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,u,.,,'l.-‘.. t 9."; . . , ...'. 3.; . ‘ ““:‘.‘A"' c3“
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-'¢¢' 'z‘:
. .

 

 

Ira-135i:

 

This is to certify that the

thesis entitled

Intorgonorio hybridization in crosses involving
barley and its wild relatives

presented by
Rye-Ho Huang

has been accepted towards fulfillment
of the requirements for

Ph. D. Gr 8
degree in op cionco

/%Z%

 

 

Major professor

 

0-7639

§

INTERGENERIC HYBRIDIZATION IN CROSSES
INVOLVING BARLEY AND ITS WILD RELATIVES

by

Rye-Ho Huang

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

DOCTOR OF PHILOSOPHY

Department of Cr0p and Soil Sciences

1978

ABSTRACT

INTERGENERIC HYBRIDIZATION IN CROSSES
INVOLVING BARLEY AND ITS WILD RELATIVES

by

Rye-Ho Huang

Karyotype analysis of Agropyron trachycaulum, Hordeum

jubatum, Hordeum vulgare and their hybrid series (AHV442,

 

AHV444, and VH-35) were investigated for chromosome exchange

and potential transfer genes from either agropyron trachycaulum

 

or Hordeum iubatum into Hordeum vulgare. Cytological tech-

 

 

niques and light microscopic observations were unsatisfactory
in detecting chromosome exchange. This was partly due to the
similarity of karyotypes of the genomes involved.

Since the amphidiploid of Agropyron trachycaulum and

 

Hordeum jubatum as female was crossed with diploid and

 

tetraploid Hordeum vulgare, the first backcross was achieved

 

using one of the restored AHV-35 derivatives as female and

diploid Hordeum vulgare as the pollen source. Chromosome

 

association observed in the pollen mother cells of the back-
cross showed a significant decrease in univalents. Based on
comparative cytological observation in AHV442, AHV444 and
AHV-Bl, a hypothesis of genetic control on genome interaction
was derived to elucidate the phenomenon of preferential

chromosome elimination. The first backcross, by genome

61‘.

VB.

 

 

formula was composed of at least two dosages of Hordeum vul—

gare genome, therefore genome balance between Hordeum vulgare

 

and the others was achieved by adding one Hordeum vulgare

 

genome at a time to the other combined genome of Hordeum

jubatum and agropyron trachycaulum. The significance of

 

introgressive hybridization between cultivated barley and

its wild relatives was discussed. Since some cultivated germ-
plasm is vulnerable to epidemics and limited in adaptation

due to man's disturbance, the highlight of this study was
emphasizing the reconstruction and replenishing of the culti-

vated germplasm through hybridization to its wild relatives.

ACKNOWLEDGMENTS

My appreciation is expressed to Dr. J. E. Grafius,
my major professor for his patience, encouragement and
assistance throughout the course of study and manuscript
preparation; to Dr. D. H. Smith for his all-out assistance;
to Dr. W. Tai for his research training; to Dr. P. Carlson
for his encouragement and to Dr. W. Magee for his phi1030phy
of self discipline.

Thanks also to Dr. C. M. Harrison for his regard and
manuscript review; to Mr. D. E. Wolfe for his friendship.
To my field and laboratory colleagues, A. M. Maddur, B. H.
Zakri, R. P. Steidl, A. Al-Shamma, J. Shyte and J. E. Nelson
go my thanks for friendship, and G. D. Starks, L. E. Murry
and J. Whallon for their suggestions in cytogenetics.

Financial support for my study and research was
managed by Dr. J. E. Grafius. This is very gratefully

acknowledged.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . .
LIST OF FIGURES. . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . .
LITERATURE REVIEW. . . . . . . . . . . . . . . . .

Interspecific and Intergeneric Hybridization. .
Chromosome Elimination. . . . . . . . . . . . .
Karyotype Analysis. . . . . . . . . . . . . . .

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

Plant Material. . . . . . . . . . . . . . . . .
Methods of Hybridization. . . . . . . . . . . .
Embryo Culture. . . . . . . . . . . . . . . . .
Tissue Culture. . . . . . . . . . . . . . . . .
Cytological Techniques. . . . . . . . . . . . .
Feulgen-Aceto Carmine Differential

Banding Technique. . . . . . . . . . . . . .

RESUIJTS O O O O O O O O O O O O O O O O O O O O O O

Karyotype analysis. . . . . . . . . . . . . . .
Results of Hybridization and

Cytological Studies. . . . . . . . . . . . .
Results of Genome Interaction and

Chromosome Elimination in AHV444 . . . . . .

DISCUSSION . . . . . . . . . . . . . . . . . . . .

InterSpecific and Intergeneric Hybridization
as a Means of Improving Barley Winterhardiness

Chromosome association as an indicator of
the course of chromosome elimination . . . .

Karyotype analysis. . . . . . . . . . . . . . .

SUMMARY. . . . . . . . . . . . . . . . . . . . . .

iii

Page

vi

15
15
15
16
19
19
23

23
46
47
68

68

73
79

83

Page
APPENDIX. 0 O O O O O O O O O O O O O O O O 0 0 O O 84
Literature Cited . . . . . . . . . . . . . . . . 98

BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 101

iv

Table

1A

2A

LIST OF TABLES

A SUMMARY OF BARLEY INTERGENERIC
AND INTERSPECIFIC HYBRIDIZATION. . . . . .

ORIGIN AND DESIGNATION OF RESEARCH MATERIALS.

THE ARM RATIO, DIFFERENCE AND DESCRIPTION
OF A. TRACHYCAULUM KARYOTYPE . . . . . . .

 

DESIGNATION OF ALL CROSSES ATTEMPTED. . . . .
THE CHROMOSOME ASSOCIATION OF PLANTS. . . . .

CHROMOSOME NUMBER AND ITS VARIATION
IN MICROSPORE IN AHV444. . . . . . . . . .

Number of Seeds and Plants Obtained
in Hybridization . . . . . . . . . . . . .

The Chromosome Association of Plants. . . . .

30
46

47

55

87

88

Figure

10

11
12
13

14

15.

1A

The karyotype
The karyotype
The karyotype
The karyotype
The karyotype
The karyotype

Histogram for

Distribution of chromosome index versus
ploidy in hybrids AHV444, AHV442 and VH-35 .

Plate A.

Plate B.

LIST OF FIGURES

of

of

of

of

of

of

the ploidy and chromosome
of A. trachycaulum, H. jubatum and H.

H. vulgare with 2n=l4,

I212

A. trachycaulum-

 

AHV442 with 2n=35.

AHV444 with 2n=42.

VH—35 with 2n=35

AHv-Bl o o o o o o o o o a

Plate C.
Plate D.

Plate E.
number

Plate F.
number 19,

Plate G.

16,

15.

Metaphase II in AHV444.

16 and anaphase .

Pollen cell in AHV444

. jubatum with 2n=28.

Pollen mitosis with chromosome
21, 14,

Pollen mitosis with chromosome
18,

Cytological study of AHV442 and AHV444.

vi

vulgare

Chromosome association in amphiploid
(A.t.XH.V.), AHV442, AHV444, and AHV-Bl. . .

Spike of A. trachycaulum, natural
hybrid (A.t.XH.v.), AHV442, AHV444 and

Metaphase II and tetrad in AHV444

Page
25
28
30
34
37

39

42

51

53
59

61

63

65
67
92

INTRODUCTION

For several major crops, genetic vulnerability in
terms of a lack of genetic variability for stress adapta-
tion has been called to the attention of plant breeders.
Germplasm improvement of cr0ps can be achieved more easily
by introgression through hybridization than by induced
mutation of genetically exhausted cultivars. The domain
of introgressive hybridization includes wide crosses be-
tween similar Species and backcrosses to cultivated progeni-
tors. Triticale is an example of a crOp synthesized by

wide hybridization between Triticum spp. and Secale cereale L.

 

The gene pools of wheat and corn can be and have
been replenished by backcrossing to their respective pro-
genitors. In the genus Hordeum wild species have been con-
sidered a useful source of genes carrying specific attri-
butes for possible transfer to the cultivated forms. A
list of hybridizations of barley with its wild relatives
has been summarized (Table 1). Genetic transferring has
not been satisfactory since pollen incompatibility, hybrid
lethality, hybrid sterility and infrequent recombination

block successful hybridization on a practical scale.

2
The basic task of introgression in barley depends
largely on the strategy of genetic manipulation at the
genome level. This study presents a case for using the

complex genome derived from H. jubatum, H; trachycaulum

 

and H. vulgare. The hybrids have created the potential
for a wide scale genetic association between H. jubatum

and H. vulgare or A. trachycaulum and H. vulgare. The

 

impact of a complementary influence of a third genome is
significant. A series of intermediate hybrids has been
synthesized during the course of a backcross to H. vulgare.
Karyotypic analysis of plants and their hybrids might lead
to an understanding of their relationship in terms of
genome compatibility and chromosome association. The
possible mechanism of the phenomenon of chromosome elimi-
nation was investigated for its significance in genetic

transfer.

