bowden,
1965) indicate a chromosome number of (2n = 6x = 42). The second

species was barley, Hordeum vulgare L. (2n = 2x = 14) and three

 

cultivars were used; lCoho', 'Larker', 'Beacon'.

CROSSING TECHNIQUES

 

In all crosses performed as a part of this study, H, procerum
was the female parent, and one of the three barley cultivars was the
male parent. No reciprocal crosses were done because earlier
experience with these plants indicated that the cross was more likely
to be successful with H, vulgare as the male parent. This was also
the case for similar interspecific Hordeum crosses (Steidl, personal
communication).

For all crosses, the female parent was emasculated by first
removing all spikelets above and below those judged to be at the
right stage of development. Generally, this meant that only florets
with pale-green to yellow anthers were used and the rest discarded.
Lateral florets were removed from the remaining spikelets. The lemma

and palea of each remaining floret was then cut off just above the

anthers with scissors, and the anthers removed with fine forceps. The
spikes were then covered with a thin, cylindrical cap of aluminum

foil. Aluminum foil was superior to other coverings, because it

keeps the spikes cool, prevents dehydration, and prevents contamination
by stray pollen.

For a source of fresh pollen, barley spikes were selected in
which the anthers were just about to dehisce. The tops of the florets
were cut off just above the anthers and the spike was placed in
strong light, either sunlight or in close proximity to an incandescent
bulb (about 50 cm.). This caused the anthers to be extruded from
the florets and to dehisce. Pollen was collected by inverting spikes
inside a small bag fashioned from dialysis tubing and tapping it to
release pollen into the bag.

Previously emasculated spikes with receptive stigmas (stigmas
spread out and florets open) were then inverted in one of these bags
containing pollen, the opening of the bag was held closed, and the
spike was returned to an upright position. This inversion-reversion
process was then repeated until most of the pollen had come to rest
on the florets; this resulted in the deposition of massive amounts of
pollen on the stigmas, thus insuring that each stigma had an ample
supply of viable pollen. This is also the quickest and simplest way
to do these pollinations. After pollination, the aluminum foil cap

was replaced.

EMBRYO CULTURE

 

Spikes were cut from the plants and taken to the laboratory for
embryo culture 12 to 14 days after pollination. The percent seed set
was also scored at this time.

All embryo culture work was done in a laminar flow hood using
standard sterile techniques. The seeds were removed from their
respective florets and sterilized in a 20% solution of commercial
bleach (corresponding to a solution of roughly 1% sodium hypochlorite)
for 20 nfinutes, then washed briefly in sterile, double-glass-distilled
water. The embryos were dissected from the caryopses, and placed
scutellum side down on a modified Norstog's medium, (Norstog, 1973;
Taira and Larter, 1978; Appendix A) in slanted culture tubes. The
embryo cultures were incubated in the dark at 27°C, then transferred
to a lighted culture room at 25°C when they showed definite root and
shoot (cotyledon) development. Some embryos produced only very
short roots and shoots (about 2 - 5 mm) in the incubator, as if
development was arrested at that point. These were transferred to
a callus regeneration medium, designated B-5R (Orton, 1978; Appendix B),
and placed in the lighted growth room to induce them to germinate
further.

When the seedlings reached the three leaf stage, they were
potted in plastic cups filled with vermiculite and placed in a growth
chamber with a 16 hour day length and a 19° - 14°C day-night

temperature regime. The plants were later transplanted to soil in

clay pots and placed in the greenhouse.

CYTOLOGICAL METHODS

 

Mitotic material was pretreated in two ways. Healthy root tips
of growth chamber-grown plants were excised and pretreated in a
solution of .01% colchicine at room temperature for 4 hours. Root
tips from greenhouse-grown plants were excised and pretreated in
distilled water at 2 - 4°C for 24 - 48 hours. In all cases, the
root tips were fixed in 3:1 ethanol-acetic acid (Farmer's solution)
at room temperature for 12 - 24 hours and then rinsed and stored in
70% ethanol at 4°C.

Root tips were prepared for squashing by hydrolyzing in 1N HCl
for 10 minutes at 60°C, rinsing with distilled water, and staining
in Feulgen's solution for 2 hours in the dark. Root tips were then
rinsed with distilled water and further softened in 5% pectinase
(SIGMA) for one hour at 30°C in a water bath. The pectinase was rinsed
out with distilled water. The root tips were either squashed
immediately or stored in 70% ethanol at 4°C until needed.

For observation of meiosis in pollen-mother-cells (PMC's), anthers
were dissected from central florets and squashed in aceto-carmine
stain.

Good cytological preparations were documented by photographing
them with a Zeiss Photomicroscope II using Kodak Panatomic-X film in

the built-in 35 mm camera.

Pollen stainability was determined by squeezing pollen out of
mature anthers in a drop of IzKI stain. Only darkly-stained grains

were scored as stainable; a minimum of 300 grains per plant was scored.

