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This is to certify that the

. ;thesis entitled

Hybridization of Barley (Hordeum Vulgare L.
Emend. Lam.) with Its Wild Relatives.

 

presented by
Robért‘Parkef Steidl
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hasbeen. accepted towards fulfillment
of the requirements for

Ph .0. degree in Wee

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Major profesfl

 

Date (3/0/7é

0-7639

   
   
   

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ABSTRACT

HYBRIDIZATION OF BARLEY (HORDEUM VULGARE L.
EMEND. LAM.) WITH ITS WILD RELATIVES

 

BY

Robert Parker Steidl

Hybrids with Hordeum jubatum L. (4x) and the a.

 

gubatum amphiploids g. jubatum X g. compressum Griseb.

(6x) and Agropyron sericeum Hitchc. X g. jubatum (8x) were

 

made to transfer to barley (fl. vulgare L. emend. Lam.)
economically useful improvements from g. jubatum in these
traits: winterhardiness, resistance to the cereal leaf

beetle (Oulema melanopus L.), tolerance to salty soils

 

and tolerance to wet soil conditions. To transfer these
traits, attempts were made to produce an intermating pop-
ulation containing the desired variation from the wild
species, expressed in the genetic background of commercial
barley. Recurrent selection and intermating could be
practiced in this population until transfer of the desired

variation was achieved.

Robert Parker Steidl

Chromosome elimination and cytoplasm from‘fi. lu-
batum were both instrumental in blocking production of
this intermating population by standard crossing methods.
Cytoplasm from g. jubatum conferred gross lack of vigor
and male sterility to g. vulgare derivative lines. Chrom-
osome elimination greatly reduced the frequency of produc-
tion of certain genomic constitution hybrids. Chromosome
elimination also was shown to be the most logical explana—
tion for the complete loss of wild chromosomes in second
5. vulgare backcross progenies of (HJ X HC)(6x) X VV.
Thus, it appeared chromosome elimination partially to
completely blocked production of certain genomic consti-
tution hybrids and backcross progenies. Problems with
the g. jubatum cytoplasm were circumvented by making the
hybrids with E. vulgare as female.

A different approach was undertaken, utilizing
chromosome elimination in conjunction with callus culture
and other techniques to produce the desired parental ma-
terial for the intermating population. This would either
substitute for or be a part of the more traditional back-

cross method of producing the desired parental material.

Robert Parker Steidl

This approach involved several intervening steps
with the eventual production of a population consisting
of plants with different chromosome number and chromosome
constitution. Then, hopefully, selection of the desired
parental material could be made. If not, the most promis-
ing material could again be run through the intervening
steps of callusing, subculturing and regenerating large
numbers of plants to eventually produce the desired
parental material.

All intervening steps were worked out so that pro-
duction of large numbers of plants by callus culture of
one hybrid have already been achieved. Thus, I am confi-
dent that by taking advantage of chromosome elimination,
techniques including tissue culture can eventually produce

the desired parental material.

HYBRIDIZATION OF HARLEY (HORDEUM VULGARE L.

EMEND. LAM.) WITH ITS WILD RELATIVES

BY

Robert Parker Steidl

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1976

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to my
co-advisors Dr. J. E. Grafius and Dr. D. H. Smith for
their encouragement and guidance throughout the course
of this investigation and for their critical review of
the manuscript. My deep appreciation is also extended
to Dr. Carter M. Harrison for his critical review of the
manuscript.

My deepest thanks go to my fellow graduate stu-
dents Gil Starks, Lynn Murry, Rye Ho Huang, and Tom Orton
for their suggestions and collaboration on this research
problem and to their major professors William Tai and
Peter Carlson.

A special thanks to a fellow graduate student,
Barb Steidl (my wife), for her assistance in review and

typing and for her understanding.

ii

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . . . . .

LIST OF FIGURES O O O O O O O O O O O O O O O 0

INTRODUCTION 0 O O O O O O O O O O O O O O O O O

LITEMTUE “VIEW 0 O O O O O O O O O O O O O 0

MATERIALS AND METHODS . . . . . . . . . . . . .

I.

II.

III.

IV.

V.

RESULTS

I.

II.

III.

PLANT MATERIALS. . . . . . . . . . . .
METHODS OF HYBRIDIZATION . . . . . . .
EMBRYO CULTURE MEDIA . . . . . . . . .
EMBRYO CULTURE . . . . . . . . . . . .
CHEMICAL TREATMENT OF PLANT MATERIAL .
AND DISCUSSION. . . . . . . . . . . . .
CROSSES MADE BY CHUNG LEE. . . . . . .

SEQUENCE AND OBJECTIVES OF CROSSES AND
COLCHICINE TREATMENT . . . . . . . .

ICOLCHICINE TREATMENT OF PLANT MATERIAL
CROSSING AND PROGENY PRODUCED. . . . .

HYBRID SEED DEVELOPMENT. . . . . . . .

iii

Page

vi

15

15

15

22

25

27

31

31

42

52

55

77

TABLE OF CONTENTS (cont'd.) Page

VI. CHROMOSOME ELIMINATION AND EVIDENCE FOR
THE E. VULGARE BACKCROSS OF (HJ X HC)
(6x) X VV AND THE PENTAPLOID HYBRID FROM
(HJ X HC)(6X) X VVVV HAVING THE SAME
BASIC GENOMIC CONSTITUTION. . . . . . . 81

VII. BREEDING IMPLICATIONS OF THE HYPOTHESIS
THAT THE Bl.2 HAS A PENTAPLOID

CONSTITUTION. . . . . . . . . . . . . . _93
FUTURE DIRECTION O O O O O O O O O O O O O O O O O 98
LITERATURE CITED 0 O O O 0 O O O O O O O O O O O O 101
APPENDIX 0 O O O C O O O C O C O O O C O O O O O O 109

iv

LIST OF TABLES

Table Page
1. The designation and source of plants. . . . . 16
2. Crosses made by Chung Lee . . . . . . . . . . 32

3. Designation of all crosses attempted and

mature progenies produced. . . . . . . . . 45
4. Order in which crosses were made. . . . . . . 47
5. Colchicine treatment. . . . . . . . . . . . . 48
6. Stability of hybrid progeny . . . . . . . . . ' 65

7. Starks' metaphase I counts of progeny from
(HJ x HC) (6X) x W o o o o o o o o o o o 68

8. 'Starks' metaphase I counts of the pentaploid
and B102 0 O O I O O O O O O O O O O I I O 69

9. Orton's preliminary embryo squash counts of
(HJ X HC)(6x) X VVVV . . . . . . . . . . . 34
Appendix
A. Norstog's agar medium II. . . . . . . . . . . 109

B. Wick embryo culture medium. . . . . . . . . . llO

LIST OF FIGURES

Figure
1. 32.2 spike, crossing technique, B2.2 seed
and flat with plants. . . . . . . . . . . .
2. Embryo culture . . . . . . . . . . . . . . . .
3. Spike morphology . . . . . . . . . . . . . . .

4. Parthenocarpic fruit and other cross results .

vi

Page

21

24

35

6O

INTRODUCTION

Varietal improvement can only be achieved by
utilizing useful genetic variability. This variability
can be obtained from induced mutations or by introgres-
sion within or between biological species. Induced muta-
tions have provided only a relatively minor portion of
varietal improvements.

Variability within a crop's biological species is
usually readily transferable. Transfer of a single gene
into the desired genetic background is most easily
achieved by the backcross method of plant breeding.
Transfer oftquantitatively inherited improvements is much
more effectively achieved by recurrent selection and in-
termating. If the appropriate genetic background is par-
tially fixed by one or two backcrosses, then selection
can be mostly limited to the desired trait for transfer.

The basic approach is the same for transfer from
related species. That is, transfer of a single gene is

usually most easily achieved by the backcross method while

transfer of a quantitatively inherited improvement is
usually most successfully achieved by selection in an
intermating population containing the desired trait ex-
pressed in the crop's genetic background. Plants with
the desired chromosomes or translocations from the donor
parent can be more easily recognized during the selection
phase of cyclic intermating and fertility is generally
high enough so the next cycle can be produced.

The greatest difference in transfer of a quanti-
tatively inherited improvement from a related Species as
opposed to the same species involves problems in obtain-
ing the desired intermating population. These problems
include everything from greater sterility and lower re-
combination in hybrid* and backcross generations to com-
pletely unexpected phenomena operating in the material.
Cytogenetics can be an extremely useful tool in coming
to an understanding of these phenomena and in determining
the best approach to deal with them.

Ease of transfer from a related species is deter-

mined by the closeness of species relationship and the

 

*Throughout this thesis, the term "hybrid" refers to a
species hybrid.

ploidy level of the recipient species. Closely related
species have greater chromosomal pairing and more similar
functions of corresponding genes. Thus recombinant chrom—
osomes will tend to produce changes in a smaller number of
gene products and the changes will result in smaller devi-
ations in enzymatic products. Slight deviations in pro-
duction of enzymatic products for relatively few genes
usually result in relatively minor imbalances in gametes
and zygotes. Polyploids from closely related species
have repeated copies (one copy per chromosome set) of
eSSentially the same gene. Thus, in polyploids from
Clo sely related parents chromosome changes such as addi-
tion, substitution and recombination will result in
Smaller changes in production of enzymatic products.
Diploids are generally weakly buffered. The vast
majority of genes producing essential enzymes are not re-
peated throughout the genome. Thus each enzyme and its
corresponding gene must perform its function at the cor-
1'th rate for the biological system. Translocation of a
non<=orresponding chromosome piece,when fixed, results in
esaentially four copies of some genes and no copies of

other genes. This type of translocation has little chance

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of survival because it results in biologically profound
changes in the enzymatic rates of all enzymes involved.
Transfer of a short and corresponding chromosome piece

will hopefully be within the buffering capacity of the

diploid species.

Transfer of traits requires production of hybrids
and subsequent generations with transfer at some point.
Many hybrids between species of the Triticeae (Triticum,
Aegilops, Hordeum, Secale, . . . ) have been produced.
Generally these hybrids were relatively easily obtained.
But production of specific combinations can not be effec-
tively predicted, because phenomena such as chromosome
elimination, ploidy level and inhibition of fertilization
determine ease of production of certain hybrids. Thus
certain combinations can be difficult to obtain.

Dr. J. E. Grafius chose to use a g, jubatum-g.
compressum amphiploid, which was generously supplied by
Dr. A. B. Schooler, as the donating parent of g. jubatum
traits to g. vulgare (Schooler, 33 al., 1966). §.'jubatum
a perennial tetraploid species, is native to much of the
North American continent. g. compressum is a perennial

diploid species native to South America.

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The main objective of this work was to transfer
useful levels of variability from g. jubatum for any of
the following characters: winterhardiness, resistance to
cereal leaf beetle, tolerance to salty soils, and toler-
ance to wet soil conditions. g. jubatum has very high
levels of useful improvements for all these traits. Al-
though extensive efforts have been made to find good re-
sistance to the cereal leaf beetle within g. vulgare,
only moderate levels have been found. Useful levels of
variability for salt tolerance do exist, but are only
moderate when compared with the tolerance of g. jubatum.

Secondary aspects of this study deal with various
problems and techniques arising from attempts to transfer

improvements from g. jubatmm.

LITERATURE REVIEW

Transfer of genes is the major objective of this
study, therefore it is important that we understand the
most important factors controlling ease of transfer.
These factors are ploidy level of the recipient parent
and closeness of species relationship. Their importance
to gene transfer can be better understood by thinking of
their effect on the buffering capacity of a Species (Har-
lan, 1966). Closely related species generally have rea-
sonable levels of pairing and correSponding genes on the
chromosomes perform the same function at essentially the
same rate. Thus, transfer in‘closely related species
generally involves a very small translocation which per-
forms essentially the same functions at the same rates.

When the parents of a polyploid are closely re-
lated, there are repeated copies (one copy per chromosome
set) of essentially the same gene. Thus polyploids from
closely related parents are much more buffered against

biologically important changes in enzymatic rates.

Hexaploid wheat is a beautiful example of the
greater buffering capacity which polyploidy confers.

Comparison of hexaploid wheat, Triticum aestivum, with

 

its diploid progenitors T. monococcum (AA), Aegilops

 

speltoides (BB), and A. squarrosa (DD) and its inter-

 

 

mediate progenitor, tetraploid T. turgidum (AABB) pro-
vides an Opportunity to compare the buffering capacity
of polyploid levels of this polyploid series.