LITERATURE REVIEW

IntersBecific and
Intergeneric Hybridization

 

 

Unconventional hybridization of economic crops is
an ideologic means for plant breeders to marshall a new
array of germplasm capable of increasing adaptation as
well as production. The feature of wild hybridization
occasionally advocated and substantiated in some cases
has intrigued both plant and animal breeders.

Gordon and Raw (1932) crossed wheat and barley and
obtained a wheat-like fertile plant. No barley-type pro-
genies appeared in the subsequent F2 to F4. They postu-
lated the cause as parthenogenic stimulation and an
accompanying doubling of the maternal chromosome complements.
More likely, the progeny was the result of an accidental
self-pollination.

The idea of using chemical agents to circumvent
crossing barriers between wheat and barley was prOposed
by Bates (1974), but the results failed to produce a true
hybrid from the immunosuppressant treatment.

H. vulgare crossed with Secale yielded somewhat
similar results. Quincke (1940) reported that fertilization

evidently took place in the hybridization between Hordeum

4

vulgare hexastichum x Secale cereale but the seed ceased

 

 

to develoP in early stages. In addition, Thompson and
Johnson (1945) further proved that fertilization took place
in 90 percent of the ovaries. Embryos develoPed normally
in the early stages but disintegrated following endosperm
breakdown. Cytological study showed an abnormal size and
number of nuclei in the endosperm. Similarly, the pheno-
menon was observed in certain other species by Huskins
(1948); Thompson (1962); Bammi (1965), and Rizzoni et al
(1974). They suggested that the cause of separation and
grouping of chromosomes was related to genome origin. The
genetic make-up and the development of the endosperm are
complex features because of the double fertilization
involved. The relationship between embryo and endosperm
in the course of seed develOpment is not understood. At the
present time, embryo culture has been develOped to serve the
practical need of rescuing the embryo before it aborts.
Brink and CooPer (1947) used embryo culture to obtain a
hybrid between Secale and Hordeum species. They indicated
that mitotic irregularities were the result of malfunction—
ing of the antipodals. Chromosome abnormalities were pre-
valent in the form of lagging chromosomes, split univalents.
bridges, micronuclei etc.; therefore, the hybrid was sterile
and no further backcross was obtainable.

Fedak (1977) reported obtaining a haploid barley in
the cross between barley and Secale with no clue to the

treatment during the course of hybridization. Selective

5
chromosome elimination was given as the cause for producing
haploid barley.

Hybridization between Hordeum vulgare and Elygus Spp.

 

has been uneXpectedly successful. Although Smith (1942)
and Reznicuk (1939) failed in their attempt to produce a

hybrid between Elymus Spp. and Hordeum vulgare, Bakhteiev

 

and Darevskaya (1945) were able to hybridize Hordeum vulgare

 

with H. arenarius and H. giganteus. Morphologically, this

 

 

hybrid had intermediate characteristics and 21 somatic
chromosomes. Bakhteiev and Darevskaya (1975) also reported

a cross between Hordeum spontaneum C. Koch em. Bacht. (2n=l4)

 

and Elymus arenarius L. (2n=56). Unfortunately, that hybrid

 

failed to reach complete deve10pment. Ahokas (1973) speci-
fied evidence of cytOplasmic influence in a barley and

Elymus hybrid. When using Hordeum vulgare as female, four-

 

teen viable hybrids were obtained, but none survived when
Elygus was the female. Subsequently, the barley chromosomes

were eliminated resulting in monOploid H. arenarius in

 

Hordeum vulgare cytoplasm and the plant finally produced a

 

fertile tiller. Earlier, Pissarev (1945) even obtained a
haploid barley instead of a haploid Elygus from a barley and
Elymus hybridization. Korablin (1937) reached the point of
obtaining 107 seeds from a barleyéElyEus cross. Schooler*
obtained promising winter-type barley derived from a barley

(Dicktoo 2x) X Elygus (4x) hybridization.

 

*Personal communication with Dr. J. E. Grafius

6
Hybridization of barley with wild Species was extended
to AgrOpyron spp. by Smith (1942). Since then no success
has been reported. But natural hybridization between
AgrOpyron and Hordeum has frequently been discovered, such

as North American ngohordeum G. Camus ex A. Camus, (Stebbins

 

et al 1946); Elymus Macounii complex of Agropyron trachycaulum

 

(Link) Malte and foxtail barley; Hordeum jubatum by Keller

 

(1948); Booher and Tryon (1948); Forsberg (1953), AgrOpyron

 

pilosilemma x Hordeum jubatum by Mitchell and Hodgson (1965a);

AgroPyron, Oschense. Roshev., and Hordeum turkestanicum by

 

Nevski (1934).

Eventually, a few cases fulfilled the breeding pur-
pose of transferring agronomic traits from wild Hordeum spp.
into cultivated barley. Two successful genetic transfers
for disease resistance are from crosses between H. vulgare
and H. leporinum by Hamilton et al (1955), and from H. 1222'
EIEEE by Hamilton et al (1955), and from H. bulbosum by

Schooler (1964).

TABLE 1

A SUMMARY OF BARLEY INTERGENERIC
AND INTERSPECIFIC HYBRIDIZATION

 

 

Interspecific

 

Crosses

(H. brachyantherum
x H. bogdanii (6x))
x H. vngare (4x
Traill)

H. vulgare x H.

Californicum

 

H. vulgare (4x)
x H. bulbosum

. vulgare x H.
rachyantherum

. vulgare x H.

gpressum

. vulgare x H.

ubatum

 

 

IE CHE Cflm

i

H. vulgare x H.
leporinum

 

H. vulgare x H.
murinum

. vul are (4x) x
. Bngosum (4x)

IEEItI:

H. Lechleri (6x) x
vulgare (2x)

(H. jubatum x H.
compressum (6xT) x

H. vulgare (4x)

 

* The phenomenon of

Results

Hybrid & progeny

Hybrid died before
maturity*

Hybrid
Hybrid
Hybrid

Hybrid

Hybrid*

Hybrids died in the
seedling*
Parthenocarpic seed
Hybrid died before
maturity

Haploid H. vulgare*

Hybrid and haploid*
H. lechleri

Hybrid and haploid
(3x)

Literature

 

Schooler (1976)

Davies (1956,
1960)

Davies (1958)
Schooler (1964)

Morrison et a1
(1959)

Morrison (1959)

Morrison &
Rahathy (1959)
Steidl (1976)

Hamilton, Symko
and Morrison
(1955)

Malloch (1921)
Forlani (1950)

Morrison et a1
(1959)

Lange (1969,
1971a)

Kao & Kasha (1970
1969) Konzak et
a1 (1951)

D. H. B. Sparrow
(1974)

Steidl (1976)

Chung Lee (1970)
Schooler (1964)

chromosome elimination involved

8

Table l-Continued

 

 

 

 

 

Intergeneric
Crosses Results

H. vulgare x H. cereale Two hybrid plants
and a barley
hybrid*

_. vulgare x H. jubatum Hybrid but no
chromosome asso-
ciation

H. vulgare x H. cereale Haploid barley*

H. vulgare x Unconfirm hybrid*

H. vulgare

H. vul are x Elygus Hybrid and progeny

TZx)

H. vulgare x H. aren- Haploid Elymus in

arius H. vulgare cyto-
plasm

Literature

 

Thompson, W. P.
D. Johnston
(1945)

Steidl (1976)

Fedak (1977)

Bates (1973)
Fedak (1977)

Schooler (1976)

Ahokas (1970,
1973)

* The phenomenon of chromosome elimination involved

 

Chromosome Elimination

 

The phenomenon of chromosome elimination is ubiqui-
tous in unconventional hybridization in both plant and
animal cells. The effort to unfold the mechanism of
chromosome elimination has practical and theoretical value.

Chromosome elimination refers to the cause of select-
ive loss of chromosomes of one genome. It is characterized
by a complex interaction involving genetic and physiological
control. Earlier, insect researchers encountered the
phenomenon in the family Diptera. The phenomenon was first
described by Kahle (1908).