RESULTS
A total of 19 spikes of H, procerum were pollinated by 3 different

barley cultivars. The results of these hybridizations are presented
in Table I. H, vulgare cv. 'Larker' was the male parent in 7 crosses,
and the cultivars 'Coho' and 'Beacon' were used in six crosses each.
A total of 242 florets was pollinated, 172 of which set seed; this is
an average seed set of 71%. All of these seeds contained embryos,
indicating there was no parthenocarpic fruit set. In all cases, also,
the endosperm which developed was watery, and had begun to collapse
by the time the embryos were excised for culture. One hundred and
forty embryos were placed on embryo culture medium, and 21 were placed
on callus initiation medium as part of another study. Eleven embryos
were either too small to culture, or were damaged in the process of
excision from the caryopsis. Seventy embryos, or 50% of the total,
germinated almost immediately and were transferred to the lighted
growth room for further development. Seventeen embryos seemed to stop
development after only a day or two. Previous experience indicated
that these would simply remain dormant, and eventually die. These
were transferred to the B-5R medium, normally used for regeneration of
plantlets from callus. All seventeen of these embryos germinated.
In all, 87 embryos, or 62% of those cultured, germinated and were
transplanted to vermiculite as seedlings, and placed in a growth
chamber.

In the late summer of 1980 a growth chamber malfunction, resulting

in extremely high temperatures, killed approximately one-half of these

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plants. All surviving plants were then transferred to the greenhouse
and subsequently repotted in clay pots.

The mitotic chromosome numbers of 27 interspecific cross progeny,
which resulted from five different crosses, are presented in Table 2.
All ten plants which resulted from crosses 10 and 11, (H, procerum X
H, vulgare cv. 'Larker') had 21 chromosomes in mitotic metaphase plates
of root tip squashes (Figure 1, Table 2). This corresponds to the
haploid chromosome number of the female parent. Morphologically,
these plants closely resemble H, grocerum; however, they are shorter
in stature and their spikes and spikelets are smaller (Figure 2).

All of these plants are sterile.

The eleven plants which resulted from crosses l and 4 (H, grocerum
X H, vulgare cv. 'Coho') had chromosome numbers which varied. Some
plants had the same number of chromosomes in all cells studied, but in
other plants, the number was variable. Overall, the number of somatic
chromosomes in the plants from the Coho crosses ranged from 23 to 29
(Figure 1, Table 2), with a mean of 26.9. These plants have spike and
spikelet morphology intermediate between the parents, and all are
taller than either parent (Figure 2). A notable exception to this is
plant number 4-2, which has 23 chromosomes (Figure 1). While its
spike and spikelet morphology is intermediate, its stature and gross
morphology more closely resembles that of the polyhaploids from the
Larker crosses. All of these plants are sterile.

The six plants studied from cross 14, (H, grocerum X H, vulgare

cv. 'Beacon') also have variable chromsome numbers (Figure 1, Table 2),

12

TABLE 2

Chromosome Numbers of Progeny of Interspecific Crosses

(H, procerum (6x) X H, vulgare (2x))

 

 

Plant No. Male Parent

Number of Cells

 

Chromosome Number

 

21

23 26 27 28 29

 

Total Coho
10-1 Larker

Total Larker

14-1 Beacon

Total Beacon

15 11

25
5 3
29

25
21

33
29

34

19
19 24 1

25
53 17

 

 

*Includes 2 cells with 42 chromosomes each.

13

with a range of 27 - 29 and a mean chromosome number of 27.4. These
plants also exhibit morphology which is intermediate between the
parents, (Figure 2), are taller than both parents, and are sterile.

Seven plants, judged to be a representative sample of the
variation in chromosome numbers and genetic background, were selected
for meiotic studies. In addition, meiosis was also studied in
H, procerum and H, vulgare cv. 'Larker'.

A summary of metaphase I chromosome associations in H, procerum
and in H, vulgare is presented in Table 3.

Meiosis in H, vulgare cv. 'Larker' was essentially nominal
(Figure 3). Metaphase I plates exhibited seven bivalents in 98% of
the PMC's observed. Anaphase I was without laggards in the fifteen
cells observed; segregations were the expected 7 - 7 in well—spread
PMC's.

Metaphase, anaphase, and telophase II were also apparently normal
and without bridges or laggards, (Table 4). 0f the 320 tetrads
observed, only one (0.3%) had micronuclei. No supernumerary dyads or
tetrads were observed.

H, procerum appears to be a relatively normal hexaploid (Figure 3).
In the 36 metaphase I cells studied, (Table 3), there was a mean of
19.6 bivalents and 2.4 univalents per cell. The maximum number of 21
bivalents occurred in 54% of the cells studied. Trivalents and
quadrivalents were relatively rare, each occurring with a frequency of

.06 per cell. All metaphase I cells had 42 chromosomes. Post-metaphase

14

I chromosome behavior, (Table 4) was relatively normal, with only

a few bridges and laggards at anaphase I and telophase I, and a few
micronuclei at the dyad stage. The second meiotic division, however,
was characterized by bridges and laggards at anaphase and telophase,
resulting in micronuclei in 24% of all tetrads studied. Pollen
stainability was 76% (Table 5).

The plants which resulted from the crosses with 'Larker' as male
parent were polyhaploids with 2n = 3x = 21. The metaphase I
chromosome behavior of the two plants studied, numbers 10-5 and 11-1,
is presented in Table 6 and in Figure 4. Both plants had mean
univalent frequencies of just over seventeen, and mean bivalent
frequencies of just less than two. Multivalent frequencies were
exceedingly low.