All members of this series have only bivalent
formation due to genetic suppression of homoeologous
pairing. Suppression or removal of SBL (gene causing
the greatest suppression of homoeologous pairing) allows
considerable multivalent formation at the tetraploid
and hexaploid levels (Riley and Kempanna, 1963). Species
crosses at the diploid level when SBL is either not pres-
ent or suppressed have considerable bivalent formation.
Based on pairing data, these species are relatively
closely related.

Chromosomes of these three genomes (AA, BB, and
DD) can be grouped into seven sets of three chromosome
pairs. The degree to which one of the three homoeologous

chromosome pairs can compensate for another pair of the

homoeologous group also indicates the degree of chromosome
relation. If considerable compensation occurs, then the
homoeologous chromosomes have largely the same genetic
content.

Lilienfeld (1951) showed substitution did not
occur at the diploid level between the A and B genomes.
Substitution lines at the tetraploid level were difficult
to obtain and there was good reason to assume many of the
substitution chromosomes were actually recombinant chrom-
osomes (Sears, 1969).

At the hexaploid level there was enough buffering
capacity that the compensation potential of all 42 pos-
sible homoeologous substitution lines (e.g. 1A tetrasomic,
1D nullisomic) could be observed (Sears, 1966). Compen-
sation was measured by comparing the substitution line
with its correSponding nullisomic line. Compensation
ranged from some restoration of vigor but no restoration
of fertility, to near normal size, vigor and fertility of
all six homoeologous substitution lines of group III.

Although pairing configurations showed these

species were fairly closely related, they diverged to

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the extent that considerable buffering was needed to
allow reasonably easy chromosome substitution.

A more dramatic expression of the substitution
potential of hexaploid wheat was demonstrated by the nu-
merous intergeneric substitution lines. Disomic substi-
tutions of rye homoeologous groups 1, 2, 3, 5, and 6
chromosomes were made for part or all of their correspond-
ing homoeologouschromosomes (Sears, 1968; O'Mara, 1947;
Riley, 1965; Lee, etpal., 1969; Mettin, 23.31., 1973).
Compensation was generally good. The commercial varie-
ties ‘Saladin,’ 'Orlando,‘ 'St 2153/63,‘ 'Zorba' and
'Salzmunder' are lR/lB substitution lines (Zeller, 1973).
1R imparts high resistance to rust and moderate resis-
tance to powdery mildew.

Disomic substitution of Agrqpyron elongatum homo—
eologous chromosome groups 1, 3, 4, 5, 6 and 7 have been
produced (Sharma and Knott, 1966; Larson and Atkinson,
1972; and Dvorak and Sosulski, 1974). Compensation was
very good. Disomic substitution lines with several other
species have been produced.

Monosomic and disomic addition or substitution

lines are very useful in transferring traits because

10

they provide continued Opportunity for producing recombinant
chromosomes. Most of the early transfers also utilized ir-
radiation to break chromosomes. Transfers utilizing irradia-

tion include leaf rust resistance from Aegilops umbellulata

 

(Sears, 1956), stem rust resistance from Agropyron elongatum

 

(Knott, 1961), leaf rust resistance from Agropyron elongatum

\

 

(Sharma and Knott, 1966), leaf rust and powdery mildew resis-
tance from rye (Driscoll and Jensen, 1963), leaf rust resis-

tance from Agropyron intermedium Wienhues, 1966), and leaf

 

rust resistance from rye (Mukade, at 31., 1970). All of
these are well documented.

Transfer has also been achieved from rye by natural
translocations (Driscoll and Sears, 1963; and Mettin, 23 31.,
1973). These translocations involve 4A/2R and lB/lR. Nat-
ural breakage and transfer is the most likely explanation
for both of these lines, because wheat and rye chromosomes
do not appear to pair even with SBL suppressed (Riley and
Kimber, 1966). Furthermore, most of the chromosomal tranST
locations in the literature appear to be of this type.
Larsen (1973) showed that of the tWenty reported chromo—
somal translocations in wheat only three involved homoeolo-

gous chromosomes. Thus stable interspecific addition and

11

substitution lines can be very useful in obtaining the
desired transfers even without any appreciable pairing.
Recently, transfer from species with some pairing
affinity with wheat has been very successfully achieved
by interfering with the genetic control over homoeologous
pairing. Riley (1968) used genetic suppression of SBL to

transfer yellow rust resistance of Aegilops comosa to

 

wheat. Athwal and Kimber (1972) showed genetic suppres—
sion of SBL could be used to transfer traits from Aegilops

umbellulata. Sears (1973) used nullisomic SB plants to

 

transfer leaf rust resistance from Agropyron elongatum
chromosomes to their homoeologous wheat chromosomes.
Many 3Ag/3D and 7Ag/7D transfer lines were produced. For
both translocation types, male transmission was excellent
for some lines. Pairing of 3Ag with 3D and of 7Ag with
7D was about 30% and a corresponding percentage of the
progenies had translocations.

These results are so spectacular that it would
seem reasonable to conclude transfer from many other more
closely related species could be just as easily achieved

by utilizing similar methods.

12

Hexaploid wheat's highly buffered genetic system

can handle considerable genetic manipulation. Thus trans-

fer from species like rye, with essentially no pairing

homology, is possible. The weak buffering of diploids

is demonstrated by the failure of fairly extensive

attempts to transfer traits from wheat to rye (Deorikar,

l 9 6 3) .
Attempts to transfer traits to H. vulgare is

another good example of fairly extensive efforts to trans-

fer traits to a diploid species. No properly verified

examples of transfer to Ii. vulgare exist in the litera-
ture. The only possible examples of transfer are from E.

W (Hamilton, 5-33 §_1_., 1955) and from g. bulbosum

(Schooler, 1964) . Transfer of improvements in any one of

Se\reral traits to g. vulgare could result in substantial

improvement in overall productivity of the crop.

Many of the previous attempts to transfer improve-

meI'lts to g. vulgare have been made with g. jubatum as the

doIlor parent. The main objective of these previous

WOt'kers was to transfer improved levels of winterhardi-

he as from g. jubatum. g. jubatum has excellent winter-

haII:'diness. Transfer of winterhardiness would result in

13

a large increase in overall productivity of this crop,
because a winter hardy barley can utilize considerably
more of the environmental resources than spring barley.
Improvements in winterhardiness within _I_I_. vulgare have
plateaued and little progress has been achieved in the
last 10 years. (Personal communication, Dr. J. E.
Grafius)

The B. jubatum-Ii. vulgare cross was first made by

Q11 incke (1940) . Since then several researchers—~Morrison,
$ 3;. (1959), Vinogradova (1946, cited in Smith, 1951,
and Price, 1968), and Kerber (unpublished)-—have success-
fully repeated this cross, but none have gotten beyond the
high levels of F1 hybrid sterility. Cytogenetic results
of Kerber's cross were cited by Wagenaar (1960) .

My crosses all have 3. jubatum or a g. jubatum
a1TlE>hiploid as one of the parents. 5. jubatum, like wheat,
is a member of a polyploid series (Rajhathy and Morrison,
19 61) . Relatively little work has been done on this poly-
ploid series. When using polyploids either as the recip-
ient or donor species, the possibility of genetic control
over homoeologous pairing must be seriously considered.

MI1:1:‘ry (1975) found essentially no pairing in the hybrid

14

from the cross diploid _Ii. vulgare X g. ju’bat’um. This

could be caused by either genetic control over pairing,

no homology, or physiological problems inherent in this

hybrid.
Some data exist on the pairing homology of the

Species in the g. .jubatum amphiploids I used. Starks and

Tai (1974) found It. compressum has some homology with g.

jl—lbatum. _Ii. jubatum also has some homology with one of

the genomes of Agropyron sericeum (Murry, 1975) . The

on 13; species reported to have some pairing with the bio-

l‘Dgical species 31. vulgare is _I_I_. bulbosum (Kasha and

Sadasivaiah, 1971) .

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MATERIALS AND METHODS

I . PLANT MATERIALS

The stock of the Hordeum jubatum X _}_I_. compressum

 

amphiploid was obatained from Dr. A. B. Schooler. The

natural hybrids AgrOpyron trachycaulum X g. jubatum and
A. sericeum X g. jubatum were collected in Alaska by Dr.
J- E. Grafius with help from Dr. W. W. Mitchell and Dr.

R- Taylor of the Alaska Experiment Station Staff (Table 1).

II. minions OF HYBRIDIZATION

Good growing conditions must be maintained. Hy-
brids are much more successfully produced from vigorously
QrOWing parental material. Frequent repotting and high
fertility levels are very helpful in maintaining good
Vigor, The most vigorous growth was obtained with day-
time temperatures between 15C and 29C. Hybrid material
in E- jubatum cytoplasm required warmer conditions (gen-

erally between 22C and 29C) for most vigorous growth.

15

TABLE 1 .

16

The designation and source of plants.

 

PLANT

DE S IGNAT ION.

SOURCE

 

Hordeum lubatum X
g. compressum
amphiploid

Wren trachycaulum X
_11- jubatum (spontaneous
hybrid)

A. sericeum X g. jubatum
(spontaneous hybrid)

Hordeum j ubatum

5.9m vulgare
(tetraploid)

m vulgare
(diPIOid)

(HJ x HC)(6x)

ATX HJ

AS X HJ

HJ

Selfed seed from Dr. A. B.
Schooler, North Dakota
State U., Fargo, N.D.

Clone from Dr. W. W.
Mitchell, Palmer, Alaska

Clone from Dr. W. W.
Mitchell, Palmer, Alaska

Seed from Dr. W. W.
Mitchell and Dr. R.
Taylor, Palmer, Alaska

A. B. Schooler, North
Dakota State U., Fargo,
North Dakota

Several varieties, bulks
and populations from
Dr. J. E. Grafius, Michi-
gan State University,
East Lansing, Michigan

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In some crosses temperatures above 26C were detrimental
to good hybrid seed development. When using I_I_. m
as the male parent, night temperatures of 15C or below
were necessary to allow the spike to protrude from the
boot before the pollen was shed.
Procedure Used When Female
Is Male Sterile

1. Healthy heads of the female parent with some
open florets were selected. Florets were clipped, remov—
ing 1/3 to 1/2 of the lemma and palea. Several heads were
then grouped together and loosely wrapped with aluminum
foil. If possible, the flag leaves were kept outside the
aluminum foil; otherwise, the flag leaves were removed.
When necessary, the head wrapped in aluminum foil was sup-
PortEd with an iron rod.

2(a) . When _I-I. ‘12.];9252. was used as the male parent,
Spikes with undehisced yellow anthers were selected. Flor—
ets Were clipped, removing 1/3 of the lemma and palea, and
crosses made by the approach method (Curtis and Croy, 1958)
or the anthers were allowed to fully extrude and pollen was
disseminated by gently tapping the spike of the male parent

above the female .

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18

2(b). When wild species were used as the male
parent, spikes with freshly extruded anthers were gently
tapped above the female. This required luck in guessing
when anther extrusion would occur. Clipping of florets
to cause anther extrusion met with little success.
Abundant fresh pollen was obtained by cutting spikes with
a few extruded anthers and storing with stems in water at
5C for two days. After storage, spikes were transferred
to the greenhouse in a cold (5C) box. Rapid anther extru-
sion occurred after removal of spikes from the box.

3. Greatest success was achieved when plants were
P°1linated with massive amounts of pollen for two or three
daYs- Good success in producing hybrid or backcross seed
required much higher pollen loads than species selfing.
migres Used When the Female
\e Fertile

1. Healthy spikes, which were to be used as the
female, with yellow-green anthers were selected. The
older the spike, the greater the chance of selfing, but
emas<21.11ation and drying out caused much more injury to
y("inger spikes. Spikes with pollen shed during emascula-

tlon or having several obvious selfed seeds were usually

its: 2
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19

discarded. This precaution and the obvious morphological
differences between interspecific hybrid and selfed seed
helped avoid mistaking a selfed seed for a hybrid seed
during embryo transfer.

In all the species crosses, selfed seed had plenty
of starch deposition before endosperm collapse occurred in
hybrid seed. At this time, hybrid seeds usually took on a
shrunken or thinner appearance from reduced starch deposi-
tion. Hybrid seed with H. vulgare as the female remained
Plump with a watery sac-like endosperm with little or no
StarCh deposition.