Later on, attempts to interpret the phenomenon of
chromosome elimination were made by Huettner (1934),
Kraczkiewicz (1936) and Metcalfe (1935). Chromosome elimi-
nation occurs immediately after fertilization and continues
through embryo deve10pment. Generally, a set of daughter
chromosomes was excluded from the major group without recon-
stitution in the daughter nuclei. Kraczkiewicz (1936)
extended this explanation to include the state of cytOplasm
reaction and specified the function of the Spindle while in
mitotic cell division. A similar assumption was suggested

by Du Bois (1933) for Sciara CoprOplila. On the other

 

hand, Geyer-Duszynska (1959), working with Wachtliella

 

persicariae embryos, pinpointed errors in the functioning

 

of the centromeres of germ line chromosomes as the direct
cause of their elimination from somatic nuclei. Bantock

(1970) even detected chromosome elimination occurring at the

lO

fifth division of Mayetiola destructor embryos. Camenzind

 

(1974) concluded that the predictable loss of Specific
chromosomes during embryological develOpment was due to
complex genetic and physiological controls. Chromosome
elimination prevailing in the families (Diptera); the

Ceciodmyidae, the Sciaridae, and the Chironomidae was attri-

 

 

 

buted to the heterogeneous genetic makeup and diverse origin
of family.
In the plant kingdom, the phenomenon of chromosome
elimination is assumed to play a unique role in evolution
as well as Speciation because it acts as a screening mecha—
nism for compatible genomes in natural hybridization. It
was a frequent event in wide hybridization under controlled
hybridization between certain species and genera which
puzzled plant breeders. Nevertheless as a mechanism per se
it has been neglected by plant breeders due to its complexity.
Intensive study of chromosome elimination has been
characterized by interSpecific hybridization in the genus
Hordeum (Hamilton et a1 1955, Cauderon and Cauderon 1956)

and in the genus Nicotiana (Gupta and Gupta 1973). In corn,

 

Rhoades and Dempsey (1972) reported that B chromosomes might
cause the elimination of knob bearing members of the regular
chromosomal complement at the second microspore mitosis.

The hypothesis is derived from the regulatory gene system
extended to the difference in DNA replication in the hetero-
chromatic segment of knobbed chromosomes.

Alternatively, Kasha and Kao (1970) hypothesized that

11
Specific chromosomes in H. vulgare might carry factors con-
trolling genome balance and stability in the hybrid with
H. bulbosum L. Further investigation suggested that chromo-
somes 2 and 3 of H. vulgare contain major genetic factors
which are critical to the chromosome balance and stability
in interspecific hybrids between H. vulgare and H. bulbosum
(Ho 1975). These factors function only in the condition
that both are dominant and present in sufficient dosage to
overpower the Opposite factors in H. bulbosum. In other
words, chromosome elimination is preferential as well as
directional. In plants, evidence of chromosome elimination
can be obtained indirectly by morphological observation;
Gupta and Gupta (1973) demonstrated that directional chro-
mosome elimination has a high visual correlation with a
phenotypic shift from hybrid intermediacy in the direction
of the parent whose chromosomes remain. In addition, Starks
(1976) and Steidl (1976) observed a concomitant loss of
phenotypic characters associated with H. vulgare in a complex

plant; H. jubatum x H. compressum amphiploid crossed with

 

H. vulgare.

Karyotype Analysis

 

A karyotype is defined as the particular chromosome
complement of an individual or a related group of individuals
in terms of the number and morphology of the chromosomes in
mitotic metaphase. Karyotypes of different species and
'genera may differ with reSpect to their basic chromosome

number, shape and relative size as well as the number and

12
size of constrictions. Recently, chromosome banding patterns
of hetero and euchromatic chromosomal segments have been used
as a technique for karyotypic identification.

Karyotypes of barley chromosomes were first described
by Tjio and Hagberg (1951). They assigned Roman numerals to
each of the chromosome pairs, I to V designating the non-
satellited chromosomes arranged in order of decreasing total
length, VI the chromosomes with the large satellite and VII
the chromosomes with the small satellite. The accompanying
linkage groups in barley were elucidated by a group of bar-
ley cytogeneticists and this system has been further summar-
ized by Ramage et a1 (1951) and by Nilan (1964). Barley
may be one of the most genetically well-studied crOp plants
due to its special chromosome behavior and its economic value.

Studies such as induced mutation, linkage map con—
struction, genome relation and chromosome association in
barley have been facilitated by the series of translocation
stocks in cultivated species. But karyotypic study is the
cornerstone for all related studies. Kunzel and Nicoloff
(1975) called for a necessary revision of the barley karyo-
gram. They indicated that the interchangeable chromosomes
for revealing the linkage groups are associated with differ-
ent chromosomes. Although the banding system for chromosome
identification is under intensive study in certain plant
species, so far the results are still inconsistent (Sharma
et a1 1974, and Gill and Kingber 1974, Murry 1975).

Tuleen (1973) suggested that chromosome I might not

13
be the longest chromosome of the standard barley karyotype.
This view was later agreed to by Kunzel and Nicoloff (1975)
who studied a multiple translocation line. By employing
the C-banding Giemsa staining technique, Nada and Kasha
(1976) came to a similar conclusion that, namely, the karyo-
type identified from a trisomic line was different from that
of the standard karyotype.

Karyotypes have also served as a key to classify taxa
as a supplement to systematic classification based on gross
morphology of plants. Chromosomes are the physical bases
of heredity, their karyotype characteristics of species com-
paratively unique from one another. Richards (1972) reported
that extensive karyotypic variation was formed in the inter—

specific taxa of Taraxacum. He developed the karyotypic

 

similarity between taxa. Richards and Booth (1977) employed

this comparison formula within taxa of the Hordeum murinum

 

group and indicated that mitotic chromosomes in this group
showed a continuous variation in length from four to nine
microns. The karyotype transformed into a parameter might
reflect similarity as well as compatibility between species.
In a practical approach, Smith et a1 (1972) proposed screen-
ing the genus Secale for taxa with chromosome karyotypes
more like those of wheat. Then the use of wheat-like Secale
taxa as a parent in crossing with wheat might increase
chromosome association in Triticale combined genomes.
Superficially, the conventional chromosome association

in meiosis could also reflect on karyotypic similarity in

14
the hybrid. Wagenaar (1959, 1960) perceived the chromosome

size difference of Secale from Hordeum jubatum in the form

 

of univalents at Metaphase I.

Hordeum jubatum chromosome morphology was well

 

described by Morrison (1959). Covas (1949) and Hitchcock
(1950) made chromosome counts in terms of taxonomical clas-

sification. The relationship of Hordeum jubatum within the

 

genus Hordeum (by karyotypic study), especially to the
cultivated form of barley, implied both practical and evo-
lutionary Significance. Karyotypic analysis has even served
as a basic tool in describing the relationships within the
genus Hordeum (Rajhathy and Morrison 1959, 1961). By means
of karyotype analysis Schulz-Schaeffer (1960) were able to
demonstrate specific relationships in Bromus using satellite
chromosomes as indicators. In the same manner, Rajhathy

and Morrison (1961) Specified the satellite chromosome of

Hordeum jubatum in a tetraploid form. In addition to under-

 

standing Species relations, through karyotype analysis,
Morrison (1959) reported that the diploid Species Hordeum

marinum, H. maritimum, H. hystrix and H. gussoneanum possess

 

 

a similar karyotype and are probably conspecific. Similarly,
Rajhathy and Morrison (1961) also indicated by means of

karyotypic analysis that Hordeum jubatum and Hordeum brachy-

 

 

antherum are conspecific. With respect to breeding, Elkington
et al (1976) coincidently reported that the range of banding
styles is correlated with well marked breeding barriers

between most species in the genus Allium (Liliaceae).

MATERIALS AND METHODS

1. Plant Material

 

The stocks of AgrOpyron trachycaulum, Hordeum jubatum

 

 

and the spontaneous hybrid used in this study were collected
in Alaska with the help of Dr. W. W. Mitchell and R. Taylor,
Alaska experiment station staff. The amphiploid was obtained
with colchicine treatment by Dr. R. P. Steidl.

Diploid H. vulgare; Larker (CI 10648), Coho (CI 13852)
Dicktoo (CI 5529) and one tetraploid H. vulgare from Dr. A.
B. Schooler were used to cross with the amphiploid and the

first backcross. (Table 2)

2. Methods of Hybridization

 

In the winter, plants were grown in the greenhouse
with daytime temperatures between 15-25°C while in the sum-
mer, stock was kept in a growth chamber. Frequent repotting
and regular fertilizing were necessary in maintaining plant
vigor and healthy heads. During the pollination period, a
relatively low night temperature was necessary to prolong
pollen dehiscence. The crosses were made by the approach
method (Curtis and Croy 1958) and better results were
achieved when the plants were pollinated with massive amounts

of pollen for two to three consecutive days. It was generally

15

16
observed that the lodicule ceased to Open the lemma and
palea after fertilization had taken place. The pollinated
head was covered loosely with an aluminum foil envelOpe and

was unwrapped and eXposed to light for 20—30 minutes every

day.

3. Embgyo Culture

 

Embryo culture was necessary to rescue the hybrid from
degenerated seed. Transfer to media was essential for the
immature embryo to be germinated before reaching full develop-
ment.

Two kinds of media were generally used in this study;
Norstog's medium II (Norstog 1973) and wick medium (see
appendix for the content of media). Obtaining hybrids from
proembryos grown in either media was unsuccessful. Well
develOped embryos did best in wick media Since the liquid
later surrounds the scutellum which functions as an absorbing
appendate of the embryo. The poorly develOped embryo might
have suffocated from the liquid layer in the wick medium and
therefore a solid medium was preferred.

The procedure of excising the embryo and transferring
it to the media was as follows:

a. Spike and seeds were sterilized for one minute

with commercial clorox diluted with an equal
part of distilled water and then rinsed three
times with distilled water.

b. The transfer was made under the transfer hood

and dissecting microsc0pe. The immature

17
caryopsis was held by pointed tweezers and
the embryo taken out by dissecting needles.