The combined post-metaphase I chromosome behavior is presented
in Table 4 under the heading, 'Larker Crosses', and in Figure 4.
Anaphase I is characterized by a high frequency of laggards, many of
which divide precociously, as well as unequal segregations (other than
10 - 11) and tripolar division figures. Telophase I also exhibits
bridges, laggards and tripolar divisions, resulting in dyads with
micronuclei, as well as triads (dyad stage with three cells). Meta-
phase II seemed to occur as scheduled, despite the now-jumbled nature
of the nuclei in a high proportion of dyads. Anaphase and telophase II
were characterized by even higher percentages of bridges and laggards

than anaphase and telophase I. There were no tripolar figures

15

observed at the second division; however, over 58% of the tetrads
observed showed micronuclei, and over 10% of the "tetrads" had
supernumerary cells (more than 4). Pollen stainability was 0.2%
(Table 5).

Three plants were studied from the crosses with 'Coho' as the
male parent. The metaphase I chromosome behavior of these plants,
numbers 1-4, 4-2, and 4-8 is presented in Tables 7, 8, and 9,
respectively, and in Figure 5. Note that the number of chromosomes in
metaphase I PMC's varied within each plant. Plant number 1-4, which
was determined to have a somatic chromosome number of 2n = 28, had a
mean chromosome number in metaphase I cells of 25.9, with a range of
24 - 28. There was a mean number of 2.2 bivalents and 18.2 univalents
per cell, with a trivalent frequency of .12. Plant number 4—2, with
a somatic chromosome number of 2n = 23, had a mean metaphase I
chromosome number of 23.5, with a range of 23 - 26. Mean bivalent and
univalent frequencies were 1.8 and 19.9, respectively; no trivalents
were observed. Plant number 4-8, with a somatic chromosome number of
2n = 27, had a mean chromosome number of 26.9 in metaphase I cells,
with a range of 26 - 28. This plant had a mean of 19.9 univalents,
3.1 bivalents, .26 trivalents, and .02 quadrivalents per cell; this
represents the highest frequency of multivalents of the five hybrid
plants studied.

The post-metaphase I chromosome behavior of these plants is

presented in Table 4 under the heading, 'Coho Crosses', and in Figure 5.

16

In many respects the meiotic behavior of these plants closely resembled
that of the polyhaploids discussed previously. While tripolar first
divisions seemed to decrease, the frequency of bridges and laggards
increased at anaphase and telophase of both the first and second
division and both dyads and tetrads had more micronuclei than the poly-
haploids. The number of supernumerary cells at the tetrad stage was
also greatly increased, and a few "tetrads" were observed with only
three cells, as if the second division did not occur in one half of a
dyad. Multinucleated cells were frequent. Pollen stainability was
0.19% (Table 5). -

Two plants were studied from the crosses with 'Beacon' as the
male parent, and these also had variable chromosome numbers in PMC's.
Plant number 14-2 (Table 10), with a somatic chromosome number which
varied from 27-29, (mean 27.6) had a mean metaphase I chromosome number
of 26.9, and a mean of 22.7 univalents, 2.0 bivalents, and .06
trivalents per cell. Plant number 14-7 (Table 11) with a somatic
chromosome number of 27-28, (mean 27.2) had a mean metaphase I
chromosome number of 27.0. There was a mean of 23.4 univalents and
1.8 bivalents per cell. Photographs of meiotic phenomena from these
plants are included in Figure 5.

The post-metaphase I chromosome behavior of these plants is
presented in Table 4 under the heading "Beacon Crosses". Although
the data in this case is rather sparse, the pattern established by

the Coho crosses emerges. Tripolar first divisions, bridges and

17

laggards at anaphase and telophase of both divisions, tetrads with a
high frequency of micronuclei, supernumerary cells, and multinucleated

tetrads were all observed. Pollen stainability was 0% (Table 5).

18

TABLE 3

Summary of Metaphase I Chromosome Associations in Hordeum vulgare cv.
'Larker' and in Hordeum procerum

 

 

 

 

 

 

Plant Chromosome Association
Number
I II III IV of cells %
H. vulgare 2 6 * l 2.2
Ev. Larker' 7 44 97.8
Total 45
Mean per
cell 0.04 6.9
H, procerum 21 19 52.8
2 20 5 13.9
4 19 2 5.5
6 18 3 8.3
8 17 2 5.5
12 15 l 2.8
9 15 l 1 2.8
8 15 l l 2.8
4 l7 1 1 2.8
l 19 1 1 2.8
Total 36
Mean per

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A.p=oov a m4m<~

21

 

 

 

 

 

 

 

 

 

 

NO OOO OOO OOO ONm NONON
O._ O O
N.O N N
N.O N N.NN NO m.N Om O
N.ON .N m.ON NO O.N ON mNN. N O
N.OO ON O.OO NNm 0.00 ONO O.OO OOO OON ONm O
N.N N O.O ON m

mppmu No Nagasz

ONN NNO OOO ONN NOOON

O.ON ON. N.OO ONN 0.0N ONN m.O N NONOOOONONZ ONNz

O.ON NO N._O NON 0.0N NNO N.OO ONO Nmecoz

much. m.—

ON ON NO NO ON NOOON

O.OO_ ON N.mN O O.ON ON NONOOOOO

can mwmuwgm

O.NN N O.ON NN N.Om Om OONNOOOO

O.NN O 0.0m NO m._ N OOOONNO

N.OO MN O.ON ON O.ON Nm OON ON Nasaoz

NH mmmzqmpmh

N N N N N N N N N N OOOOEOOONN OOO

mammogu coommm mammogu ocou mammogu megmg Ezemooga .z megmg macaw oNNNomz

 