2. Each floret was gently opened by holding the
tip Of the palea with the thumb and using tweezers to pull
open the lemma. Anthers were then removed. Usually the
bottom and top spikelets were too immature and were re-
moved. In material with more than one floret per spike-
let, e.g. (AS X HJ) (8x) , all florets past the second were
remOVed. |

3. Crosses were made when the female stigma was
featihery (Figure 1B) . Sometimes this was the same day as
ema~'°'<=ulation. Florets either opened on their own or were

gently opened before pollination.

20

FIGURE l.--BZ.2 spike, crossing technique,
B2.2 seed and flat with plants.

A. 32.2 plant with approximately 40%
self-fertility

B. Emasculated spike of (HJ X HC)(6x)
with open receptive florets

C. Crossed spikes wrapped in aluminum foil
D. B2.2 seed

E. Flat of colchicine treated 69-101

H oudmflm

 

'1”-
ill:

Hr?

III

22

III. EMBRYO CULTURE MEDIA

Embryo culture should allow nOrmal embryonic
growth and differentiation; then normal germination should
occur. Normal development and germination are two separ-
ate stages. At present, the Optimum media for normal de-
velopment of the two stages is quite different. I had no
particular success with transferring embryos to a second
medium to Optimize development in both stages. Thus the
best results were Obtained by using a wick medium system*
for well differentiated embryos and Norstog's Medium II
(Norstog, 1973) for poorly differentiated embryos (See
Tables A and B Of the appendix for a complete listing of
chemicals in these media.) Embryos near completion of de-
velopment (having a well-developed scutellum) were very
successfully grown on a wick medium (Figure ZD), while
embryos with poor development could only be successfully
grown to mature plants on Norstog's Medium II. Even Nor—
stog's medium resulted in a low production of mature
plants from poorly differentiated embryos. NO mature
plants were produced by embryos lacking in differentia-

-tion (proembryos). Both media were moderately successful

 

*Wick.medium system is hereafter referred to as wick medium.

23

FIGURE 2.--Embryo culture.

Chung Lee's material on wick medium
with cotton stOpper wrapped in alum-
inum foil.

Chung Lee's transfer technique;
1/2 caryopsis placed on wick medium.

Seedling Of [(HJ X HC)(6x) X HJ] X VVVV
near death.

82.2 seedling on wick medium.

HJ X VV seedling with tillers, auricles
and roots on agar medium.

- an: Ewan-1... __:...;-

'_. . - _ . .___.

 

“e“‘fir‘an -.“—_—.A_- "‘

is. .4... _._"r—."_-

 

'

24

N wusmwm

 

It“
:4

7n
1 l

()

(J

x

25

in the production of mature plants from embryos with in-

termediate development .

IV. EMBRYO CULTURE

Embryos were dissected from the seeds after col-
lapse of the endosperm occurred. This stage was deter-
mined by noting when the caryopsis changed from green to
white or yellow. The best success was achieved by waiting
until this stage, because of better development of the
embryo. The only exceptions were caused by dormancy and
chromosome elimination for the second _H_. vulgare backcross
of (HJ X HC) (6x) X W and W X (AS X HJ) (8x) , respectively.
In the case of the second H. vulgare backcross, embryo
dormancy occurred after normal starch deposition began
(early dough stage) - I

The procedure of excising the embryo and transfer-
ring to media was as follows:

1. The transfer hood was turned on and allowed to run
for several minutes. The transfer area was sponged down
with 95% alcohol. Tweezers and dissecting needles were

flame-sterilized before transfer of each embryo.

26

2. Seeds were surface sterilized by immersing for 2
minutes in a 20% Clorox solution. When possible, the
lemma and palea were removed before sterilization. If
not, they were removed in the first step of dissection
Of the embryo.

3. The embryo was dissected out under a dissecting
microscope with a tweezer in one hand and a dissecting
needle in the other. The embryo is at the basal end
of the seed on the lemma side. Thus the most effective
method of dissection was to hold the distal half of the
seed in the tweezers with the lemma side facing up and
carefully slit the lateral side of the seed, starting in
the middle and working toward the basal end. The embryo
would usually then pOp out. If it did not, it could be
easily found just below the seed coat at the basal tip
Of the seed on the lemma side. The location of the embryo
and its distinctive appearance make even very small em-
bryos easy to find after a little practice.

4. The excised embryo was then transferred to the
Culture medium. This required flaming the culture bottle

IMNIth after removing the foam stopper and again just be—

fore reinserting the stopper. To transfer onto wick

27

medium required careful orientation of the embryo with the
scutellum facing the surface of the filter paper and the
radicle oriented downward. Failure to orient the radicle
correctly can be corrected later, but incorrect orienta—
tion of the scutellum is fatal. On agar media, either

the embryo was placed on the medium surface and oriented
with the scutellum facing down or the embryo was submerged

slightly and orientation ignored.

V. CHEMICAL TREATMENT OF
PLANT MATERIAL

 

Colchicine Treatment

Due to the high levels of hybrid sterility, use of
chromosome doubling agents is common in interspecific
crossing programs. For doubling purposes I chose to use
nine different colchicine treatments. Treatments one and
two were the method Wellensiek (1947) used and a modifica-
tion of it, respectively. (1) Near single-tiller divi-
sions were alternately immersed (dipped) in a .05% colchi-
Cine solution during daytime and in water at night for
four consecutive days; and (2) several consecutive daily

sPrayings of leaves of vigorously growing clones were

28

done using a .05% colchicine solution with 4% DMSO (di-
methyl sulfoxide, a surfactant).

Treatments three through nine were all modifica-
tions of POpe and Love's (1952) treatment. In these
treatments washed tiller divisions were dipped in .5%
colchicine. Plants were wounded by making a short slit
in the leaf base near the meristem with a dissecting

needle. These treatments were:

 

 

TREATMENT TIME
(3) nonwounded root dip 6 hours
(4) wounded root dip 6 hours
(5) nonwounded root dip 12 hours
(6) wounded root dip 12 hours
(7) horizontal treatment on 7 hours

paper towels with roots
protected from colchicine

(8) wounded horizontal treatment 7 hours
(9) inverted plant dip with roots 12 hours
protected from drying with
wet paper towels
After colchicine treatment, divisions were planted
in flats at the rate of approximately 200 per flat in '

lirree or four rows per flat (Figure 1E). Many treatments

resulted in 50% mortality. A few hybrid seedlings were

.K
is

also
'n'lC
.o..
fen

I
‘

t

an.

n .l K

\n h \, nu
-

, §

in
tall

29

also colchicine treated using Pope and Love's method. The
wick embryo culture medium was poured Off at the two leaf
stage and .05% colchicine solution added to the test tube.
Seedlings were treated for 8 hours, the colchicine solu-
tion was carefully washed off, and the seedlings trans-

ferred to soil.

g-Amino Caprioic Acid
(EACA) Treatment

EACA is a relatively cheap, highly water—soluble
animal-effective immunosuppressant. Bates, 23.. a_1_._., (1974)
concluded EACA appeared to be the most effective of four
immunosuppressants tested at blocking a hypothetically
Similar immuno-system in plants. The immunosuppressants
were injected daily into the leaf enclosing the spike
Starting before meiosis and ending with emasculation.

In a personal communication, Dr. Bates suggested a modifi-
Cation for narrow leaf barley material. This modification,
which I used, involved spraying spikes with 1 mg/ml EACA
with .l% X-77 (a surfactant) from before meiosis to near
trarleer time. The surfactant was used to insure adequate

uptake of the immunosuppressant.

C‘T

C)

(I)

("I

(‘14

30

Gibberellic Acid Treatment

Gibberellic acid (6A3) treatment of the maternal
plants during seed development was started by Larter and
Enns (1960). Their objective was to maintain more normal
post—fertilization development of the seed tissues of
hybrids. They treated tetraploid H. vulgare crossed with
diploid H. vulgare by clipping the flag leaf tip and plac-
ing the flag leaf in a shell vial with 100 ppm GA. Treat-
ment was continuous from time of emasculation to embryo
transfer. This treatment produced near-normal development
Of the endosperm and embryo, while untreated seeds were
shrunken and had much smaller embryos.

Kruse (1967, 1969, 1973) found this method time
consuming and not particularly effective. He placed one
drop Of 75 ppm GA on the stigma of each floret for each
of the two days directly following pollination. Using
this technique, he had remarkable success in producing
interspecific and intergeneric hybrids. I modified this
technique, because I found it extremely tedious for large
numbers of crosses. Spikes were immersed once daily in
75 ppm gibberellic acid for a few seconds the next one or

two days after pollination.

RESULTS AND DISCUSSION

The enormous phenotypic plasticity of weeds (Har-
lan, 1975) is very similar to the plasticity in the "weed"
X cultivated barley crosses. Many of these plants vary
from an extremely robust and large to a very small plant.
Thus phenotypic criteria used in describing these plants
must be sharply limited. I have, however, found these
criteria useful: overall plant type and vigor in several
environments, vigor in cooler environments, presence and
relative size of auricles, relative size of lemmas and

overall head type.

I. CROSSES MADE BY CHUNG LEE

Chung Lee, a former graduate student of Dr. J. E.

Grafius, crossed the H. brachyantherum X H. depressum

 

amphiploid with diploid H. vulgare in the spring of 1969.
NO further work was done with this cross. In the spring
of 1969 he also crossed the H. jubatum X H. compressum

amphiploid with diploid H. vulgare (Table 2). His embryo

31

H.
‘11
I?

l

I".

m.
the

.uw

 

1
‘u.

.h,
.clu

'12-

32

TABLE 2. Crosses made by Chung Lee.

 

 

HYBRID DATE PED IGREE
PARENTAGE
DESIGNATION CROSS MADE DESCRIPTION
(HJ X HC)(6x)X CI 12528 12-1* \ March 1969 69-101**
12-3 69-102
12-4 69-103
(HJ X HC)(6x)X CI 12518 11-2 March 1969 69-104
11-3 69-105
(HJ X HC)(6X)X CI 12518 14-3 March 1969 69-106
(HJ X HC)(6x)X Manchuria 16-1 March 1969 69-107
(HJ X HC)(6x)X Manchuria 18-1 March 1969 69-108
(HJ X HC)(6x)X CI 12518 10-1 March 1969 69-109
(HJ X HC)(6x)X CI 12518 26-1 March 1969 69-110
(HJ X HC)(6x)X Paragon 12-1/Bl Dec. 1969 69-101-Bl.2***
(abbrev.:
Bl.2

 

*12-1 stands for a particular mature F

1 plant
culture tube number
designation Of each mature hybrid from that tube

_ first generation of selfing
lZ-l-l—ldfsecond generation Of selfing

year Of crossing
** 69-10111; individual F1 from 100 series cross
L—-first selfing generation

*** Bl.2 is a simplified notation used by Mather and Jinks

(1971) to denote a first backcross to parent two (BCIPZ)

311*.
he <

'1
«an.

t .
'W'
Us

@471
\o
l

h

u.

#2.

.‘l

33

culture techniques are shown in Figure 2A,B. At that time
he only cut the seed in half and didn't dissect out the
embryo. This method is not particularly reliable and only
produces mature plants from very well-developed embryos.

He was successful in backcrossing one Of these hy-
brids, 69-101, to diploid H. vulgare. This clone,
69—101-Bl.2 (abbreviated Bl.2) , is morphologically and
CYtologically similar to the "pentaploid" hybrid, 74-405,
from (HJ X HC) (6x) X W (Figure 3E and Table 8), which
leads to speculation that Bl.2 is the progeny from a normal
5: lulgare pollen grain and either an unreduced egg or a
dOllbled sector Of 69-101.

Some of the F1 hybrids produced selfed seed. In
many cases, subsequent generations were essentially com-
Pletely fertile. I grew plants of 69—101-1, 69-101-1-1,
69-3|.Ol-1--1-1, 69-102—1, 69-105-1, 69-106-1, 69—106-1-1,
69-107-1, 69-107-1-1, 69-109—1, and 69-109-1-1.

.Only 69-101-1 had any morphological differences from

the E- j'ubatum-fi. compressum amphiploid (Figure BB). This

 

plant was also extremely different cytolog‘ically. 69-101-1
had a highly variable chromosome number at metaphase I of

elosis and multipolar cell diViSions 1n melOSlSo Starks

34

FIGURE 3.--Spike morphology (left to right)

A. H. compressum
H. jubatum
(HJ x HC)(6x)

(HJ X HC)(6x) X HJ

B. 69-101-1
69-101-B1.2 (Bl.2)

Spike and tiller with auricles Of
VV X (HJ X HC)(6x) hybrid

69-101

VV spike and tiller with clasping auricles

 

 

 

 

 

Figure 3 A,B

 

36

FIGURE 3. Spike morphology (cont'd.)