3. The excised embryo was then transferred to
the culture medium with careful orientation
of the embryo with the scutellum facing the
media.

4. The embryos were grown in the dark in an
incubator at 24°C. After 7-10 days some
embryos started develOping into tiny plantlets
and were then given artificial light until
they reached the two-leaf stage. The seedlings
were then transferred to sterile soil under
high humidity conditions until they were

established.

4. Tissue Culture

 

Tissue culture techniques were employed to induce a
meristem point in the callus from which develOpment of the
plant takes place. The apprOpriate media (B-5) was experi-
mentally selected by Dr. Carlson's laboratory. B-5 medium
develOped at the Prairie Regional laboratory for growing
soybean tissue culture has also been used successfully to
grow cells of a large variety of plant tissue. B-S medium
refers to the basic medium with no growth hormone or organic
supplements. In this study we used 2-B5 medium, referring
to the basic medium plus 2 ppm of 2,4-D. Kao and Kasha
(1969) used B-5 medium (omitting 2,4-D) for barley embryo

culture. X42 media is a modified B-5 media with no 2,4—D

18

which was employed to regenerate a plantlet from callus. The

procedure for tissue culture is as follows:

a.

The immature Spike wrapped deeply in the boot
was a suitable tissue to be induced to produce
callus.

Boots with the Spike inside were sterilized

by 95 percent alcohol for 30 seconds and then
transferred to a petri dish for dissecting.
Using the tweezer held at one end of the boot,
the boots were Opened by dissecting needles

to dissect out an immature spike. The Spike
was laid on a scratch media surface. The
petri dish was sealed with parafilm.

The culture was incubated in the growth cabinet
in the dark at 28°C. The callus grew rapidly
in fresh media and was regularly subcultured
once a week.

When the callus reached a certain Size, it

was transferred to X42 regeneration media and
was eXposed to light. A clone of plantlets
emerged from small meristem domes. Colchicine
(.05%) was incorporated in the media (B5 and
X42) and subcultured for a range of two days
to one week.

Plantlets removed from the media were washed
off the agar and transferred to sterile soil
under high humidity conditions before they

were established.

l9

5. Cytological Techniques

 

For determining the chromosome numbers, plants were
put in the cold room around 2°C overnight and then treated
in .05% colchicine for 10 minutes at room temperature. In
some cases plants with root systems were pretreated in .05%
colchicine solution in the cold room (2°C) overnight and
then fixed in Newcomer's solution (Newcomer, 1953). The
root-tips were then rinsed in distilled water, hydrolysed
in 1N HCL at 60°C for 20 minutes. Hydrolysis is essential
to subsequent Feulgen staining; therefore, a range of time
from 10 to 30 minutes was used for hydrolysis of the small
root-tip samples to find the Optimum time.

Slides were prepared by a squash technique. Photo-
micrographs recorded by a ZeiSS photomicrOSCOpe II with a
built-in 35 mm camera and Linholf Technika 4x5 attached to
the photomicrOSCOpe II using Kodak panatomic-X film (FX402)
and contrast process ortho film, reSpectively. This system
was SOphisticatedly developed in Dr. Tai's laboratory.

Karyotypic chromosomes were prepared from a photo-
graph print with well-Spread metaphase. Karyotypic treatments
as chromosome length, ratio and index were referred to the
nomenclature from Levan et a1 (1965).

6. Feulgen-Aceto Carmine
Differential Banding Technique

 

 

Aceto-carmin solution was prepared as a standard solu-

tion by the formula in the Darlington and La Cour book (1966).

20

The Feulgen preparation is in the appendix and the

staining procedures were as follows:

a.

For arresting mitotic cell division at metaphase I
stage, intact root tips from the whole plant were
put in cold jars with ice in the cold room for
overnight and then the root tips were cut to 2-3
cm lengths.

The root tips were fixed with Carnoy's solution
and rinsed with distilled water and placed in

1N HCL at 60°C for an average of eight minutes

(a range of time is necessary to cover the Opti-
mum hydrolysis).

The root tips were transferred to Feulgen solution
and placed in a dark place during the staining
process. In order to get maximum or overstaining
by Feulgen stain, the root tip must remain in
Feulgen solution for at least one hour.

The root tips were sampled to assure the maximum
Feulgen staining which shows dark, shining
(florescent-like) chromosomes.

The root tips were squashed in a drOp of aceto-
carmine and the aceto-carmine was added to the
cover Slip while gradually heating the slide.
This process will allow the aceto-carmine to
stain differentially on tOp of Feulgen stain.

The slide was sealed with wax, stored overnight

and then rinsed with 45% acetic acid.

21
The Slide was exposed under the light so the
Feulgen stain would fade out. The slide was
checked every day to choose the best differential
banding pattern Showing on the chromosome.
Pictures were taken before the slide was perma-

nently mounted.

22

 

wpfimuo>flco mumum
cmmH20flz .mmocmfium HflOm pom

 

 

 

mono mo Lamenumomo .HOAmum 1x83
.3 so omnmmun mannoaoo lam x e<v mmmammmemaaaa< om lam x eacsefloadflnae<
saunas
mxmmad .Hmfiamm Housumcv Esumosn .m
.Hamnouflz .3 .3_Soum maoao mm x ea mm¢~<H4 mm x Seasmosnomuu .<
muoxmo nuuoz .omuom
.3 mumum muoxmo euuoz I
.HOHoonom .m .¢ EOHH comm >m >>>> mm mummaa> .m
mmmm Ho oouxoflo
memoa Ho umxnmq I
Nmmma H0 0:00 >3 >> «a mummas> .m
mxmmam
.HOEHmm .Hoawme .m can I
aamnouflz .3 .3 Scum comm am mmm¢m< mm esflsmomnomuu .m
mxmmH<
.Hmfiamm .Hoamme .m can I
Hamnounz .3 .3 Sega comm we m¢m<~¢a¢ mm saunas“ .3
:flmfluo oofiumcmflmma ucoecmwmm< mfiocww Cm woman

 

 

wqfiHmMde m0m<mmmm m0 ZOHBGZUHme 02¢ ZHUHMO

N OHQMB

RESULTS

Karyotype analysis

 

 

Karyotypes of H. vulgare, H. jubatum, H, trachygaulum,
H. vulgare x H. jubatum, (H. trachycaulum x H. jubatum)
(8X) x H. vulgare (2X), and H. vulgare (4X) x (H. trachy-

caulum x H. jubatum) were Obtained.

H. vulgare (Fig. l)

Chromosome types and karyotypes. Generally,

 

three chromosome types were recognized in the somatic
metaphase stage: (1) Chromosomes without satellites
(chromosomes 1, 2, 3, 4, and 5). Within this group,
chromosomes 1 and 2 were difficult to distinguish from
each other by conventional cytological techniques such
as relative length and arm ratio. The only difference
is that chromosome 2 is slightly longer in the long arm.
Chromosomes 3, 4, and 5 are relatively shorter in both
arms. (2) Chromosome 6 has a large satellite and (3)
Chromosome 7 has a short satellite. The arm difference
(difference between long arm "1" and Short arm "s"; d=l-s)
ranged from 1 to 8, the arm ratio (r=%) from 1.1 to 2.1,
and the index (i=§;§x 100) from 32 to 62. Chromosomes

l and 4 were median, chromosomes 2, 3, 5, and 6 were

submedian, and chromosome 7 was subterminal.

23

Fig.

l

24

The karyotype of H: vulgare with 2n=l4.
The chromosomes are arranged in order of
decreasing length from 1 to 5. Chromosome
6 has a large satellite chromosome and
chromosome 7 has a small satellite.

d: arm difference

r: arm ratio

i: chromosome index

s!

25

Hordeum vulgare (2 I: H)

o 5 ‘ )
a o ) , s:
2‘ 2 :o H ‘6
32 J, o? ‘8 )0

Figure 1

I66

no

 

26
H. jubatum (Fig. 2)

The chromosome morphology of H. jubatum has been
described by Morrison (1959), and Rajhathy and Morrison
(1961). There are three types of chromosomes in Figure 2:

(1) Large satellites with a constriction cutting the
long arm (chromosome 1).

(2) Small satellites on a Short arm (chromosomes 5
and 10).

(3) Subterminal chromosomes (chromosomes 4 and 13).

(4) Submedian chromosomes.

The chromosomes are not stained uniformly by
the Feulgen-Carmine double staining technique. A dicen-
tric chromosome and a plant with 42 chromosomes were
reported by Morrison (1959). In this study, H. jubatum
has been confirmed as a segmental allotetraploid with
a chromosome number of 28. It has been used as a major
entry for interSpecific and intergeneric hybridization in

the genus Hordeum.