 

N.NOOOV O NONNN

22

TABLE 5
Pollen Stainability

 

 

 

Plant Stainable Not Stainable Total % Stainable
H, procerum 1514 486 2000 75.7
'Coho' Crosses:
1-4 0 300 300 0.0
4-2 3 297 300 1.0
4-8 .9 L00_0 M Q
Total 3 1597 1600 .2
'Larker' Crosses:
10-5 0 300 300 0.0
m 1. .222. 1% 9.3.3.
Total 1 599 600 .1
'Beacon' Crosses:
14-2 0 300 300 0.0
14-7 Q_ 309_ 399, 0.0

Tota1 O 600 600 0.0

 

 

23

TABLE 6

Summary of Metaphase I Chromosome Associations in Two Polyhaploids from
the Cross H, procerum X H, vulgare cv. 'Larker'

 

 

 

 

Plant Chromosome Association Number

10-5 21 -14 15.7
19 1 18 20.2
17 2 36 40.4
15 3 14 15.7
13 4 4 4.5
14 2 1 2 2.2
16 2 1 1 1.1

Total 89

Mean per cell 17.4 1.7 .03

11-1 21 19 13.6
19 l 35 ' 25.2
17 2 42 30.2
15 3 21 15.1
13 4 17 12.2
11 5 2 1.44
9 6 l .72
16 1 1 1 .72
13 2 l 1 .72

Total 139

Mean per cell 17.01 1.91 .007 .007
Combined Total 228

Combined
Mean per cell 17.2 1.8 .017 .004

 

 

24

TABLE 7

Summary of Metaphase I Chromosome Associations in 1-4
(H, procerum X H, vulgare cv. 'Coho')

 

 

Chromosome Association

 

 

Chromosome Number
I II III IV Number of Cells %

22 l 24 1 2.8
23 1 25 2 5.7
21 2 25 6 7.1
19 3 25 2 5.7
17 4 25 1 2.8
20 1 1 25 1 2.8
18 2 1 25 l 2.8
26 26 l 2.8
24 l 26 5 4.3
22 2 26 3 8.6
18 4 26 1 2.8
14 6 26 1 2.8
25 1 27 2 5.7
23 2 27 2 5.7
21 3 27 1 2.8
19 4 27 1 2.8
17 5 27 1 2.8
20 2 1 27 1 2.8
24 2 28 1 2.8
15 5 l 28 1 2.8

Total 35

Mean per

Cell 18.2 2.2 .11 25.9

 

 

25

TABLE 8

Summary of Metaphase I Chromosome Associations in 4-2
(H, procerum X H, vulgare cv. 'Coho')

 

 

Chromosome Association

 

 

Chromosome Number
I II III IV Number of Cells %

23 23 4 11.8
21 l 23 8 23.5
19 2 23 8 23.5
17 3 23 5 14.7
15 4 23 1 2.9
20 2 24 l 2.9
23 l 25 l 2.9
21 2 25 2 5.8
17 4 25 1 2.9
15 5 25 1 2.9
22 2 26 1 2.9
26 26 1 2.9

Total 34

Mean per

Cell 19.9 1.8 23.5

 

 

26

TABLE 9

Summary of Metaphase I Chromosome Associations in 4-8
(H, procerum X H, vulgare cv. 'Coho')

 

 

Chromosome Association

 

Chromosome Number

 

I II III IV Number of Cells %
24 1 26 2 4.6
22 2 26 l 2.3
20 3 26 2 4.6
18 4 26 l 2.3
17 3 l 26 l 2.3
27 27 2 4.6
25 1 27 1 2.3
23 2 27 4 9.3
22 1 1 27 2 4.6
21 3 27 5 1.6
20 2 1 27 4 9.3
19 4 27 3 6.9
17 5 27 4 9.3
17 3 l 27 l 2.3
17 2 2 27 1 2.3
16 4 l 27 1 2.3
15 6 27 4 9.3
10 7 l 27 1 2.3
22 3 28 2 4.6
20 4 28 1 2.3

Total 43
Mean per

Ce11 19.8 3.1 .26 .02 26.9

 

 

27

TABLE 10

Summary of Metaphase I Chromosome Associations in 14-2
(H, procerum X H, vulgare cv. 'Beacon')

 

 

Chromosome Association

 

 

Chromosome Number
I II III IV Number ‘ of Cells %
26 26 4 8.2
25 l 26 l 2.0
22 2 26 3 6.1
20 3 26 2 4.1
27 27 2 4.1
25 l 27 7 14.3
23 2 27 10 20.4
22 1 1 27 l 2.0
21 3 27 12 24.5
20 2 l 27 1 2.0
19 4 27 3 6.1
24 2 28 1 2.0
21 2 l 28 1 2.0
20 4 28 l 2.0
Total 49
Mean per

Ce11 22.6 2.0 .06 26.9

 