C. 69-101
69-lOl-Bl.2 (Bl.2)
Selfed progeny of B2.2

B3.2

D. VV X (HJ X HC)(6x)

Haploid H. vulgare spike from VV X HJ
progeny

Normal spike of same progeny from VV X HJ

HJ X VV progeny

 

D
C
3
e
r
u
g
.1
F

 

38

FIGURE 3. Spike morphology (cont'd.)

E. Polyhaploid from (HJ X HC)(6x) X VVVV
Intermediate progeny from (HJ X HC)(6x) X VVVV
Pentaploid from (HJ X HC(6x) X VVVV

Bl.2

F. (HJ x HC)(6x)

VVVV Spike and tiller with clasping auricles

 

F
E
3
e
r
u
g
.1
F

 

40

FIGURE 3. Spike morphology (cont'd.)

G. Agropyron sericeum

H. jubatum
(AS X HJ)

(AS X HJ) (8x)

H. W
vv X (AS x HJ)(8x)
(AS X HJ) (8x) x VVVV

H
G
3
e
r
u
g
.1
F

 

42

(unpublished) did not find multipolar cell divisions in
either 69-101 or Bl.2 although both were studied exten-
sively. The other hybrid selfs could easily be contamin-
ant selfed progenies from the H. jubatum-H. compressum

amphiploid. This amphiploid and H. jubatum Often produce

contaminant plants in greenhouse pots and field clumps.

II. SEQUENCE AND OBJECTIVES OF
CROSSES AND COLCHICINE
TREATMENT

This section contains a sequential description of

the crossing and colchicine treatment program. At present,

several of these crosses are essentially worthless for

transferring traits to H. vulgare, but their production was

useful in coming to a much better understanding of the

problems involved in making these transfers. Problems such

as lack of good pairing and hybrid sterility are expected

barriers to transfer of traits in this type of material. I

also found H. jubatum cytoplasm imparted gross lack of vigor

and cytOplasmic sterility to H. vulgare derivatives. Chrom-

osome elimination also made certain combinations difficult

and relatively useless in a standard backcross intermating

program .

43

Kasha (1974) listed several criteria as evidence for
chromosome elimination in interspecific or intergeneric
crosses: 1) genome balance in hybrids; 2) high seed set,
but seeds were small and usually non-viable; 3) chromo-
some instability of hybrid leading to mosaics, chimeral
sectors and variable chromosome number in pollen mother
cells; and 4) production of hybrids and haploids.

In this section and preceding sections I have
attempted to describe my results so that they might be of
help in interpreting other crossing results.

In Michigan, the greenhouse crossing season begins
in September and lasts through June. Many of the potential
parents (species and hybrids) do not produce good heads un-
til November. By June, greenhouse conditions are too hot
for most hybrid seeds to develop properly. Summer green-
house use is mostly restricted to maintaining stocks in
as good condition as possible.

My crossing program began with making the second H.
vulgare backcross of (HJ X HC)(6x) X VV. Our Objective was
to produce parental material for an intermating population
containing the desired genetic variation. Then cyclic in-

termating and selection would be practiced with the hope

44

of achieving the desired recombination and ultimately a
commercial variety with the desired improvements. Although
this cross was made during the first two greenhouse seasons
(1972-73 and 1973-74), it failed to produce the desired
parental material (Tables 3 and 4). Figure 3A to 3H shows
spike morphology of progeny and parents of all crosses.

During January to March of the first crossing sea-
_ son, clones of AT X HJ and AS X HJ were divided to tiller
divisions and were colchicine treated (Table 5). The ob-
jective was to produce higher level amphiploids as poten-
tial donor parents to H. vulgare. Amphiploids of both hy—
brids were Obtained. An extensive attempt to double
(HJ X HC)(6x) X VV was also begun. The objective was to
produce an amphiploid parent which, when crossed with
diploid H. vulgare would produce progeny with two sets of
H. vulgare chromosomes. It was hoped these hybrids would
be more fertile and could produce the parental material
for an intermating population. Attempts to double this
cross were terminated in late 1974 after 10,000 tiller
divisions were treated without success.

In May of the first greenhouse crossing season,

another cross-~VVVV X (HJ X HC)(6x)--was attempted with

45

movlvh

woolen

mow 0» Hovlvh

Nam OD Homlmn
How 0» Hmmlmh
mom on Homlvo

N.leaoalmo

Am.am .>ounbac
N.Hmuaoaume

«we Ou HmHImh

«NNH OU.HHHI¢>
OHH Ou HOHImo

NH

Ha

NH

ea

NH
0H

Aeeoaemueoev
>> ea .aa

anemone sunfloofiuoucfl

Amofloadmrmaomv
=4-E

:4 ed .dfl
:fi .< .<<¢
.<d>
>.<¢
>>

>>=4.¢¢
mmouoxoon oncoHs>

=<.<¢>

> :¢.¢¢
> =<.¢¢

>>>> x Axeciom x ems
or x AxeCAom x are
mm x Axeciom x are
mm x >>

>> x we

>> x A>> x Hoaumec

>> x HOHImo

AerAom x ems x >>

>> x Axmciom x ems

 

ZOHBdZUHme
mmmOHQmm BZ<Am

wzmwomm mmafiéz
m0 mmmSDZ

ZOHBfiZOHmMQ WSOZMU

ZOHEdZUHme mmomo

 

.ooosoonm meecomond ensues one owedaouum mommouo Ham mo

coeumcowmoo .m mqm<9

.Bmmadoumo mummHs> mm sues
moans»: How oo>uomou one mmIHm dawns .Emmadouho ourmadb nmlsos
sues oranges How couscmflmoo our moeuom concede moo muo>o mo meldo muonsdz
mmouo umasowuumd menu scum Hm comm on» we «NH
some mw3 mmouo oru use» or» we we «malwba

 

46

.l: o .5geam as x Ga: Ga x m5
Selma H 222? one :5 x 9: x >>
ST: A >>.<<2m >>>> x :81; x 9:
an- o Erma >25 x mm
in e in E x semi?
111 o 2.525 33c 8m x 2: x E

A.U.HGOOV m mqmda

47

TABLE 4. Order in which crosses were made.

 

 

CROSS TIME . PROGENY
Bl.2 X VV Nov. l972-June 1973 6
VVVV X (HJ X HC)(6x) May 1973 0
(HJ X HC)(6x) X HJ June 1973 12
(HJ X HC)(6x) X HC June 1973 0
Bl.2 X VV Nov. 1973—June 1974 6
(HJ x HC)(6x) x vvvv Feb.-April 1974 5
[(HJ x HC)(6x) x HJ) x vvvv Feb.-April 1974 f 0
(HJ X HC)(6x) X VV Feb.-April 1974 6
(AS X HJ)(8x) X HJ April 1974 0
(AS X HJ)(8x) X VVVV April 1974 1
HJ X VVVV Summer 1974 0
HJ X VV Summer & Fall 1974 9
(HJ X HC)(6x) X VV Summer & Fall 1974 6
VV X HJ 3 Oct. 1974 to Feb. 1975 ll
VV X (HJ X HC)(6x) Feb.-April 1975 14
VV X (AS X HJ)(8x) March-May 1975 1

[VV X (HJ X HC)(6x)] X VV Aug. 1975 to March 1976 0

48

 

 

o ooo.oa same .>oznmema seesaw >> x AerAom x ems
m oooa mama eoumznsumseme Lem x sac
om OOOH meme rouezusumseme Lem x mac
omosoomm omeamme mmmque
moHoqumeza so mmmzoz oonme ezmSeamme omeamme anmmemz

 

.usofiumouu

oseownoaou

. m mqmdfi

49

the hOpe that two sets of H. vulgare chromosomes and H.
vulgare cytoplasm would greatly increase fertility of the
hybrid and backcross progeny. It was hoped this would
provide a better chance to form an intermating population
containing the desired variation. Attempts to produce
this cross were stopped after the first Six heads failed
to Show Signs of floret closure, stigmatic change from a
receptive appearance, or development of seeds.

In June of the first crossing season another cross
--(HJ X HC)(6x) X HJ--was successfully made. Progeny of
this cross were used by Starks and Tai (1974) to better
elucidate the genomic relationship of H. jubatum and H.

compressum.

 

At that time, six heads of the H. jubatum X H.

compressum amphiploid were also backcrossed to H. compres-

 

EBB for the same Objective. Although the amphiploid was
massively pollinated, no signs of floret closure, stigmatic
change, or seed development were observed.

During February, March, and April of 1974, (HJ X HC)
(6x) X VVVV, 73-304 X VVVV and (HJ X HC)(6x) X VV were at-
tempted. The first two crosses were made with the hopetmat
two sets of chromosomes from H. vulgare would greatly in—

crease the fertility of the hybrid and backcross progenies.

50

True hybrids and progenies with partial and complete elim-
ination of the chromosomes Of H. vulgare were obtained
from (HJ X HC)(6x) X VVVV. Seedlings of 73-304 X VVVV
died before transfer to soil, probably due to extreme
chromosome elimination (Figure 2C). (HJ X HC)(6x) X VV
was made to obtain seedlings for colchicine doubling treat-
ment. None of the Six treated seedlings was doubled.

Two other crosses were also attempted in April of
1974--(AS X HJ)(8x) X HJ and (AS X HJ)(8x) X VVVV. Four

heads of the ngopyrog sericeum X H. jubatum amphiploid

 

were pollinated with H. jubatum. Five days later, after

no signs of floral closure, stigmatic change, or seed set,

these heads were pollinated with VVVV. Four seeds rapidly

developed, producing one (AS X HJ)(8x) X VVVV hybrid. The

H. jubatum backcross was attempted to elucidate the genomic
relationship of this cross. The H. vulgare hybrid was made
for trait transfer purposes.

By the summer of 1974, healthy H. vulgare plants
were repeatedly obtained under growth chamber conditions.
Therefore, I attempted a cross during the summer. H. 13-
EEEET X VVVV gave a respectable seed set ranging from 0%

to 90% on different Spikes, but only proembryos--globular,

51

undifferentiated embryos--resulted from this cross. Crosses
with good seed set, but with a very low number of mature
hybrid plants produced is good evidence for chromosome elim—
ination (Kasha, 1974). Again, this cross was attempted to
produce a hybrid with two sets of H. vulgare chromosomes
with the hope of greater hybrid fertility.

Two other crosses--HJ X VV and (HJ X HC)(6x) X VV-—
were started during the summer and completed in the fall
with the crossing taking place in the growth chamber. These
crosses were made to determine the effects of EACA, an im-
munosuppressant, which Bates, SE.21° (1974) claimed spec-
tacular effects from. The HJ X VV cross was also made for
breeding purposes and to help elucidate the genomic rela-
tionship Of H. jubatum and H. vulgare.

By this time, the deleterious effects on vigor and
male fertility of the H. jubatum cytoplasm on H. vulgare
derivatives were quite apparent. Thus reciprocal crosses
were attempted. Between August and late December, spring
habit, normal H. vulgare and genetic male sterile H. vulgare
plants were used. Both failed to produce any hybrid seed.
Starting in early January, hybrids were produced from winter

genetic male sterile diploid barley X H. jubatum. (HJ X HC)

52

(6x) and (AS X HJ)(8x). During August and September of 1975
seventy heads of the VV X (HJ X HC)(6x) hybrid were back-
crossed with VV. Only one lateral floret developed, and
this had an endosperm with normal starch deposition but no

embryo.

III. COLCHICINE TREATMENT OF
PLANT MATERIAL

During January to March of 1973 clones of AT X HJ
and AS X HJ were divided into tiller divisions. For colchi-
cine treatment, I chose to use eight colchicine treatments
and no attempt was made to keep track of which were effec-
tive, because many factors such as vigor of material, par-
ticular species, stage of growth, temperature and lighting
determine the best colchicine treatment (Elliott, 1958).
One thousand vigorous tillers of each spontaneous hybrid
were divided into eight nearly equal groups. Each group
received one of the treatments used. Colchicine treatments
used were 1 and 3-9. After treatment, tillers were care-
fully washed and planted in flats. Several treatments

caused 50% mortality.