H. trachycaulum (Fig. 3)

 

The karyotype of H. trachycaulum has not yet been

 

fully described in the literature. The chromosomes are
arranged according to length, and in this study two pairs
of satellited chromosomes were found: one pair of chromo-
somes had a very small satellite (chromosome No. 2); the
other (chromosome No. 7) is relatively larger. One pair
of the subterminal chromosomes, and the rest of the pairs,

are median and submedian chromosomes. The classical

Fig.

2

27

The karyotype of H. jubatum with 2n=28. The
chromosomes are arranged in order Of decreasing

length.

 

 

Hordeum jubatum (2!: 28)

ccflfllit‘IMMl

o\(333)2)i\i((

F iiiiii

 

)i

 

Fig. 3.

29

The karyotype of H. trachycaulum with 2n=28.

 

The chromosomes are arranged in order of

decreasing length.

 

 

s1

‘66

375

30

AQVOPYYOH trachycaulum (2n=28)

 

MKSQWQC

S!

2

O

C | j .
Ho :01 12 1. H
‘2. 482 45) 3.. 46.

Figure 3

25

"

‘76

n0

31

descriptions of fourteen pairs are as follows (Table 3):
The tip and proximal regions of chromosomes were stained
darker and the region between the tip and centromer was
stained lighter comparatively. In relation to stain
reaction, the chromosome of the darker stained region
was more compressed, and the region where they were of
a puffy texture was stained lighter.

Canderon (1962) found three pairs of satellited

chromosomes and the rest were median and submedian in

H. junceum karyotypes.

AHV 442 (2n=35) (Fig. 4)

 

This plant was Obtained from a cross between amphi-
ploid-(A.t. x H.j.) and diploid H. vulgare. By the
genome formula, it is composed of A1A2A3BV (2n=35);
fourteen chromosomes as AlA2 from H. jubatum, fourteen

chromosomes as A3B from H. trachycaulum and seven chromo-

 

somes as V from H. vulgare.

Chromosome types and karyotype. The chromosomes of
AHV 442 do not occur in identifiable pairs. The karyo-
types were made by sequence of their relative length.
By observation, the chromosomes from H. vulgare might be
identified under the micrOSCOpe by their staining texture.
A preliminary result Of Feulgen-Carmine staining shows
differential staining similar to banding. Further
studies are needed to use the chromosome banding technique

as a tool to classify chromosome in Hordeum.

32

Table 3

THE ARM RATIO, DIFFERENCE AND DESCRIPTION
OF H. TRACHYCAULUM KARYOTYPE

 

 

 

 

No. r d Designation
2.5 median
1.2 4 subtelocentric
with large satellite
3 1.45 6 submedian
4 2.5 10 submedian
5 2.1 11 subtelocentric
5 1.81 9 submedian
7 1.66 9 median with a small
satellite
2.2 11 submedian
1.1 2 median
10 1.8 8 submedian
11 1.2 3 submedian
12 1.07 l median
13 1.36 4 submedian
14 2.16 7 submedian

 

As can be seen in Fig. 3, it is impossible to classify all 14
pairs on the basis of chromosome morphology. However, using
the arm ratio (4) and designation of centromer position plus
the presence or absence of satellite it is possible to dis-
tinguish some.

33

Fig. 4 The karyotype of AHV 442 with 2n=35. There
are 14 chromosomes from H. jubatum, 14 from

H. trachycaulum and 7 from H. vulgare.

 

1.5 ‘40
O O
35 009

‘25 155
2 s
“4 J"

I”

'7‘ :5
S 4
)6. .o
2 «1
° 3
J) J “I

‘ st

16 2,
5 o
1’ I so

34

AHV—442 ( INIJS)

I"!

301

E

00-

170

1., 1“ ‘2,
o a ,
)5 no 44
'5 :42 s
‘ 1 n
Go 41: ,..
‘ 3 i 31.
1 o 1
05‘ so 315
Q s :5!
" i .33
‘ 0 IO
009 so :31
‘9 U 2 to
J I ,
5“ “9 H a

Figure 4

no

I ' .
212 f
v d
32 i

s;-

J‘

'tSS

39.1

 

35

AHV 444 (2n=42) (Fig. 5)

 

This plant was produced by using the amphiploid
(A. t x H.J.) as female and tetraploid H. vulgare as the
pollen parent. It was composed of A1A2A3BVV (2n=42) with

AlAZ from H. jubatum, A3B from H. trachycaulum and VV

 

from H. vulgare. No identifiable pairs could be found
with confidence. Satellited chromosomes might selectively
be assigned with confidence to each parent. By their
origin, there should be 14 satellite chromosomes in this
hybrid, only 10 satellites were recognized. Based on
morphology and satellites chromosomes 13 and 15 might be
from H. vulgare, chromosomes 5 and 21 from H. jubatum,

and chromosomes 6 and 26 from H. trachycaulum. Median and

 

submedian chromosomes were prevalent in nonsatellited
chromosomes. One chromosome was comparatively small; it
might be similar to a B chromosome.

VH__'___35 (Fig. 6)

This plant was produced by using a winter type male
sterile H. vulgare (Zn-14) as female and H. jubatum as the
pollen parent. The hybrid was somatically unstable Since
various chromosome numbers were counted in the root tip,
pollen mother cell and root tip from callus regenerated
plants. A karyotype was made from a callus regenerated
plant with chromosome 2n=35 which might be a partial chromo-
some doubled plant. At a glance, chromosome configuration
and staining texture showed different patterns in compari-

son to either H. jubatum or H. vulgare. Originally, they

36

Fig. 5 The karyotype of AHV444 with 2n=42. There are
14 chromosomes from H. jubatum, 14 from H.

vulgare and 14 from H. tracHycaulum.

 

 

 

1' :""'(\mt))

2K

285

37

AHV 444

I 1? 11

325 16

HTHH"

IO 9 I 1 6

“I ‘3 too :1
° ‘ O 2 9 l o v
2) 5 no )5 436 Joe 04 n I no

in :57 26 :91 :1 no :u :1: :1!
2 a 4 s 1 0 5 s
a: ”I 277 )0. u. 315 coo u. so.
‘45 :5 157 1 ‘7! ‘57 '25
1 J 4 o 5 0 2
4 « u. we :0 mu m g «4

Figure 5

5

SIS

)9

(2I242)

S!)

Q)
:57

H2

47

l

.34

ll2

J 2
:21 I
3 o
u so
:2 1.2
2 S
u 3.
st
I37 no
.I 2
u: on

v noy
~—
116 f
. d
00 ) i
8'
)5
:0
222

 

Fig.

6

38

The karyotype of VH-35 with 2n=35. There are

mixed ploidy of both H. vulgare and H. jubatum.

42.

375

2

\a...

133

428

i 5

‘0

50

2O

30

2‘6

125

“4

39

VI!

N‘nt

fl

3 (

25

12.

037

15

40

I.

375

335

Figure 6

17‘

)6.

’8'

'71

(‘5'

t.)

152

1.5

15

t)?

‘2‘

2‘6

5‘5

B”).

157

38.

21.

313

‘51

3|.

2|o

J|5

no

40
were 7 chromosomes from H. vulgare and 14 chromosomes
from H. jubatum at the zygotic stage. Chromosome con-
formation and ploidy were severely changed through
callus cell differentiation. This karyotype showed at
least 8 satellited chromosomes, only one of them
(chromosome 21) can be assumed to be similar to H. vulgare.
Also, no identifiable pairs could be selected. Differ-
ential staining of Feulgen-Carmine was recorded in some
of the chromosomes. Generally there is one arm stained
darker than the other arm. Banding only Shows in four

chromosomes.

Chromosome Index Similarity
Faced with a situation of great karyotypic complexity
in parental entries and series of hybrids, a simple numer-
ical method of comparing karyotypic similarity is
strongly needed for certain reasons:
1) There is no single karyotypic character which
absolutely differentiates the two species.
2) Karyotype of species can be stratified on
their Similarity.
3) No karyotypic relation was ever established
between genera.
Based on the ploidy and chromosome index, karyotypic

Similarity of H. trachycaulum, H. jubatum, and H. vulgare

 

shows certain differences (Fig. 7):
1) H. trachycaulum has a broader chromosome index

which covers the range of both H. jubatum and

 

41

Fig. 7 H. trachycaulum Shows broader chromosome index

ranging from 17-52 with a peak at 37. The

range covers both H. jubatum and H. vulgare but

 

the peak at 37 has fallen next to H. jubatum.

(bro.

I):
H;

II;

i
i
will:

42

 

 

 

 

37

3+|mmmW

t.
3

Chro. Index

Figure 7

H Vu'

43

H. vulgare. H. jubatum has a wider chromosome
index than H. vulgare.

2) H. trachycaulum chromosome index has a peak

 

at 37 which is second to H. jubatum. H. vulgare
has the same peak as H. jubatum.

3) Based on the chromosome index comparison, H.
vulgare has a higher karyotypic similarity
with H. jubatum than the similarity with H.

trachygaulum.

 

In the hybrid series, the genomes and chromosomes are

derived from H. trachycaulum, H. jubatum and H. vulgare.