 

28

TABLE 11

Summary of Metaphase I Chromosome Associations in 14-7
(H, procerum X H, vulgare cv. 'Beacon')

 

 

Chromosome Association

 

 

Chromosome Number
I II III IV Number of Cells %
27 27 11 13.9
25 1 27 25 31.6
23 2 27 21 26.6
21 3 27 11 13.9
19 4 27 9 11.4
24 2 28 l 1.3
22 3 28 1 1.3
Total 79

Mean per
Cell 23.5 1.8 27.0

 

 

29

Figure l: Mitotic chromosomes of the polyhaploids and hybrids

a Plant #10-5 (H, procerum X 'Larker') with 2n=21. 830X.
b+c Plant #4-2 (H, grocerum X 'Coho') with 2n=23. 830X.
d Plant #1-7 (H, procerum X 'Coho') with 2n=26. 830X.
e Plant #1-6 (H, procerum X 'Coho') with 2n=28. 830X.
f Plant #4-8 (H, procerum X 'Coho') with 2n=27. 830X.
9 Plant #14-7 (H, procerum X 'Beacon') with 2n=27. 830X.
h Plant #14-7 (H, procerum X 'Beacon') with 2n=28. 830X.

3O

 

 

 

31

Figure 2: Spike and Spikelet Morphology.

a H, vulgare cv. 'Beacon' spike.
b H, vulgare cv. 'Coho' spike

D
II

. procerum X 'Beacon' F1 spike

O.
|I

. procerum X 'Coho' F] spike

(D
II

. procerum spike

f H, procerum polyhaploid spike
'Beacon' spikelet

b' 'Coho' spikelet

c' H, procerum X 'Beacon' F1 spikelet
d' H, procerum X. 'Coho' F] spikelet

e' H, procerum spikelet
f' H, Ergggggm_polyhaploid spikelet

32

 

Figure 3:

33

Microsporogenesis in H, vulgare cv. 'Larker' and in

H, procerum.

a 'Larker', metaphase I with 711. 830X.

b 'Larker', normal tetra. 830X.

c H,

(D D.
n: p:

'1'!
II

:- «3
F: K:
O O

procerum,
procerum,
procerum,
procerum,
procerum,
procerum,

. procerum,

prophase. 550X.

diakinesis. 550X.

metaphase I with 211. 830X.
telophase I with bridge. 830X.
metaphase II. 550X.

anaphase II. 550X.

tetrad with micronuclei. 550X.

34

 

 

 

35

Figure 4: Microsporogenesis in polyhaploids from the cross,

H, grocerum X H, vulgare cv. 'Larker'

a Plant #10-5; metaphase I with 111 + 191. 830X.

b Plant #10-5; metaphase I with 1111, 111, + 161. 830X.

c Plant #10—5; metaphase I with 411 + 131. 830X.

d Plant #11—1; metaphase 1 with 411 + 131. 830X.

e Plant #11-1; metaphase I with 511 + 111. 830X.

f Plant #ll-l; metaphase I with 611 + 91. 830X.

9 Plant #11-1; anaphase I with laggards, some showing
precocious division. 830X.

h Plant #10-5; anaphase I, tripolar showing 9-1-5
segregation. 660x.

i Plant #10-5; telophase 1 showing formation of 3 nuclei

and bridge following a tripolar segregation.

35

 

 

37

Figure 4: continued.

j Plant #10-5; tripolar telophase I. 830X.

k Plant #ll-l; three unequal-sized nuclei forming at
telophase I. 830X.

1 Plant #10-5; dyad with micronucleus and microcell.
660x.

m Plant #10-5; metaphase II in a triad, the result of a

tripolar first division. 660x.

n Plant #10-5; telophase II with bridges. 660x.

0 Plant #10-5; telophase II with laggards. 660x.

p Plant #11-1; tetrad with micronuclei. 830X.

q Plant #11-1; tetrad with microcell. 830X.

r Plant #11-1; tetrad stage with 6 cells. 830X.

38

 

 

 

Figure 5:

a Plant
b Plant
c Plant
d Plant
e Plant
f Plant
9 Plant
h Plant
i Plant

39

Microsporogenesis in H, procerum X. H, vulgare hybrids

#4-8;

metaphase I with 611 + 151. 660X.

#14-2; metaphase I with 271. 660X.

#4-2;

#1-4;

#4-2;
#4-2;

#4-8;
#4-2;

anaphase I with laggards and precocious
division. 660X.

dyad with cytomixis, micronuclear, and microcell.
830X.

telophase I with laggards. 830X.

tetrad with multiple nuclei and micronuclei.
660X.

tetrad stage with 6 cells. 830X.

tetrad stage with 8 cells. 830X.

#14-2; Pollen abnormalities. Note supernumerary

pollen pores in large irregular-shaped

pollen grain. 660X.

4o

 

DISCUSSION

 

There has been some confusion concerning the correct name for
Hordeum procerum. According to Bowden (1965) the species was first
described by Nevski (1941), but later independently described by

Covas (1950) under the name Hordeum hexaploidum. Reports published

 

prior to Bowden's article, eg. Rajhathy gt_gl, (1964) refer to the

material studied as H, hexaploidum. Later reports (Subrahmanyam, 1977)

 

refer to the material as H, procerum. The sample used in this study,
PI No. 266196, appears to fit the description of Covas (1950). Based
on this and the priority of the name under which the plant was first

described, PI No. 266196 is identified as Hordeum procerum Nevski.