53

By June, the surviving plants were growing vigor-
ously and beginning to head. The heads were cut Off and
the plants transplanted to the field. Field conditions
were good for rapid tiller production and vigorous growth,
but these plants failed to produce large numbers of heads
over the next two years in the field. Evidently the right
conditions for floral initiation never occurred. These
hybrids seemed to require very long days and a moderate
temperature for floral initiation.

For a few weeks after transplanting to the field
a few plants continued to head. Several of these spikes
produced a few seeds.

Three amphiploids of AT X HJ were Obtained from the
seed (Table 5). They all had good fertility under faVorable
growing conditions. Only one of these plants has been main-
tained. See Huang (1975) for cytological investigation of
this amphiploid.

Thirty doubled plants of AS X HJ were obtained from
the seed. Some of these were completely sterile under fimmmh
able growing conditions, while others had very good fertil-
ity. Only those with 56 chromosomes at anaphase I and a

good fertility level were maintained. Only one of these

54

was used in crossing and chromosome studies. See Murry
(1975) for cytological investigation of this amphiploid.

From the Spring of 1973 to the fall of 1974, all
nine doubling techniques were extensively used to double
the hybrid (HJ X HC)(6x) X VV. Ten thousand plants were
treated with no fertile seeds produced.

With a very Similar cross (HJ X VV) three Canadian
research groups (personal communications from Dr. Eric R.
Kerber, Steve Symko, and Dr. Bryan Harvey) also failed to
produce fertile doubles. Extensive attempts were made by
all three research groups. Both Bryan Harvey and Eric
Kerber used all the standard colchicine doubling techniques
for several years.

There are many possible explanations for the fail-
ure to produce fertile seeds or visibly doubled sectors in
these crosses. Some of these are: failure to produce
doubled cells; lack of competitive ability of doubled tis—
sue; rapid reversion of doubled cells; and sterility of
doubled tissue. Lindstrom (1966), working with colchicine—
treated tetraploid rye, concluded that failure to produce
octaploid seeds and/or octaploid meiosis was probably due

to lack of competitive ability of the octaploid tissue.

55

IV. CROSSING AND PROGENY PRODUCED

 

Crosses vary greatly in seed set, transfer survival,
response to environmental stresses, etc. An understanding
of how several crosses behave can be very useful in making
further crosses and in interpreting the results of crosses
made by other researchers. The crossing method and results
of crosses including progeny produced and environmental in-
fluences are herewith discussed.

Second and Third Backcross
Generations of (HJ X HC)
(6x) X VV

Extensive efforts were made to produce the second
backcross to H. vulgare of (HJ X HC)(6x) X VV. This in-
volved careful selection and backcrossing of 2000 healthy
Bl.2 heads over two greenhouse seasons (1972-73 and 1973-
74). Good heads Of Bl.2 were seldom produced before late
November and by late May, greenhouse conditions were too
hot for further successful crosses. Crossing was performed
on a regular basis, but success was very dependent on
weather conditions. Depending on environmental conditions,
from 5% to 50% of the Spikes would begin development of

Small seed(s). Usually these would live for 6 to 10 days

56

and cease development at the proembryo (no differentiation)
stage.

In an attempt to provide a better environment for
embryonic development, thirty spikes were treated with
gibberellic acid by the Larter and Enns (1960) method.

The treatment delayed collapse of some seeds until they
were twenty—five days old, but only proembryos were pro-
duced by this treatment. This lack of success caused me
to abandon gibberellic acid treatment until I used Kruse's
(1967) methods on crosses with H. vulgare as female parent.

On untreated plants, chance and the combination of
environmental factors, particularly temperature and light-
ing, allowed continued development Of the embryo on rare
occasions. Many of these continued growth and development
until they were completely differentiated and similar to
small mature embryos. Much Of the chance factor probably
involved elimination of certain chromosomes to produce a
diploid H. vulgare (see section on chromosome elimination).

Twenty B2.2 seeds developed to the normal H. vulgare
seed size (Figure 1D) and contained completely differenti-
ated embryos comparable in size to those of H. vulgare.

These were all transferred to wick type embryo culture

57

media (Figure 2D). Five developed abnormally on media and
died. Three remained dormant and white with an appearance:
of viability for at least six weeks. These embryos were
left on the plant until early dough stage and dormancy
seems to have been induced during starch deposition.

The remaining 12 B2.2 embryos produced mature
plants (Table 3). Only one of these had a relatively high.
self—fertility of approximately 40% (Figure 1A). A few of
the remaining plants set an occasional selfed seed. All
selfed seed occurred in a hot summer greenhouse. Selfed
seed and third backcross seed from all plants had no self-
fertility when grown in cooler environments.

Only seven normal H. vulgare bivalents were Observed
in selfed andthird.backcross H. vulgare plants. These plants
closely resembled Dr. Schooler's restorer lines (H. vulgare
in H. jubatum cytoplasm) except, due to their lack of re-
storer genes, they failed to set selfed seed in cooler en—
vironments. Other than for cytoplasmic effects, these
plants only exhibited H. vulgare characteristics.

H. jubatum cytoplasmic effects were the most distic-
tive features of the.restorer and.second.and third backcross

H. vulgare plants. These plants were far less vigorous than

58

normal H. vulgare, even under ideal growing conditions.
Under less than ideal growing conditions the plants often
failed to head normally and died at any age from one to
nine months. Under extremely unfavorable conditions,
plants produced many abnormal tillers, probably due to
failure of the development of main tillers. Figure 4D
Shows a representative examplecfifweak and abnormal tiller
production.

Extreme lack of vigor made it very difficult to
use the restorer lines as parents in crosses. Thus forma—
tion of an intermating population among the restorer lines
would have been nearly impossible. Therefore the H.'jubatum
cytoplasm seems a dead end even with restoration genes
present.

(AS X HJ)(8x) X VVVV and
(HJ X HC)(6x) X HJ

 

These crosses are representative of the two dis-
tinctly different seed set groups of my crosses. (HJ X HC)
(6x) X HJ, like several other crosses had a very good seed
set, ranging from 0 to 95% on individual Spikes. (AS X HJ)
(8x)x VVVV had one of the highest seed sets of the low seed

set group. It set a total of four seeds on four Spikes.

59

FIGURE 4.-—Parthenocarpic fruit and other cross results

Large parthenocarpic fruits (normal seed
size) in central florets of six row

‘H. vulgare

Smaller parthenocarpic fruit in all florets.
Stigmas still feathery

Four hybrid seeds from VV X HJ. Other
florets still with feathery stigmas 18
days after pollination

Selfed progeny from 82.2 with yellowing
of leaves (nicofume injury) and many weak
and often deformed tillers

Seed from [VV X (HJ X HC)(6x)] X VV

Seedling from HJ X VV on Norstog's
agar medium II

60

 

Figure 4

61

None of the species X species or species X amphiploid
crosses fell into an intermediate category.

Seed set of the high seed set group was comparable
with that expected for a cross within a species, but some-
what more susceptible to environmental influences. Crosses
of the low seed set group are either extremely susceptible
to environmental influences (like oat crosses in the green-
house in Michigan) or they have fairly effective isolation
mechanisms preventing fertilization.

Seed Of the cross (HJ X HC)(6x) X HJ produced a
reasonable amount of endosperm starch and could possibly
have germinated normally without embryo culture. Thirty
well-differentiated embryos were transferred to Norstog's
agar medium. Only twelve of these developed into mature
plants (Table 4). Probably many more mature plants would
have developed on the wick medium from these completely
differentiated embryos. Self fertility was 70%, which is
very high for pentaploid hybrids. Morphologically, the
plants were intermediate between their parents in spike
characteristics and overall morphology (Figure 3A). For
cytological verification and discussion of pairing, see

Starks and Tai (1974).

62

Seed of the cross (AS X HJ)(8x) X VVVV also pro-
duced well developed embryos with some starch deposition
in the endosperm. Only one mature plant developed from
these embryos. Morphologically, this plant more closely
resembled its female parent, although the head was somewhat
different and auricles were definitely more pronounced. For
cytological verification and discussion of pairing, see
Murry (1975).

Other Crosses Resulting in
Embryos with H. jubatum

Cytoplasm

 

 

The following crosses, (HJ X HC)(6x) X VV, HJ.x VV,
HJ X VVVV, [(HJ X HC)(6x) X HJ] X VVVV, and (HJ X HC)(6X)X
VVVV all had a relatively high seed set, which can be favor-
ably compared with the seed set of (HJ X HC)(6x) X HJ. All
crosses had a seed set ranging from 0 to 95% on different
spikes. This group of crosses demonstrated the effects
chromosome elimination had on the ease of obtaining mature
progeny.

It was relatively easy to obtain mature progeny
from crosses of HJ X VV and (HJ X HC)(6x) X VV. The crosses

produced fairly completely differentiated embryos. Seed of

63

the cross (HJ X HC)(6x) X VV had some endosperm starch depo-
sition, while HJ X VV produced enough starch that mature
plants were produced without embryo culture (Kerber, per-
sonal communication). I also produced one mature HJ X VV
hybrid without embryo culture while eight hybrids were
produced using embryo culture. Twelve mature (HJ X HC)

(6x) X VV hybrids were produced (Table 3). Transfer suc—
ceSs was around 30% for both crosses on agar or wick media.
Lack of complete embryo development probably restricted
transfer success.

Phenotypically, the hybrids Of both crosses more
closely resembled their female parents. Hybrids of both
crosses can be easily distinguished from their respective
female parents on the basis of overall plant type, slightly
more H. vulgare-type heads and presence Of auricles (Figure
3A—D). All the hybrid plants produced from these crosses
were self-sterile.

Approximately l/2 of the hybrids of each.cross were
produced using EACA, an animal—effective immunosuppressant,
to test the effects of this chemical. No morphological
differences were noted between the treated and nontreated

hybrids. All treated progenies were just as sterile.

64

Colxchicine treated seedlings and divisions of (HJ X HC)(6x)
X VNJ seemed just as stubborn in producing seeds as divi-
Sions Of 69-101 were. Thus in contrast to Bates, EE.El°'S
(1974) claims, I was unable to Observe any effects of EACA.

Metaphase I chromosome counts Of 69-101 were all
28, thus the genomic constitution AA'A"V seemed quite
stable (Starks, unpublished). This agreed with Tom Orton's
preliminary embryo squash data (Table 9). Kerber (unpub-
lished) found AA'V metaphase I counts varied from 16 to 23,
so it would appear AA'V was not quite as stable.
While crosses of (HJ X HC)(6x) X VVVV, [(HJ X HC)

(6x) X HJ] X VVVV and HJ X VVVV had just as high seed set,
production of mature plants seemed to be strongly controlled
by chromosome elimination. See Table 6 for genomic stabil-
ity of this crossing series. Crosses of HJ X VVVV produced
only globular undifferentiated embryos (proembryos) which
were not successfully cultured on Norstog's medium. Crosses
of [(HJ X HC)(6x) X HJ] X VVVV and (HJ X HC)(6x) X VVVV
produced many reasonably well differentiated embryos.
These embryos were essentially at the same stage of devel-
opment as (HJ X HC)(6x) X VV embryos. Approximately 100

embryos of each cross were transferred to embryo culture

65

TABLE 6. Stability of hybrid progeny.

 

HYBRID CROSS METAPHASE I

PROGENY

 

AA' A“ V (HJ )( HC)(6x) X VV Stable--
all counts 28

VAA' VV X HJ variable--
l6-22*
AA' V HJ X VV l7-23**

AA' A" VV (HJ )( HC)(6x) X VVVV stable 21

22-28

variable

all hybrids

8 hybrids--3 of which
produced some haploid
tillers

all hybrids

3 polyhaploids (AA'A")
1 intermediate progeny

1 pentaploid hybrid

 

*Murry (1975)
**Rajhathy and Morrison (1959)

66

media. If the stage of embryo develOpment were the only
factor controlling production of mature plants these crosses
should have produced many mature plants. The more differen-
tiated embryos of both crosses were transferred to a wick
medium. Only a small percentage of the embryos of both
crosses had no development on culture media.

Approximately 1/2 of the remaining embryos of each
cross produced only abnormal growth ranging from undiffer-
entiated growth or hypocotyl development only, to abnormal
roots and shoots. The other 1/2 for a brief time developed
nearly normal shoots and roots. [(HJ X HC)(6x) X HJ] X VVVV
embryos only maintained near-normal growth for a short time
before dying. Thus no mature plants were produced from this
cross. Figure 2C is a good example of how long near-normal
growth usually occurred.