 

The karyotype of each hybrid is a temporary set. Alteration
of ploidy and chromosome will follow the subsequent cross
and genetic instability. Therefore, a comparison between
hybrids and their progenitors would not be a solid assessment
for a genetic relationship. Plotting the ploidy and chromo-
some index of hybrids, AHV444, AHV442 and VH 35 shows (Fig. 8):
1) Generally, hybrid groups have chromosome index
peaks between 37 to 42. This is in accord
with the parental group.
2) AHV444 with a high, ploidy nature has a wider
range of karyotypic index.
3) VH-35 increases the variation of chromosome

index in comparison to the scope of its parents.

 

 

Fig.

8

44

The chromosome index peaks in hybrid group
VH-35 show co-center between 37-42 which is
in accord with their parental group. Chromo-
some rearrangement in terms of translocation

might be the cause of index change.

45

 

 

 

“I
I!)
H)

Index

Chum

Figure 8

46

II. Results of Hybridization
and Cytological Studies

 

 

Interspecific and Intergeneric Hybridization

 

The crosses and hybrids Obtained are shown in Table
4. Their Spike morphology is Shown in Plate B, Figure l, 2,
4, 5, 6. Embryo culture was used to rescue the premature
embryo before abortion and to stimulate embryo germination
in order to establish the hybrid develOpment (Plate B,
Figure 3.

In the first backcross, AHV-B, the embryos develOped
without endosperms. This indicated that a certain indepen-
dence between embryo and endosperm develOpment was initiated
during the course of early zygotic differentiation. The
seedling of AHV-B, was vigorous and morphologically related
to H. vulgare, concomitant to increasing H. vulgare genomes
due to the backcross (Plate B 4, 5, 6). Generally, the
characteristics of AHV-Bl could be described as follows:

1) Three Spikelets per node.

2) More than two florets per spikelet

3) Disarticulated below the glume

4) Facultative pubescence on the leaf

5) A shortened Spike Of a similar size to culti—

vated form.

Certain characters could not be defined within the
general morphology of the genus Hordeum Since the hybrid still

contained portions of the AgrOpyron genome and was maintained

 

in the Agropyron cytOplasm (Plate B, Figure l, 2.)

 

47

The meiotic chromosome association of plants was
observed in the pollen mother cell. These data are compared
with those of other plants in Table 5 and Plate A, Fig
2, 3, 4. In AHV-B, univalents averaged 4.49, bivalents
averaged 14.41 and trivalents averaged 16. The univalents
were significantly decreased in comparison to other plants
studied, while the multivalents were slightly increased.
III. Results of Genome Interaction

and Chromosome Elimination
iE’AHV444

 

A systematic micrOSCOpic observation of microsporo-
genesis was carried out to investigate a possible cause of
chromosome elimination in plant AHV444.

A. Pr0phase. In prophase stages, chromosomes could

not be traced back to their genome of origin by stain-

ing and morphology. AS prOphase is a progressive
sequence, it is hard to discern these stages due to

the condition of abnormal condensation associated with

the sticky formation of chromosomes. Superficially,

all chromosome complements seem to congregate within

the nucleus in a major group. When the mucleus envelope

is disrupted, small chromosome groups gradually segre-
gate from the major group during the condensing process.

The univalents segregated randomly throughout the

Spindle in most of the plates before reaching metaphase

I. Uncoiled chromosomes expelled from the major chromo-

some group in early prophase were occasionally observed.

 

48

Table 4

DESIGNATION OF ALL CROSSES ATTEMPTED

 

 

 

Cross Genome Plants

Designation 2n Designation Obtained Remark

AT x HJ 28 A1A2A3B l8

HJ x AT 28 A1A2A3B l

AHV442 35 A1A2A3V 7

AHV444 42 A1A2A3VV l

mix-octOploid 56 A1A1A2A2A3A3VV l

AHV—B 35 A A VVV 10 OctOploid was

1 x y

used as female
to cross tetra-
ploid H. vulgare

VAH444 42 A1A2A3VV 2

VAH442 35 A1A2A3V 5 Winter type male
sterile H. vul-
gare was used as
female in crosses
with the amphi-
ploid (A.t.XH.j.)

Amphiploid-

(At x Hj) x

Amphiploid-

(Hj x Hc) 20

VH 35 8 Hybrid made by

R. P. Steidl

 

THE CHROMOSOME

49

Table 5

ASSOCIATION OF PLANTS

 

 

 

 

Chromosome Association No. of
Plants I II III IV cells
A. trachycaulum Range: 0 l4 0 0 100
(2n=28) Mean : 0 l4 0 0
H. jubatum Range: 0 l4 0 0 100
(2n=28) Mean : 0 l4 0 0
(A.t. x H.j.) Range: 7-24 2-8 0-2 0 50
Natural (2n=28) Mean 16.38 5.44 0.14 0
(A.t. x H.j.)*** Range: 7-18 5-10 0-3 0 31
Artificial
(2n=28) Mean 11.51 7.22 0.67 0
(H.j. x A.t.)*** Range: 7-22 3-7 0-2 0-1 96
Artificial
(2n=28) Mean 15.80 5.60 0.32 0.11
Amphiploid-
(A.t. x H.j.) Range: 0-6 22-28 0-2 0—1 71
(2n=56) Mean 2.36 25.63 0.71 0.06
AHV 442* Range: l-l4 9-17 0-1 0 66
(Zn-35) Mean 6.53 13.98 0.16 0
AHV 444** Range: 5-15 12-17 C-1 0 112
(2n=42) Mean 7.89 15.92 0.86 0
AHV—B Range: 1-7 11-17 1-2 50
(2n=35) Mean : 4.49 14.41 0.56
Amphiploid -
(A.t. x H.j.) Range: 2-8 10-17 1-3 0-1 30
x Amphiploid -
(J.j. x H.c.) Mean 6.64 15.56 2.76 1
* Amphiploid - (A.t. x H.j.) x H. vulgare (2n=l4)
** Amphiploid - (A.t. x H.j.) x H. vulgare (2n=28)

*** Reciprocal crosses

 

Fig.

Fig.

Fig.

Fig.

50

Plate A

Metaphase I in amphiploid - (A.t. x H.j.) with
2n=56

Metaphase I in AHV 442 with 13 I + 8 II (3 ring)
+ 2 III (arrow) (x915)

Metaphase I in AHV 444 with 13 I + 13 II (7 ring
by arrow) + 1 III (x 915)
Metaphase I in AHV-B with 3 I + 13 II + 2 III

(arrow) 1

 

 

 

 

 

PPPPPP

 

Fig.

Fig.

Fig.

Fig.
Fig.

Fig.

Spike

and H.

Spike

Young
towel

Spike
Spike

Spike

52

Plate B

of H. trachycaulum (right) hybrid (center)
jubatum (left)

of Amphiploid-(A.t. x H.j.)

 

seedling of hybrid plant grows on paper
in liquid media

Of AHV 442
of AHV 444

of AHV—Bl (left) and H. vulgare (right)

 

 

53

 

 

Plate B

54
These cells seemed to be aborted before reaching meta-

phase I Since no reduced chromosome number was recorded

at metaphase I.

B. Metaphase I. Selected metaphase I was the main

 

stage used to record chromosome associations in the
form of their pairing patterns. The chromosome number
was in accord with the expected complements of 2n=42
in most cases. Multivalents were Observed in Spite
of a confusing sticky association which usually involved
more than three chromosomes.

At metaphase I, the chromosomes of H: vulgare pre-
sumably in form of rings seem to be larger and stained
deeper than the chromosomes Of either H. jubatum or

H. trachycaulum (Plate A, Fig. 3). But such a dif-

 

ference was not clearly discernible and consistent in

all the cells.

C. Anaphase I. An anaphase bridge was frequently
observed; this might be the result of belated separation
of chromosome association due to sticky association or
to a deletion and a translocation. The stickiness of
the chromosome in conjunction with spindle abnormality
might be a physical mechanism of chromosome differ-
entiation such as the centri fission-fusionl process

which was significantly seen in metaphase II.

 

region

lGross chromosomal rearrangement involving in centromer

55

D. Interphase I. Incursion chromosomes, or chromo-

 

some fragments in the process of uncoiling, were
observed in interphase I. (Plate D, Fig. 7). The
impact of chromosome association resulting in chromo-
some differentiation after first division was signi-
ficantly revealed through abnormality in chromosome
number and behavior after first division. The process
in two daughter cells was not synchronized since they

were independent, with different genetic make-up.

E. Metgphase II. A variety of chromosome numbers in

 

metaphase II were commonly Observed. The majority of
chromosome complements in metaphase II were under 21,
the expected number under the normal reduction process
of the first meiotic division. Mainly the chromosome
number after reduction ranged from 20-15. Due to the
lack of a technique for individual chromosome identi-
fication, this selective reduction process could not
be specified by different genome of origin. Secondary
chromosome association in the form of a sticky con-
nection at the terminal and centric region between
chromosome was a common phenomenon. Obviously, chromo-
some fragments derived from centric fission can be
identified (Plate C, Fig. 1, 2, 3, 4, Plate D, Fig. 5).
Unequal segregation in proceeding anaphase II was
recorded (Plate D, Fig. 6). Chromosome enclosed in

microspore cells by abnormal furrowing separation from

56
the major cytOplasm was an alternative to individual

chromosome degredation (Plate D, Fig. 8).