 

One of the major stumbling blocks in an interspecific hybridiza-
tion program is the low success rate of the crosses. Since these are
usually hand pollinations, the production of even a few hybrids for
further study is an exhaustive, labor intensive, frequently expensive
endeavor. One of the original goals of this study was to attempt to
define those conditions which would yield higher numbers of hybrid
seedlings, with a minimum amount of effort. Comparing the results of
this limited study with those of a previous one provides a basis for
judging the success of these efforts.

Subrahmanyam (1977) was able to obtain seedlings from the cross
H, procerum (2n=6x=42) X H, vulgare (2n=2x=14). He reported a seed
set of 6.9%, and an embryo culture success rate of 51.9%; his overall

yield of seedlings per floret pollinated was .03. In the present

41

42

study, an overall seedling per floret yield of .36 can be calculated
(Table 1). This is a ten-fold improvement over the previous report.
The major difference seems to be in the percent seed set obtained in
this study, which averaged 71%; the embryo culture success rate was
also slightly higher, at 62%. What, specifically, is the cause of
these higher success rates?

The spike of H, procerum typically has in the neighborhood of 50
spikelets, which mature sequentially from top to bottom. On any given
day, only 10 or 20 of the florets may contain newly mature, receptive
stigmas which are prime candidates for fertilization. By selecting
only these florets, time wasted in emasculation is saved and unnecessary
eyestrain is avoided. This also results in a more favorable
nutritional status for the developing caryopses. Another problem
area is trying to get an ample amount of viable pollen onto receptive
stigmas. Pollination is assured by simply shaking massive amounts of
pollen on receptive stigmas.

The higher success rate of the embryo culture technique in the
present study can be directly attributed to the 17 embryos which were
forced to germinate on the B-5R medium. Similar cessation of germina-
tion on regular embryo culture media was reported by other workers
(Steidl, 1976; Morrison, gt_gl, 1959). The 17 recalcitrant embryos
transferred to the regeneration medium, B-5R, in this study all went
on to germinate completely and all plants which survived the growth

chamber malfunction and were transferred to the greenhouse have reached

43

maturity. Whether this was due to the difference in sugar concentration
(2% in B-5R vs. 5.1% in Norstog's) or to the presence of plant hormones
in the regeneration medium (GA3, IAA, Kinetin) is not known at this
time. Schooler (1960) reported on the use of gibberellins in embryo
cultures. It is known that gibberellins, auxins, and cytokinins have

a role in seed germination (van Overbeek, 1968). It is probable that
one or more of these substances may be lacking in those embryos which
fail to germinate completely, and the B-5R medium may compensate for
this deficiency.

It remains to be seen whether these procedures will produce
similar results when applied to other interspecific crosses in Hordeum.

Cytological examination of the plants produced by the interspeci-
fic cross hexaploid H, procerum X diploid H, vulgare indicates that
two basic types of plants resulted: polyhaploids with a stable
chromosome number of 2n=3x=21, and hybrids with variable chromosome
numbers which usually approach the tetraploid level (2n=23-29). This
agrees to some extent with the results reported by Subrahmanyam (1977)
for the same interspecific cross. He observed polyhaploids of
procerum and tetraploid hybrids, but he did not report any aneuploidy
or chromosome number variation in his hybrids.

The 10 polyhaploid plants closely resemble the hexaploids, with
striking similarities in morphology. No characters are apparent which
would indicate that H, vulgare has contributed in any way to their
genetic make-up. The hybrid plants, on the other hand, have morphology

44

which is intermediate between the two parents, as shown most clearly by
spikelet morphology. In the 'Beacon' hybrids (Table l), for example,
(Figure 2) the spikelets were sessile, a vulgare characteristic.

There are two possibilities by which such an interspecific cross
may result in a polyhaploid; either no fertilization takes place and
the egg develops parthenogenetically, or fertilization occurs but
chromosomes from the male parent are eliminated during embryogenesis.

The production of haploids following interspecific hybridization
has been extensively reported previously in Hordeum. The most notable
cross involves H, vulgare X H, bulbosum, which results in vulgare-like
plants (Symko, 1969; Kasha and Kao, 1970; Jensen, 1973). Using
genetic marker stocks, Subrahmanyam and Kasha (1973a) showed that these
plants did indeed include only vulgare chromosomes. Lange (1971)
suggested that either vulgare or bulbosum chromosomes were eliminated
during embryo development, but bulbosum chromosomes were eliminated
more frequently. Subrahmanyam and Kasha (1973b), however, showed that
double fertilization occurs, that there is no evidence of partheno-
genesis, and that selective elimination of bulbosum chromosomes leads
to haploid vulgare progeny. Bennett gt_gl, (1976) showed that elimina-
tion of bulbosum chromosomes is nearly complete by the fifth day after
fertilization.