In contrast, embryos from the cross (HJ X HC)(6x)

X VVVV produced five mature progeny: three “polyhaploids“

--haploids of the amphiploid; one "pentaploid“ hybrid; and

one "intermediate" progeny--intermediate between the penta-
ploid and the polyhaploid progenies in morphology and

chromosome number. Figure 3E,F shows morphological

67

comparisons of the plants and Table 7 Of Gil Starks' unpub—
lished data shows the cytogenetics of these plants.

The polyhaploids had the expected morphology of
haploids of the amphiploid, that is, more but smaller
tillers with somewhat smaller floral parts. There were no
H. vulgare characteristics in these plants. For example,
H. vulgare and all true H. vulgare interspecific or inter-
generic hybrids that I made had auricles. Like H. jubatum,

H. compressum and (HJ x HC)(6x), the polyhaploids had no

 

visible auricles. The pentaploid hybrid was intermediate

in morphology between the parents. This was more obvious
when the pentaploid was compared with hybrids from (HJ X HC)
(6x) X VV. The pentaploid had fewer but more robust tillers
with larger, often clasping auricles and a definitely more
H. vulgare-like head. Plants from the cross (HJ X HC)(6x)

x VV had small auricles that were rarely clasping.

Like most hybrids from crosses with chromosome
elimination, the pentaploid had a variable chromosome number
at metaphase I Of meiosis (Table 7 of Starks' unpublished
data). The intermediate progeny also had a variable chromo-

some number.

68

TABLE 7. Starks' metaphase I counts Of progeny from
(HJ X HC)(6x) X VVVV.

 

 

CHROMOSOME NUMBER POLYHAPLOIDS INTERMEDIATE PENTAPLOID

 

19 l
20

21 80
22

23

24

25

26

27

28 24
29

30

31

32

33

34

35

01 o ~4 a: e; H
H .s so ea a..> as as a..> to

Total
number 80 50
of cells

(A
U!

 

69

TABLE 8. Starks' metaphase I counts of the pentaploid
and Bl.2.

 

CHROMOSOME Bl.2 COUNT PENTAPLOID PENTAPLOID PENTAPLOID
NUMBER IN 1974 1974 COUNT DEC. 1975 COUNT*' DEC. 1975 COUNT**

 

15
16
17
18
19

NUII—‘NN

20
21
22
23
24
25

ubQWUlNN

26
27
28
29
3O

0090\1‘0
mepe
l-‘

O

to

31
32
33
34 l
35

w
1‘ e.a1+d.b
H H UIP‘

Total
number 74 35 19 10
of cells

 

*Count from.four or five florets from two spikes.
**All counts from one floret.

70

Both the intermediate and pentaploid progenies
changed morphologically since the 1974 data were taken.
Partly due to lack of vigor, change in the intermediate
progeny was difficult to describe, but this plant seemed
more vigorous with some changes in overall plant type and
head morphology. The pentaploid head morphology became
impossible to distinguish from [(HJ X HC)(6x) X VV] X VV
heads.

Results of these crosses are good examples of the
effects of chromosome elimination on a crossing series.

Thus these results can be useful in interpreting the re-
sults of other researchers. For example, Morrison, et 21'
(1959) transferred 1400 embryos of g, murinum X VV. These
were well developed embryos, but produced almost identical
results to those of [(HJ X HC)(6x) X HJ] X VVVV. Earlier,
this research group had produced a hybrid, which later

broke down into E, vulgare and fl} murinum sectors (Hamilton,
1955). Taken as a whole, I consider this good evidence that

chromosome elimination was operating in this g. murinum-

‘g. vulgare hybrid.

71

Crosses withgg vulgare
as Female

 

In May of 1973, the cross VVVV X (HJ X HC)(6x)
was attempted on a limited scale. Although the six heads
were massively pollinated, the florets remained open and
receptive. Both lack of floral closure and lack of stigma
collapse are good evidence of nonfertilization. Thus I
assumed that this was good evidence this cross was improb-
able. Later, when the extreme disadvantage of g. jubatum
cytoplasm became all too obvious, other crosses with g.
vulgare as the female were attempted.

Preliminary attempts with crosses of VV X HJ and
VV X (HJ X HC)(6x) gave similar results. All florets not
obviously injured by excessive drying or poor emasculation
remained open and appeared receptive for many days. Lack
of stigma collapse was taken to indicate continued recep-
tive condition (Figure 4B,C). Crossing on a large scale
was then undertaken with the aid of'a spring type, genetic
male sterile population and a winter type, male sterile
population. These populations had the added advantage of
genethzdiversity for many traits and thus possible varia-

bility for receptivity to foreign pollen.

72

Crossed heads were also treated with gibberellic
acid (6A3). Kruse (l967) was highly successful in using
this chemical in achieving interspecific and intergeneric
Hordeum hybrids.

Crossing onto spring type male sterile plants was
soon stopped after difficulties occurred with incomplete
sterility and frequent development of parthenocarpic fruit.
I use the term "parthenocarpic fruit“ for fruits appearing
to have only maternal tissue development. Sometimes small
parthenocarpic fruit were produced by spring type plants
even without gibberellic acid treatment and without pollin-
ation. Gibberellic acid treatment seemed to be necessary
to stimulate parthenocarpic fruit development in the winter
type male sterile plants. Partial genetic control of par-
thenocarpic fruit development was indicated by the more
frequent and more uniform development in the spring type
population.

The external morphology of parthenocarpic fruits
was highly variable (Figure 4A,B) with only one character
remaining constant. All develOping parthenocarpic fruits
had noncollapsed stigmas. Lack of stigma collapse could
be observed in all parthenocarpic fruit for at least ten

days.

73

Size of parthenocarpic fruits ranged from l/3 nor-
mal to normal seed size. On some heads only the central
florets developed into parthenocarpic fruits. Most park
thenocarpic fruits could easily be distinguished from nor-
mal seed, but a few were very similar to normal seeds.

Three hundred fruits were dissected. Internal
morphology was less variable than external morphology.
Fruits generally appeared to have a spongy proliferation
of ovary wall cells, which made it very easy to distinguish
parthenocarpic fruits from normal seeds. All three hundred
dissected fruits had no signs of embryo or endosperm.

Fifty winter type male sterile heads were massively
pollinated with g. jubatum. Florets remained open and
stigmas failed to collapse for many days. Only two spikes
developed hybrid seeds. One spike had ten hybrid seeds,
while the other had four hybrid seeds. Figure 4C shows
the spike with four hybrid seeds and other florets with
non-collapsed stigmas. The picture was taken eighteen days
after pollination and still some florets had noncollapsed
stigmas. Embryos were transferred immediately after this
picture was taken. The hybrid embryos were nearly as big

as g. vulgare selfs, and well—differentiated. Usually the

74

scutellum was deformed in that it curved back and partially
covered the shoot end of the embryonic axis. I assumed
this was a geotropic response, since all hybrid embryos of
this cross were in water sac-like endosperms which appeared
to allow considerable freedom of embryonic movement.

The ten most normal appearing embryos were trans-
ferred to a wick medium and all developed into mature
plants. Eight of these were normal hybrids. Three of
these hybrids sometimes produced haploid g. vulgare tillers
(Figure 3D). The other two remaining plants were neither
normal hybrids nor haploid g. vulgares. These plants re-
mained very short for many months and failed to head. They
lacked g. jubatum leaf hairs and had less distinct auricles
than true hybrid progeny. Eventually these plants produced
heads morphologically identical to normal hybrids. Prob-
ably these plants were true hybrids but had phenotypic sup-
pression of normal hybrid pubescence and auricle expression.
I based this statement on some callus culture results of
true hybrid tissue. Regeneration plantlets from callus
formed from true hybrid tissue have the same failure of
exPression of the g. jubatum pubescence and hybrid auricle

tYpe. Metaphase I chromosome counts of one of these plants

75

were mostly 2l. Thus it seemed reasonable to assume this
plant was a true hybrid with phenotypic suppression of
some characters.

The remaining four embryos were transferred to a
callus promoting medium. Three embryos soon became dis-
eased and died. The remaining embryo rapidly callused
and was turned over to Dr. Tom B. Rice for experimental
purposes.

Seventy winter type male sterile heads were mas-
sively pollinated with (HJ X HC)(6x). Four spikes set a
total of 17 seeds. Seed sets were 9, 5, 2, and 1 seed per
spike. Seeds in this cross were very similar to those of
VV X HJ. In both crosses embryos were nearly normal g.
vulgare size, well differentiated and had some overgrowth
of the shoot tip of the embryonic axis by the scutellum.
In both cases the endosperm remained a water sac with very
little or no starch deposition. All seventeen embryos were
transferred to a wick medium. Fourteen of these developed
into normal hybrid plants (Table 3). The other embryos
failed to develop into mature plants. Morphologically,
these hybrids and VV X HJ hybrids were very similar to

their respective reciprocal crosses. But these hybrids

76

were distinctly more vigorous than their reciprocal crosses
with cytoplasm from E. jubatum. Vigor is dependent on
growing conditions and thus is not a good character to
distinguish reciprocal crosses grown under different micro-
environmental conditions. The most consistent morphological
difference between reciprocal crosses was that hybrids with
cytoplasm from g. vulgare had a somewhat wider lemma.

One hundred and eighty winter type male sterile
heads were massively pollinated with (AS X HJ)(8x). Again,
essentially all florets remained open and stigmas failed
to collapse for many days. Six spikes set a single seed
per spike. Four of these seeds had large, well-differen-
tiated embryos in a water sac-like endosperm. Again some
overgrowth of the embryonic axis by the scutellum occurred.
All four of these embryos were transferred to a wick medium.
Seedling development in all four cases soon became abnormal,
resulting eventually in death.

The remaining two embryos were transferred at a
much earlier stage of development. Although embryos had
a partially develOped scutellum, they were so small that
proper orientation on a wick medium was left up to chance.

One of these hybrids developed into a mature plant.

77

Chromosome elimination could possibly explain these
results. The much lower mature plant production frequency
of some crosses in which chromosome elimination operated
seemed to indicate that embryos in the process of losing
chromosomes had a much lower survival frequency. Early
embryo transfer results in a higher percentage of hybrids
(Kasha, 1974). In a similar cross, (AT X HJ)(8x) X VV,
Huang (1975) found chromosome number in metaphase I of
meiosis was variable. Thus possibly this cross was also
unstable. Morphologically, plants of the cross VV X (AS
X HJ)(8x) were similar to (AS X HJ), but they did have
distinctly more g. vulgare-like auricles. This hybrid had
a stable chromosome number of 35 in metaphase I of meiosis.
So it was impossible to make any interpretations on whether
chromosome elimination was operating in this cross.

All the hybrids with g. vulgare cytoplasm were dis-
tinctly more vigorous than their reciprocal crosses. Thus

vigor of g. vulgare derivative lines should be good.

V. HYBRID SEED DEVELOPMENT

Normal seed develOpment involves normal development

of many tissues. This process is set in motion by

78

pollination and fertilization (Maheshwari, 1950 and Nitsch,
1971). Initially, activity is most intense in these mater-
nal tissues: the diploid integuments, nucellus and ovary

wall and the haploid antipodal cells. In the cross Hordeum

jubatum X Secale cereale, Cooper and Brink (1944) attributed

 

failure of normal endosperm development to failure of the
antipodal cells to develop normally. Thompson and Johnston
(1945) attributed breakdown of hybrid seed development
directly to irregular development of the endosperm, includ-
ing formation of giant nuclei and slowing down of the mi-
totic cycle.

These authors seem to imply normal embryo and endo-
sperm development are strongly interdependent. In actual
fact, the embryo and endosperm follow independent and auton-
omous developmental sequences and interaction between the
two systems is not a prerequisite for normal formation of
either tissue. Results of interspecific crosses support
this statement.

' Interspecific crosses in Datura sometimes produce
seeds with only endosperm or embryo. From the results of

several Triticeae interspecific and intergeneric crosses

 

Ivanovskaya (1962) concluded there was no relationship

79

between the degree of embryonic development and production
of endosperm tissue in mature seeds.