F. Photomicrographs of microspore with different
chromosome number are Shown in Plate E, Fig. 9, 10,
ll, 12; Plate F, Fig. 13, 14, 16; Plate G, Fig. 19,
20. The exine fragments of the microspore wall are
washed off during the process of slide preparation.
The chromosomes in microspores were stained darker
than usual. AS general view, chromosomes seemed to

be grouped rather than randomly dispersed, but the
chromosomes did not orient themselves on the metaphase
plate. A range of chromosomes of 13 to 23 with a peak
around 17 (Table 6). Preferential chromosome viewing
in the micrOSpore is evidently associated with the
phonomenon of chromosome elimination. Two hypotheses
are advanced to explain the distribution of chromosome
number in the microspore based on the assumption that
chromosome elimination results from genome interaction
then the data in Table 6 could support the hypotheses
by the microspore having a full set of 7V plus unknown

number from either ngOpyrom and H. jubatum versus 7A

 

(Agropyrom) + 7j (H. jubatum) with unknown number from
H. vulgare. Microspore failure to reorganize the
nucleolus, and the disoriented separation of chromo-
somes in the microspore, were Observed (Plate G , Fig.
15, Plate G, Fig. 17, 18). Thus the presence or

absence Of some factor could result in the elimination

 

57

of certain combinations of chromosomes.

Table 6

CHROMOSOME NUMBER AND ITS VARIATION
IN MICROSPORE IN AHV444

 

 

 

Chromosome Microspore Percent Expected

Number Counted Form
13 8 3.23 a
14 14 5.66 a or b
15 29 11.74 a or b
16 48 19.43 a or b
17 44 17.81 a or b
18 35 14.17 a or b
19 33 13.36 a or b
20 20 9.71 a or b
21 8 3.23 a or b
22 7 2.83 b
23 1 0.40 b

‘5?

 

a xA + yj = 7V

b 7A + 7j + xV

Fig.

Fig.

Fig.

Fig.

58

Plate C

Metaphase II in AHV 444 with
and two chromosome fragments

Metaphase II in AHV 444 with
ments (arrow)

Metaphase II in AHV 444 with
ment and sticky association

Metaphase II in AHV 444 with
breakage (arrow)

group separation
(arrow)
two chromosome frag-

one chromosome frag-

centromer transverse

 

59

Plate C

 

Fig.

Fig.

Fig.

Fig.

5.

6.

7.

8.

60

Plate D

Metaphase II with a chromosome fragment and a
centromere transverse breakage (arrow)

Telophase II with an unequal chromosome

A tetrad with a some chromosome excluded from
major group

A chromosome was eliminated by means of budding
cell in Metaphase II

 

61

    
    
    
  
  
     
  

o
.- -_
. l._ u

7o
‘
I J
C
0'1.
L‘ )

I
n
O

'0 ’
C
I
I
'. .
.
‘
'
.C
-a...‘

A
so‘0 ‘0

a

;
.1
II
\~
..,s ‘
A‘J‘

. A l]:-
a‘s‘l»; ’,.:(...
( L

I ' fie

Plate D

 

Fig.

9.

Fig. 10.

Fig.

Fig.

11.

12.

Pollen

Pollen

Pollen

Pollen

mitosis
mitosis
mitosis

mitosis

62

Plate E

with 21
with 14
with 16

with 15

chromosomes
chromosomes
chromosomes

chromosomes

(678x)
(678x)
(678x)

(678x)

 

63

 

Plate E

64

Plate F

Fig. 13. Pollen mitosis with 19 chromosomes (678x)
Fig. 14. Pollen mitosis with 18 chromosomes (678x)
Fig. 15. Pollen mitosis with disoriented Anaphase (678x)

Fig. 16. Pollen mitosis with 16 chromosomes (678x)

 

65

 

Plate F

 

Fig.
Fig.

Fig.
and

17.

18.

19
20

66

Plate G

Two pollen cell with unorganized nucleolus (590x)

Two synchronized pollen Anaphase (590x)

Synchronized pollen mitosis cells (678x)

 

67

“90!) (ON!)

 

(If. I)

Plate G

DISCUSSION

InterSpecific and Intergeneric
Hybridization as a Means of
Improving Barley Winterhardiness

 

 

Winterhardiness is the major factor limiting both
ecological range and productivity of winter barley in the
northern agricultural area. Winterhardiness is a complex
physiological trait whose improvement through the cultivated
gene pool has progressed slowly in recent years for several
reasons:

1) genetic isolation of H. vulgare from wild

species;

2) inadequate genetic variance in existing H.

vulgare gene pool;

3) concealing genetic variability by complex physio-

logical and environmental interaction, and

4) linkage with disease susceptibility or other

deleterious traits.

Thus, the introgression of an alien gene source
from wild relatives might increase the genes governing the
Winterhardiness to a higher ceiling.

The breeding program has five major steps:

1) the establishment of a hybrid population;

2) chromosome manipulation between cultivated and

wild genOmes in hybrids (Fig. 22)
68

69
3) metabolic identification and genetic modification
of differentiated and undifferentiated cell
pOpulations;
4) fertility restoration, and
5) gene accumulation through random breeding and
selection using male sterile pOpulationS.
A hypothetical outline of a possible attack on the problem
of gene introgression from wild Species is given in Figure
21 of this section. In conjunction with previous studies,
the partially fertile octOploid was obtained by colchicine
treatment on AHV442. The genetic formula of this octOploid
is presumed to be A1A1A2A2A3A3VV with a chromosome number
2n=56. It is difficult to determine chromosome associations
in metaphase I Since many multiple associations are involved.
The B genome from AHV442 is assumed to have been lost in the
process of chromosome doubling because the B genome seems
most different but we have no way of knowing that is so.
The double dosage of VV might cause preferential elimination
of the less compatible B genome. The dosage effect of VV
(genomes has been fully discussed in previous studies. Chromo-
some or genome substitution is prevalent in an intergeneric

hybrid; Knott et al (1977) reported a stem rust-resistant

wheat line derived from a wheat-nggpyron hybrid. The

 

resistance proved to be carried on an AgrOpyron chromosome

 

which had replaced the wheat chromosome 70. Apparently,

Agropyron chromosomes compensate well for 7D in both vegetative

 

and reproductive gene systems in substituted lines.

70

 

A. trachycaulum x H. jubatum

2n=28 2n:
A3A3BB AlAlAZAZ
Fl hybrid
2n=28
A1A2A3B
colchicineltreatment
H. vul are x Amphiploid x H. vulgare
2n=14 AlAlAzAzA A BB '2’n=2 8
VVVV
2n=56
AHV442 AHV444
2n=35 2n=42
A1A2A3BV AlAZABBVV
colch cine mutagenic treatment on
cell pOpulation
(mix-octOploid) (AHV444-Ml
H. vulgare x 2n=56 2n=x
n=28 A A A A A A VV A'A'A'B'V'V'
IRON! l l 2 2 3 3
(AHV442-Bl anther culture
2n=35
AxA VVV x-haploid
yl 2n=x/2 x H. vulgare
metabolic identification A'XB' V' 2 =14
and genetic modification y 2n=28
on cell pOpulation diploidization

AHV442-Bl-S

H. vulgare x AxAyVVV

2n=14 1
VV
diploidization
pollen source
Figure 21.

pollen source

Diagramatic outline of the steps and procedures

in introgressing agronomic gene source from
wild germplasm into cultivated barley

71

Interspecific hybridization Intergeneric hybridization
bigenome multiple genome
-r————incorporation incorporation

allo-polyploid

  

i)

 

 

chromosome elimination

 

amphidiploid
(non-chromosome‘
differentiation)
redundant Compile or coneospecies
diploidization (chromosome differentiation)
.. 1. gene introgression
2. chromosome exchange
I (translocation)
haploid and diploid 3. chromosome substitution

4. genome substitution

recombinant
diploidization

pollen source

Figure 22. Scheme of Chromosome Manipulation

72
Similarly, Thomas and Thomas (1974) reported that mildew

resistance of Avena barbata has been successfully incorpor-

 

ated into the cultivated oat by replacing a pair Of
chromosomes. The use of colchicine to induce chromosome
doubling in many plant Species has been well documented
since 1939. There has been speculation that colchicine
disturbs spindle formation in mitotic division yielding to
a doubling of the chromosome number. The consequence of
this doubling is to stabilize the genome in the form of
diploidization. With heterogeneous genome constitution of
AHV442, selective genome doubling in order to reach somatic
stability is one alternative. On the other hand, stability
as well as genome balance can be achieved by a regular dis-
junction of quadrivalents. Multivalents were prevalent in
this octOploid plant. Thomas (1974) reported that meiotic
stability and fertility were even accomplished by quadri-
valents instead of bivalents in an allotetraploid form of

Lolium multiflorum.