The actual mechanism of chromosome elimination is still not known;
however, several hypothesis have been put forth. Asynchrony of mitotic

cell cycle times, (Gupta, 1969; Lange, 1971; Subrahmanyam and Kasha,

45

1973), and a "modification restriction system" (Davies, 1974) have
been proposed. Bennett gt_gl, (1976) showed that chromosomes of the
species with the longer mean cell generation time either failed to
congress at mitotic metaphase, or failed to move to one of the poles
at anaphase, and were eliminated.

Haploids of other Hordeum species have been produced following
interspecific hybridization (Rajhathy and Symko, 1974; Islam and
Sparrow, 1974; Subrahmanyam, 1977, 1979, 1980), including polyhaploids
of H, procerum (Subrahmanyam, 1977). Chromosome elimination was shown
to occur in a complex interspecific Hordeum hybrid by Orton and Tai
(1977), who proposed it may be due to the malfunctioning or disintegra-
tion of a proposed cytoplasmic organelle, the "spindle organizer
(Tai, 1970)", or to a complex interaction between chromosomes and
spindle-timing determinants. This theory seems consistent with the
conclusions of Bennett et_gl, (1976).

It is apparent that fertilization of the H, procerum sample used
in this study by H, vulgare is possible, since hybrid plants were
obtained using the cultivars 'Beacon' and 'Coho' as the pollen parent.
Chromosome elimination obviously occurs in these hybrids to some extent,
because the F1 plants have chromosome numbers which vary from 23-29.
It is therefore believed that H, procerum was fertilized by H, vulgare
cv. 'Larker'. Based on chromosome numbers and plant morphology, it is
concluded that the 21 chromosome plants are polyhaploids (trihaploids)

of maternal origin, and that the plants with higher chromosome numbers

46

are hybrids. In view of the overwhelming evidence that haploids are
produced by chromosome elimination in Hordeum following interspecific
hybridization, and in view of the fact that there is no evidence to
the contrary, it is concluded that the 21 chromosome polyhaploids are
the result of elimination of H, vulgare chromosomes during embryogenesis.

The appearnce of vulgare-like characters in all plants with more
than 21 chromosomes indicates that the additional chromosomes are of
paternal origin. The lack of aneuploidy in the polyhaploids indicates
that aneuploidy in the hybrids is due to loss of individual vulgare
chromosomes, and not as the result of aneuploid procerum egg cells.

The variation in chromosome numbers between hybrids, and between
tissues in the same plant, is apparently a further manifestation of
the elimination phenomenon. Similar variation has been reported for
H, vulgare X H, bulbosum hybrids (Humphreys, 1978), as well as in a
Nicotiana interspecific hybrid (Gupta and Gupta, 1973).

There were striking differences between barley cultivars used as
the male parent in crosses. All ten plants with 'Larker' as male had
a chromosome number of 2n=3x=21, and are polyhaploids. All 11 plants
with 'Coho' as male were hybrids with variable chromosome numbers,
with a mean in all cells studied of 26.9. If the exceptional plant
with 23 chromosomes, Number 4-2 (Table 2), is excluded, the mean
chromosome number for the Coho crosses is 27.4. All six plants with
'Beacon' as male were also hybrids, with variable chromosome numbers

and a mean of 27.4. What the data point out is a difference in

47

resistance to chromosome elimination by the procerum genome, which
resides in the vulgare genome. Such a difference would have to reside
in nuclear genes, since H, vulgare is the male parent and there should
be no effect of vulgare cytoplasm. Ho and Kasha (1975) have shown
that genes for elimination of H, bulbosum chromosomes are located on
chromosomes 2 and 3 of H, vulgare; these are offset by factors on the
bulbosum chromosomes which, when in sufficientdosage, prevent the
elimination of the bulbosum chromosomes. In this study, it appears
that such factors, conditioning resistance to elimination of vulgare
chromosomes by procerum, are either wholly lacking or are of insuffi-
cient dosage to be effective in 'Larker', as opposed to 'Coho' and
'Beacon'. No such differences between varieties were reported by
Subrahmanyam (1977); it is therefore possible that there is a genetic
difference between his procerum sample and PI No. 266196. Gene
segregation in his pollen-parent population is also a possibility. He
didn't use 'Larker' in his study, and he appeared to get polyhaploids
and hybrids from the same pollinating variety.

The inheritance of this resistance to elimination could be studied,
using trisomic stocks of 'Larker', if they were available, or simply
by looking at hybrids between 'Beacon' and 'Larker'. The obvious
morphological differences between plants (polyhaploids vs. hybrids)
would make scoring the results relatively simple. If 'Larker' does,
indeed, lack a genetic factor which resists chromosome elimination,

as suggested by this study, it would be a good choice as a pollen

48

parent for the production of polyhaploids of wild Hordeum species.

Meiotic studies of H, vulgare cv. 'Larker' confirm that it is a
regular diploid (Table 3), with 711 per cell and a negligible frequency
of abnormalities at late stages of division. Meiosis of H, procerum
(Table 3) reveals that it is a nearly regular hexaploid. Metaphase I
plates studied reveal a bivalent frequency of 19.6, which approaches
the expected value of 21 II. Frequencies for trivalent and quadri-
valent associations were 0.06 each. These are so low that they may be
insignificant in the interpretation of homology between genomes.