The results of my crosses also agree with the au-
tonomous developmental idea. Completely differentiated
embryos occurred in the crosses VV X HJ, VV X (HJ X HC)(6x),
and VV X (AS X HJ)(8x) with no or only a watery sac-type
endosperm develOpment. These embryos were at least as com-
pletely differentiated as those of (HJ X HC)(6x) X HJ or
HJ X VV. In these latter crosses enough endosperm starch
deposition occurred that normal germination of the seed
was possible.

To complicate the matter even more, crosses between
different ploidy levels of the same species can result in
severe disturbances of the endosperm and embryo. Larter and
Enns (1960) found hybrid embryos of tetraploid X diploid
g. vulgare were so small that even with the aid of embryo
culture only a very few normal seedlings could be produced.

Whatever the cause of developmental breakdown,
there is no indication of incompatibility between the em-
bryo and the maternal tissues. In fact, results of crosses
between different ploidy levels of the same species is very

good evidence against incompatibility between these tissues.

80

Incompatibility due to maternal tissue also seems very un-
likely, because embryos usually have more normal develOp-
ment than the endosperm. It would seem any incompatibil-
ity with maternal tissue would more severely affect tis-
sues having a greater proportion of their genetic material
from the paternal parent.

Thus transfer should be normally delayed until
collapse of the endosperm tissue occurs. This allows for
production of more completely differentiated embryos,
which when embryo cultured usually produce many more
hybrid plants. 1

I observed two exceptions to this rule. Those ex-
ceptions were due to these intervening factors: seed dor-
mancy and chromosome elimination. Prevention of seed dor-
mancy probably only requires transfer before much endOSperm
starch deposition occurs. Transfer time in the case of
chromosome elimination must be the best compromise between
greater developmental problems with young embryos and the
increasing proportion of older embryos mixed up with
chromosome loss problems. In some crosses, e.g. HJ X VVVV,

chromosome elimination was so severe that there was no

optimum time for transfer.

81

VI. CHROMOSOME ELIMINATION AND
EVIDENCE FOR THE H. VULGARE
BACKCROSS OF (HJ X HC)(6x)
X VV AND THE PENTAPLOID HY-
BRID FROM (HJ X HC)(6x) X
VVVV HAVING THE SAME BASIC
GENOMIC CONSTITUTION

 

 

 

 

 

This section deals with the gradual change in
phenotype and metaphase I chromosome number of the penta-
ploid hybrid from (HJ X HC)(6x) X VVVV. I did not find
any similar examples in the literature on Hordeum. Ob-
viously, rapid change from hybrid to a haploid of one of
the parents is easily observed and easily documented for
publication. 3. bulbosum-E. vulgare hybrids have become
a classic example of this. These hybrids have also become
a classic example of the cause of this rapid change,
chromosome elimination. Chromosome elimination also
causes many other conditions, which Kasha (1974) has
listed as indirect evidence for chromosome elimination.
These expressions of chromosome elimination in interspe-
cific or intergeneric crosses are: l) evidence of genome
balance, e.g. triploid hybrid VBB is essentially stable,
but VB, VVBB, and VVB hybrids are unstable; 2)good seed

set, but seeds non-viable without embryo transfer; 3)

82

chromosome instability of hybrids leading to mosaics,
chimeral sectors or variable chromosome number in meta-
phase I; and 4) production of hybrids and haploids.
Obviously, some of these factors, taken individually,
could be caused by other variables.

Direct evidence for chromosome elimination can be
obtained from embryo squash counts. For example, Subrah-
manyam and Kasha (1973) made a very convincing argument
for chromosome elimination in VB hybrids from this type
of count. Their results were: 1) near hybrid counts for
most very young embryos and 2) a gradual reduction in
chromosome number of most counts over the next few days
until most counts were near haploid level. Most counts
began near a hybrid level, so production of a reasonable
number of haploids was not due to male or female parthen-
ogenesis. The gradual reduction in chromosome number of
most counts shows chromosomes were lost gradually and not
by whole genomes at a time. Embryo squash counts were
also made on the essentially stable VBB hybrids. Much
less variation in chromosome number occurred in these

embryos.

83

My crosses exhibited the following expressions of

chromosome elimination:

(l)

(2)

(3)

(4)

VAA'A" from (HJ X HC)(6x)X VV or reciprocal was
completely stable; VAA' from HJ X VV or recipro-
cal was slightly unstable; AA'VV from HJ X VVVV
produced only proembryos; AA'AFVV from (HJ X HC)
(6x) X VVVV was somewhat unstable, exhibiting
variable metaphase I counts and production of

polyhaploids, intermediate progeny and a hybrid.

Very good seed set, but very low production of
mature plants through embryo transfer, while
closely related crosses produced mature plants

relatively easily with the aid of embryo culture.

Variable metaphase I counts.

Production of hybrids and haploids.

Preliminary embryo squash results also agreed with

the chromosome elimination hypothesis (Table 9). Orton

found the mean chromosome number of (HJ X HC)(6x) X VVVV

embryos was depressed from the expected zygotic chromosome

84

.h.mm ma >> x meVHum x one no moaunam UHo >mo cm>om Hon Hones: osOmQEOHao cooza

 

 

 

m.mN o v o m o v m m m N o o N N H o N m
.m.mN N e H o m H H H . m o H o o o o N N N
m.NN H m m o N H H o N H m H m o o N v o
¢.Nm m e o o N H H H m H H H o N o o o m
0.0m H m H N N o m m H H o o o N H H N v
8.0m H v m N H o N H H H N N o o o H o m
n.0m N H o m H N N v H m N N o H o N o N
mm.mN H m o H H o N H N H H H m o H N o H

mmmzsz (mm sane mm «H mm Nm Hm om mN mN NN 6N mN «N mN NN HN HN amps
H@ mm“ mm
ozomozommo u o 6H onsazHHHom
magma muse
zmmz

EMU mmm mmuflumOSOmmU

 

.>>>> x meVAum x way no mucooo noodvm omunfim humcfleflamum m.souuo .m mqmda

85

number of 35. This suppression was significant at the
.05 level for all days except day five. Only a relatively
small portion of this decrease in zygotic chromosome num-
ber could be attributed to reduction in gametic chromosome
number of (HJ X HC)(6x) (Starks and Tai, 1974) and E. X217
ga£g(4x) (Orton, unpublished). Thus by definition, chrom-
osome elimination had occurred. Chromosome elimination
had also caused considerable instability in chromosome
number and the variance in chromosome number increased
with the age of the embryos.

Gupta and Gupta (1973) found a gradual transition
in phenotype and chromosome number in a cross of tobacco
similar to that expressed by the pentaploid. Nicotiana

suaveolens (2n=32) was crossed with N, glutinosa (2n=24).

 

In hybrid tissues, 5. glutinosa traits were dominant. For
the first few weeks the plants maintained a E. glutinosa
phenotype. Gradually, the phenotypic expression began to
shift toward the E. suaveolens phenotype. Root tip and
premeiotic anther squashes showed a high average frequency
of aberrant mitotic anaphases (12% with bridges; 11% with
fragments). Metaphase I counts showed a range of 24-29

chromosomes in a single flower. Mitotic metaphase,

86

examined in petals, also showed a similar variation rang-
ing from 24-29. The ranges in mitotic and meiotic counts
seemed to reflect prior mitotic abnormalities. Also,
metaphase I counts seemed representative of the somatic
variation of nearby tissues. By the use of mitotic kary-
otype, the authors were able to show that the transitional
change in phenotype was positively correlated with loss of

pg. glutinosa chromosomes. Their results indicated: 1)

 

metaphase I count variation corresponded to somatic varia-
tion of nearby tissues, 2) loss in characters of one
parent corresponded to loss in chromosome number, particu-
larly loss in chromosomes of that parent, and 3) mitotic
and meiotic counts reflected preceding mitotic abnormali-
ties.

It is only reasonable to assume that many of these
findings also apply to Hordeum material. Comparision of
the tobacco results with those available for the E. 231227
EEETEI vulgare material also shows evidence of actual loss
of chromosomal material. Triploid VBB hybrids haveiabout
5% of their somatic cells with micronuclei. This compara-
tively low frequency closely agrees with the small varia-

tion in chromosomal number in embryo counts of this hybrid.

87

The frequency of micronuclei in shoots of the diploid VB
hybrid was much higher and more variable, which was in
close agreement with embryo squash counts. The metaphase
I chromosome number range was large for both hybrids, but
the variance for VB was considerably higher. This seems
to reflect the preceding mitotic abnormalities of these
hybrids. Thus it would appear metaphase I counts are
representative of the somatic variation of surrounding
tissues.

The greater variability in micronuclei of the VB
hybrids possibly indicates that once the chromosome num-
ber deviated enough from the hybrid constitution more
rapid loss of chromosomes occurred. This agrees with the
cross results indicating g. bulbosum chromosomes exert a
stabilizing effect for their own retention. This would
also effectively explain the rapid change in phenotype
of some VB tillers.

Researchers working with the g. bulbosum-g. vulgare
hybrids found little or no reciprocal differences in chrom-
osome elimination. I found definite maternal effects in
g. vulgare-g. jubatum crosses. Metaphase I counts of ma-

terial with a hybrid phenotype from HJ X VV and VV X HJ

88

are essentially the same. Murry (1975) found a metaphase

I range of 16—22 for my VV X HJ hybrids and Rajhathy and
Morrison (1959) found a range of 17-23 for their HJ X VV
hybrids. Thus it appeared that the same basic somatic
variability was expressed in hybrid tissue of both crosses.
None of my nine HJ X VV hybrids produced haploid g. vulgare
tillers. Three of my eight VV X HJ hybrids produced hap-
loid g. vulgare tillers. This would seem to indicate that
in g. jubatum cytoplasm there was a strong lack of competi-
tive ability of cells, tissues and tillers losing too many
g. jubatum chromosomes. The extreme lack of vigor of g.
vulgare derivatives in a g. jubatum cytoplasm strongly
supports this conclusion.

The pentaploid (HJ X HC)(6x) X VVVV hybrid was pro-
duced in the spring of 1974. The first g. vulgare back-
cross of (HJ X HC)(6x) X VV-B1.2- had been produced by
Chung Lee in December of 1969. Thus, before I began my
study the Bl.2 had nearly three years to stabilize pheno-
typically and in chromosomal variability. This plant has
remained basically stable in phenotypic expression since

then. Its phenotype was as stable as any of these hybrids

of the wild species.

89

By the fall of 1974, these plants could be compared
cytologically and morphologically. Starks (unpublished)
found metaphase I chromosome number varied from 15-34 for
the Bl.2 and from 19 to 35 for the pentaploid progeny
(Table 8). In 1974 the pentaploid had the expected pheno-
typic characters to go with a larger culm diameter. The
pentaploid had fewer but larger tillers, which were usually
taller and had a larger head. The pentaploid also had an
more g. vulgare-type head and the auricles were usually
more pronounced. For all these characters the pentaploid
expression was more like fl. vulgare.

The pentaploid underwent a gradual shift in mor-
phology until by December of 1975, head morphology of the
pentaploid and Bl.2 were essentially identical. That is,
heads removed from both plants and mixed could not be
distinguished. Expression of the other characters was
very similar for the two plants. These characters were
much more affected by microenvironmental factors, particu-
larly within pot competition factors.

Clones of both the pentaploid and Bl.2 were divided
and paired comparisons made in two environments. Again,

other morphological traits appeared essentially identical.

90

The pentaploid expressed a somewhat greater variation in
head type indicating it was not as yet completely stable
phenotypically. From a mixture of heads of both penta-
ploid clones, some pentaploid partial reversion heads
could be separated out. The rest of the heads of the
pentaploid were still impossible to distinguish from the
heads of the Bl.2.

In the winter of 1975, another set of metaphase I
counts were made on the pentaploid by Gil Starks (Tablein.
One count involving 19 cells was from four or five florets
from two spikes. This count probably represented a chance
event of finding two spikes with essentially the same
chromosome variation. The remarkably small range also
indicated that variation within a spike was small. The
other count of ten cells came from one floret on another
Spike. Again there was a remarkably narrow range in
chromosome number. It would seem that selection for the
pentaploid's 1974 phenotype could still be partially suc-
cessful.