The meiotic studies done on the polyhaploids, however, provide
additional information about the genome relationships in H, procerum.
In these plants, there is a mean number of 1.9 bivalents per cell,
with a maximum of 6 11 observed in one cell. Further, 5 II were
observed in two cells, and 4 II were observed in 21 cells or 9% of the
total. Thus the maximum number of bivalents observed is six, which
comes very close to the basic number for the genus of x=7. Based on
this observation, the maximum association in the polyhaploids is 711
+ 71. Since it was established that the polyhaploids derived all
three genomes of chromosomes from procerum in the previous section, it
thus appears that there is a degree of homology between two of the
three genomes present in H, procerum. Since the metaphase I chromosome
pairing in these plants consisted mostly of rod bivalents, with only
a few ring bivalents, it seems likely that the two homologous genomes

are similar, but not identical. With this in mind, a preliminary

49

genome formula for H, procerum would be AAA'A'BB; thus, H, procerum
should be considered a segmental autoallohexaploid.

This conclusion may seem incongruous with the observation that
multivalents were at very low frequencies in the hexaploid H, procerum.
This is explained by either of two possibilities. It was very diffi-
cult to obtain well-spread metaphase 1 cells when working with PMC's
from the hexaploid. It is thus possible that multivalent formation
occurs more frequently than observed in this study, but such cells
weren't interpretable due to the resulting congested nature of the cell
plate. The other possibility is that there is genetic control of
chromosome pairing, which suppresses the pairing of homoeologous
chromosomes in the hexaploid. Genetic influence on chromosome pairing
has been well-documented in hexaploid wheat (Riley and Chapman, 1958;
Feldman gt_gl,, 1966) and reported in other Hordeum species (Rajhathy
gt_gl,, 1964). Starks and Tai (1974) proposed a system for control of
pairing in Hordeum jubatum: "When a single dose of this gene (or a
group of genes) is present, homoeologous pairing may prevail. When a
double dose is present, only homologous chromosomes may pair." This
same system appears to be operative in H, procerum. Previous studies

of H, procerum (mistakenly referred to as H, hexaploidum Covas) have

 

indicated that the two genomes of H, jubatum are included in H, procerum,
based on karyomorphology (Rajhathy and Morrison, 1961; Rajhathy gt_gl,,
1964). It is thus logical to assume that the same genetic system

proposed for H, jubatum by Starks and Tai (1974) is also present in

50

H, procerum. The consequences of this should be considered in future
cytogenetic studies in Hordeum, since a hybrid between H, procerum and
a diploid species should be expected to show a maximum of 711 if there
is no homology, and maximum of 1411 if there was complete homology
between the genome of the diploid and the B genome of procerum.

This same situation, that is, a hybrid between hexaploid H, 2592937
gm and diploid H, vulgare, is in question in this study. If there is
no homology between the genomes of the two parents, the hybrids should
exhibit a maximum number of 711, which coincides with the maximum
number in the polyhaploids.

The mean frequency of pairing in the five hybrids studied was
2.1 II vs. a mean of 1.9 II in the polyhaploids, a difference of only
.2. More importantly, the maximum of 7 II was observed in one plant,
and the numbers of bivalents in all five plants clearly approach 711,
the maximum number in the polyhaploids. Thus, the conclusion that

there is no homology between the genomes of H, procerum and H, vulgare.

APPENDICES

51

Appendix A

Formula for Embryo Culture Medium

mg/liter
KH2P04 910
KCl 750
MgSO4-7H20 740
CaC12-2H20 740
MnSO4-H20 3.0
H3803 0.5
ZnSO4-7H20 0.5
'CoC12-6H20 0.025
CuSO4-5H20 0.025
Na2M004 0.025
Fe-EDTA 25.0
Inositol (meso) 50.0
Thiamine-HC1 0.25
Pyridoxine-HCl 0.25
Ca-pantothenate 0.25
Casein hydrolysate (enzymatic) 2500.0
Malic acid 1000.0

g/liter

Agar 10
Sucrose 51.3
pH 5.0

Appendix 8

Formula for B-5R Medium

Major

CaC12-2H20
MgSO4-7H20
(NH4)2$04
NaH2P04-H20

Minor

KI

H3803
MnSO4-H20
ZnSO4-7H20
vNa2M004-2H20
CUSO4'5H20
CoC12-6H20
Nag-EDTA
FeSO4-7H20
Inositol
Nicotinic Acid
Pyridoxine HCl
Thiamine HCl
Kinetin

6A3

IAA

Sucrose
Glucose

Agar

mg/liter

2500
150
250
134
150

100

10.

9/1
10
10

1O

mowooo

.75

.25
.025
.025

BIBLIOGRAPHY

BIBLIOGRAPHY

Bennett, M. 0., R. A. Finch and I. R. Barclay. 1976. The time rate
and mechanisms of chromosome elimination in Hordeum hybrids.
Chromosoma (Berlin) 54: 175-200.

Bowden, W. M. 1965. Cytotaxonomy of the Eurasiatic and South American
species of the barley genus, Hordeum L. Can. J. Genet. Cytol.
7: 394-399.

Covas, G. 1950. Nueva especie de Hordeum de la flora Argentina. Rev.
Argentina Agron. 17: 230-232.

Davis, D. R. 1974. Chromosome elimination in inter-specific hybrids.
Heredity 32: 267-270.

Feldman, M. 1966. The effect of chromosomes 58, 5D, and SA on
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