Morphologically, the pentaploid hybrid had lost
its more intense expression of g. vulgare characteristics

until it could not be distinguished from the Bl.2. From

91

morphological observations it appeared that many of the
cells had lost several E. vulgare chromosomes. The 1975
preliminary cytological observations were too biased (the
sample was unrepresentative of the range and variation
present in the whole clonal population of cells) to draw
any conclusions. Development of considerable variability
in chromosome number would seem reasonable for unstable
hybrids having stable intermediate forms. For example,
the tetraploid progenies of (HJ X HC)(6x) X VV with one
set of g. vulgare chromosomes were stable. Loss of g.
vulgare chromosomes in pentaploid progenies would tend
toward this type of intermediate form. The variation
present in embryo squash counts of the (HJ X HC)(6x) X
VVVV cross strongly supported the idea that pentaploid
progenies could develOp the chromosome variation found in
the Bl.2 progeny.

I can postulate only one viable hypothesis on how
the pentaploid progeny could develop the same phenotype
as the Bl.2. Fortunately this hypothesis also has a good
explanation for the other observed results. I postulate
the Bl.2 and the pentaploid are basically both of penta-

ploid constitution. This requires that the Bl.2 was

92

produced from the fusion of a tetraploid egg from the
tetraploid hybrid with a normal pollen grain from diploid
H. vulgare. The tetraploid egg could be produced from a
doubled sector or could be an unreduced egg.

The extremely high sterility of the (HJ X HC)(6x)
X VV hybrid supports the conclusion that the Bl.2 origin-

ated from a tetraploid egg. In the cross Triticum durum

 

X Aegilops longissima, Vardi and Zohary (1967) found all

 

viable T. durum backcross progenies were pentaploids and
thus originated from unreduced eggs. The frequency of
viable progeny closely corresponded with the frequency of
restitution nuclei (all chromosomes end up in one nucleus
at the end of meiosis I). Restitution nuclei have not
been observed in the tetraploid (HJ X HC)(6x) X VV hybrid,
but this does not preclude the idea that this mechanism
or some other mechanism could not on rare occasions pro-
duce unreduced eggs. Chung Lee treated 69-101 with col—
chicine, so a small doubled sector could also have been
produced. Thus production of a Bl.2 pentaploid seems
quite reasonable; in fact, it is probably the most likely

type of progeny.

93

The pentaploid constitution hypothesis easily ex-
plains why the B1.2 and the pentaploid have the same phe-
notype now. It also fits nicely with the observed meta-
phase I chromosome variation of the Bl.2. Furthermore,
it offers a reasonable explanation for the origin of 69-
101-1. 69-101-1 and the intermediate progeny have con-
siderable gross morphological similarities. 69-101-1,
originating as a chromosome elimination product from a
pentaploid zygote seems much more reasonable than from
selfing of the extremely sterile (HJ X HC)(6x) X VV hybrid.

In summary, observation of how the pentaploid hy-
brid has slowly changed over time gives an example of how
the Bl.2 probably originated.

Since there is excellent evidence for the Bl.2
having a pentaploid constitution, the next section deals

with the breeding implications of this hypothesis.

VII. BREEDING IMPLICATIONS OF
THE HYPOTHESIS THAT THE
Bl.2 HAS A PENTAPLOID
CONSTITUTION

 

The diploid g. vulgare genomic constitution of the

12 second H. vulgare backcross progenies presents a

94

difficult obstacle to the production of the desired par-
ental material for an intermating populatiOn. The paren-
tal material should ideally be a series of plants with
reasonable fertility and all expressing part of the de-
sired improvements in the gentic background of the recip-
ient species. Thus the plants could be essentially a
diploid fl. vulgare with one or two g. jubatum chromosomes.
These plants could be cyclically intermated with selection
pressure to maintain the desired g. jubatum chromosomes
and to fix desired translocations when they occur.

There are two possible explanations for the failure
to produce the desired parental material. These possible
mechanisms must explain why the only viable mature progeny
have a diploid g. vulgare constitution. The possible mech-
anisms are: 1) All viable eggs have a haploid g. vulgare
constitution and 2) Viable eggs have some wild chromosomes
plus a haploid g. vulgare set, but the only progeny to sur-
vie to maturity have all the wild chromosomes eliminated.

Either one of these mechanisms would have profound
implications on the chances of getting the desired parental
material. Thus knowing which is operating could be ex-

tremely important. Without knowing the genomic

95

constitution of the Bl.2, it is impossible to evaluate which
is the more likely mechanism operating in the material. Since
the only viable hypothesis on the genomic constitution of the
Bl.2 is the pentaploid hypothesis, I have evaluated the re-
sults in terms of the Bl.2 beingga pentaploid.

Chromosome elimination is a logical explanation be-
cause the most likely viable egg gametes from an AA'A"VV pro-
geny are gametes with essentially two or more full genomic
sets, for example, AV, A'V, A"V, and A'V. I base this state-
ment on the genomic constitution overserved in backcross pro--

geny of other Triticeae crosses (Linienfeld, 1951; Kihara,

 

1963; Furuta and Tanaka, 1970; Vardi, 1973; and Vardi, 1974).
Fusion of these types of viable eggs with a pollen grain from
diploid g. vulgare would produce zygotes which could be com-
pared with those produced in species crosses. Kasha (1974)
showed the genomic constitution of the embryo was the most
important factor in determining embryo chromosomal stability
and the genomic constitution of the female parent had sur-
prisingly little effect.

AA‘A"V from (HJ X HC)(6x) X-VV was at the genomic
balance point (Table 6). .VAAf from VV X HJ was slightly

underbalanced (not enough wild chromosomes) and had

96

considerable chromosomal variation occurring at metaphase
I (a range of 16-22; Murry, 1975). These hybrids also
occasionally produced haploid g. vulgare tillers. Thus
progeny of this constitution tended to lose wild chromo-
somes. AA'A"VV progeny from (HJ X HC)(6x) X VVVV were
slightly overbalanced and tended to lose g, vulgare chrom-
osomes. AA'VV progeny from HJ X VVVV only developed to
the proembryo stage, suggesting excessive problems with
chromosome elimination. The most viable eggs (excluding
haploid g. vulgare eggs) would be poorly balanced genomic-
ally and chromosome elimination would be expected to be

a strong force.

At this point, it was important to determine the
chance of obtaining haploid g. vulgare eggs from AA'A“VV
hybrids. This required making some assumptions and using
the binomial expansion. I chose to first estimate the
upper limit for production of haploid g. vulgare eggs.

The most favorable circumstances for production of haploid
eggs would be to have perfect pairing of the g. vulgare
chromosomes (a 7-7 Split at anaphase I) and no pairing of

the E. jubatumHH, compressum (AA'A“) chromosomes. Thus

 

the AA'A“ chromosomes would distribute randomly to the

97

poles. The population of egg gametes would all have a
haploid g. vulgare constitution plus §.jubatum—§. £227
pressum chromosomes determined by the binomial expansion
of (1/2 + l/2)21. The chance of any one egg having only
a haploid g. vulgare constitution is (1/2)21, or approxi—
mately 5x10-7. Since approximately 80,000 florets of the
Bl.2 were pollinated, the chance of producing one diploid
g. vulgare was 80,000 (5X10)'—7 or approximately .04.

The chance of producing twelve diploid g. vulgare
progenies from pollination of 80,000 florets is 5.5 X
10-26. This comes from the Poisson distribution, where
u12/121eu is the chance for 12 such events in any one
sample (Sokal and Rohlf, 1969). U can be found by solving
u/eu=.04, where e=2.71828 and .04 is the chance for one
event. U is approximately equal to .0417. Thus, even
making these ridiculously ideal assumptions, the chance
of producing 12 haploid g. vulgare eggs is so close to
zero it can be ignored. Therefore, it would appear that
chromosome elimination played a very important role in

preventing production of the desired parental material

by classical backcrossing methods in this cross.

FUTURE DIRECTION

This study has shawlthat material having a cyto-
plasm from H. jubatum is a dead end and chromosome slime
ination is a powerful force in determining numbers and
type of mature progenies produced from many of the crosses.
Both of these factors have a profound influence on the
chances of producing the desired parental material for an
intermating pOpulation. Some progress has been made with
material having H. vulgare cytoplasm. Techniques includ—
ing tissue culture are being utilized to take advantage
of chromosome elimination in the production of the desired
parental material for an intermating population to trans-
fer traits to H. vulgare.

Dr. Tom B. Rice has found it relatively easy to
callus, subculture and regenerate large numbers of plant-
lets of these hybrids. He has used colchicine treatment
of callus cells of VV X HJ hybrids to produce amphiploid
material of this cross. Root tips of roughly one half of

the plantlets from treated callus material have the

98

99

amphiploid chromosome number. A single H, vulgare back-
cross seed was produced from a mature plant from one of
these plantlets. This seed appeared to have a completely
normal endosperm with plenty of starch, but no embryo was
present.

These results indicate there are no major barriers
to the production of callus cultures, treatment of callus
cells) and regeneration of large numbers of plantlets.
These plantlets can be very successfully grown to maturity.
These are all necessary steps in the use of tissue culture
to produce an amphiploid of VV X HJ and the subsequent use
of tissue culture to help produce the desired parental ma-
terial for an intermating population from this amphiploid.

At present, I consider two different approaches to
be the most promising prospects for the production of this
parental material from this hypothetical, at present, (VV
X HJ)(6x) amphiploid. The first approach is production of
embryos of the backcross to H, vulgare and the subsequent
production of large numbers of plants with different chrom-
osome number from these embryos by utilizing tissue culture
techniques to callus embryos and regenerate large numbers

of plantlets. Chromosome elimination will provide the

100

directional driving force for the production of material
with different chromosome numbers. Hopefully, many of the
plants will be essentially back to a diploid H. vulgare
constitution, but the direction chromosome elimination
would operate in embryos of VVAA' genomic constitution

can not be determined from the available cross data.

The second approach involves use of callus culture
of the amphiploid to produce large numbers of plantlets
with different chromosome numbers.‘ Again, chromosome
elimination will provide the directional driving force for
the production of material with different chromosome num-
bers. Hopefully, many of the plants will be essentially
back to a diploid H. vulgare constitution. Since chromo-
some elimination is operating in the VV X HJ hybrids in
the direction of loss of H. jubatum chromosomes, it would
seem quite reasonable to assume H. jubatum chromosomes
would also be lost in its amphiploid.

I am very hopeful that one of these approaches will
eventually produce the desired parental material for an in-
termating population, which will lead to the transfer of

traits to H. vulgare.

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APPENDIX

109

TABLE A. Norstog's agar medium II.

COMPONENT AMOUNT

 

g/liter medium

 

Major minerals

KH2P04 0.91
KCl 0.75
MgSO4o7H20 0.74
CaC12°2H20 0.74

Trace elements and vitamins* mg/liter medium
MnSO4'H20 3.0
H3303 ‘ 0.5
anO4°7H20 0.5
CoC12-6H20 0.025

, CuSO4°5H20 0.025
NaMoO4 0.025
Inositol (meso-) 50.0.‘
Thiamine-HC1 0.25
Ca-pantothenate 0.25
Pyridoxine-HC1 0.25

Iron source* mg/liter medium
Sequestrene (sodium ferric 16.0

diethylenetriamine pentaacetate)

Amino acids g/liter medium
L-Glutamine 0.4
L-Alanine 0.05
L-Cysteine 0.02
L-Arginine 0.01
L-Leucine 0.01
L-Phenylalanine 0.01
L-Tyrosine 0.01

Other
Malic acid** 1.0
Difco Purified Agar 6.0
Sucrose 34.2

 

*100X stock solution is useful
**Dissolve malic acid in 30 ml H20 and adjust pH to 5.0

110

TABLE B. Wick embryo culture medium.

 

 

MAJOR MINERALS‘ g/liter medium

 

 

 

 

 

 

Ca (N03) '4H20 0.58
MgSO4'7H20* 0.33
KNO3 0 . 08
KCl 0.005
NaH2P04-HZO 0.038
MICRONUTRIENTS** mg/liter medium
MnSO4 0,45
H3BO3 0.00375
KI 0.03
Glycine 3.0
Thiamine-HC1 0.1
Ca-pantothenate 2.5
CuSO4-5H20 0.025
NaMOO4 0.025
CoC12-6H20 0.25
IRON SOURCE**
Sequestrene (sodium ferric
diethylenetriamine pentaacetate) 16.0
SUGAR SOURCE q/liter medium
Sucrose 20.0

 

*Dissolve separately. .
**100X stock solution is useful.
Adjust complete medium to pH 5.4.

MICHIGAN STAT

ll WI 11))

32

mm“

1