 

 

With respect to gene introgression in the form of non-
preferential bivalents, it was unique in the genetic control
over chromosome pairing by a BL genes system between an

intergeneric hybrid of wheat and AgrOpyron (Larson, 1970;

 

Sears, 1973; Wienhues, 1973; Cauderon and Ryan 1974; Dvorak
and Knott, 1974; Sinigovets, 1974; Knott, D. R. et al, 1977)
and by a Pb gene system between wheat and Aegilops (Riley
et a1., 1968; Athwal and Kimbar, 1972; Maan and Sasakuma,

1977). The knowledge of the occurrence of genetic factors

73

affecting pairing, compels us to treat the assessment of
genetic relationship, based on the pairing behaviour of
chromosomes to a certain degree of extension. If an octo—
ploid has been reached under certain degrees of chromosome
differentiation through non-preferential multivalents, then
the probability of an ovum with a balanced chromosome com-

plement of Alsz may be higher than 1 to 221

as postulated
on a strictly random basis.

Since the fertility of the octOploid has been partially
restored, the first backcross to the tetraploid H. vulgare
(2n=28) was achieved with a chromosome number 2n=35. The
'genome formula was estimated to be AlAZVVV. The frequency
of univalents was significantly decreased in the first back-
cross. This is a promising step toward Obtaining a further
progressive backcross to H. vulgare and leading to gene
transgression. In the literature, the number of univalents
has been considered a more precise indicator than multivalents
of meiotic stability and fertility. The attempt at a pro-
gressive backcross is under way.

Chromosome association as an

indicator of the course of
chromosome elimination

 

 

 

Irregularity Of cell division and chromosome behavior
following Metaphase I might indicate the possible cause and
mechanism of chromosome elimination in the intergeneric and
interSpecific hybrid. Somatic chromosome elimination follow-
ing fertilization would have a Similar pattern in a certain

stage after Metaphase I but in zygotic differentiation it

74

would involve both genetic and physiological interation.

Plant AHV444 had an equal dosage of three different
genome origins; AlA2 from H. jubatum, A3B from H. trachx-
caulum and V V from H. vulgare. In fact, mosaic tissue and
reduced ploidy tillering are commonly produced from the
cloned population. The somatic unstability induced by V V
dosage from H. vulgare was a common phenomenon in most of
the intergeneric and interSpecific hybrids. In addition,
the somatic instability in the form of chromosome elimination
can be enhanced by a chemical agent such as gibberellic acid
during zygotic development, or colchicine at mitosis of
meristematic differentiation. Based on systematic obser-
vation from Metaphase I of pollen mitosis on this somatic
unstable plant, the phenomenon of chromosome elimination was
Speculated as a result of genome interaction. Specifically,
homoeologous association of A1A2A3 was suppressed by VV homo-
logous_genomes. Consequently, the suppressed genome of AlA2
and A3 were misgrouped and disoriented in conjunction with
spindle irregularity. The microcells containing one or two
exiled chromosomes were frequently Observed through furrowing
division in certain stages after Metaphase I.

A constant 7-ring bivalent was formulated as from VV
.genomes while AlAZ were under loosely terminal association,
A3B as free associated univalents, and eventually only the
VV genome proceeded further to pollen develOpment while the
chromosome of AlA2 and A3B genomes were randomly eliminated

along the course of microsporogenesis. In comparison with

75
plant AHV442 which lacked a V genome and was somatically
more stable since the single V dosage cannot function com-
plementarily to inhibit other genomes. Therefore an homoeo-
logous association of AlA2 and A38 were indirectly enhanced
by the V. genome. In AHV442, the V genome was assumed to
be the source of the seven univalents which were observed
scattered around the metaphase plate. When the number of
bivalents approaches fourteen, it only comes from enhanced
non-homologous pairing between A3B. On the other hand, when
VV was present in a double dosage, it inhibited the homoeo-
logous pairing of AlAZ' Bivalents were then seen as ring
shaped from VV genome and it would seem, therefore, that
directional chromosome elimination can be predicted whenever
a homoeologous association is inhibited. The impact of homo-
eologous-homologous interaction and its consequences for
directional chromosome elimination were traced from Metaphase I
to pollen mitosis. The patterns of unequal chromosome number
of Metaphase II ranged from 14-23. The variation in chromo-
some number could be explained by nondisjunction, but in this
case with such a wide range of variation and a specific inter-
val between 16-17 indicated a preferential elimination
between genomes and then, a random chromosome is lost within
the eliminated tenome. (Table 6) On the other hand, non-
disjunction due to gene disturbance would not reach such a
wide spectrum, nevertheless the cause of nondisjunction in
this complex hybrid might also link incompatibility of

chromosome association and genome unbalance. In accordance

76.

with the chromosome number in the pollen, two possible for—
mulas can be derived: 7V+xA+yJ versus 7A+7JxV. In the first
form, 7V chromosome resulted from reduced homologous pairing
of VV genomes and a random number within 7 from each of Al
and A2. In the 7A+7J+V form, a constant of 14 chromosomes
were derived from A1A2A3 and B but excluded V. The formula
of 7V+7A+xJ with randomly eliminated A3 and B would fit
rather in the range 14-21 with a peak at 16-17. In fact, the
H. vulgare type of the somatic sector and tiller were Observed.
The directional chromosome elimination was in favor of H.
vulgare in the AHV 444 plant but inferior to the other
genome in the AHV 442 plant. Using pollen cell culture and
their regenerated plantlet might further reveal the details
of the directional chromosome elimination. This approach
would be more direct than an embryo squash as well as morpho-
logical Speculation, which is difficult to insure that
normal fertilization takes place before elimination proceeds.

Kasha (1974) systematized three basic mechanistic
categories as:

l) Spindle or centromere abnormalities

2) Asynchrony in parental mitotic cycles

3) A host modification-restriction enzymatic system.

Based on these categories, chromosome elimination was
assumed to be a functionally gradual process of individual
chromosome degeneration over the period of many division
cycles. Data from a series of early embryo squashes has not

yet materialized to back up this prOposal. None of the final

77
chromosome elimination products of eight chromosome prOgenies
ever existed in the cross between the diploid trisomic H.

vulgare with tetraploid H. bulbosum.

 

A more dramatic somatic reduction would cause a
decrease in chromosome number. The process is a Spindle
oriented abnormality. Handmaker (1971) prOposed the Spindle
abnormality as the mechanical cause of chromosome elimination,
based on the study of somatic hybridization of the mammalian
cell. Groups of chromosomes are not included on the Spindle
of Chinese hamster x mouse somatic hybrid and consequently
the grouping chromosome was excluded from the daughter nuclei.
The asynchronous biogenic process due to differences in
parental cell cycle also is a special form leading to the
possible cause of chromosome elimination. Gupta (1973)
observed a high frequency of anaphase bridges concomitant
with chromosome elimination. Asynchronous cell cycles lead-
ing to insufficient time to complete replication was allotted
to the slower constituent before condensation was induced.

The chromosome bridge and the loss of a fragment was seen as
the form of chromosome elimination of the interSpecific hybrid
in the genus Nicotiana. The various forms of chromosome
elimination in mitotic cell division was similar to the form
after Metaphase I, therefore, the common cause derived from
the result of chromosome association which takes place natur-
ally in meiotic genome behavior can also be in mitotic associ-
ation in a zygote if the normal fertilization process takes
place before chromosome elimination following somatic genome

association.

78

Ho and Kasha'a (1975) hybrid pOpulation constituted
IV:2B derived from crosses between trisomic diploid H. vulgare
with tetraploid H. bulbosum from their data, univalents with
the mean around 7 and bivalents around 7 are observed. The
stability was based on homologous pairing with a B genome
as Bl-BZ; the univalent should come from the H. vulgare
genome, a case quite similar to AHV442 which has 7 univalents
from H. vulgare and 14 bivalents from the homoeologous pair-
ing of AlA2 and the non-homologous pairing A3B. Chromosome

elimination occurred dramatically when tetraploid H. vulgare

 

was crossed with tetraploid H. bulbosum since homologous
pairing was undertaken by the VV genome and eventually
inhibited the B1B2 homoeologous pairing.

As long as chromosome elimination was directional and
preferential, the strategy to bring it under control has a
significant meaning in evolution and is more important in
practical value to plant breeding. In this study, AHV442
was repeatedly treated with colchicine. As a result, genome
interactions were reconciled even doubling of the V genome
along with compatible genomes from A1A2A3. The genome
balance therefore reached a higher level with a total chro—
mosome number 2N=56 and sterility was partially restored.
Further backcrosses were subsequently achieved. The chromo-
some number in the first backcross was 35. Chromosome number
in the association significantly decreased in the univalents
with a mean around 4.49 and multivalents increased to .56

(trivalents). The constitution of this hybrid was assumed

79

to be AXAYVVV. Phenotypically the first backcross resembled
H. vulgare in dosage. The hybrid again balanced non-prefer-
ential association of either AXAy and VVV genome; AxAy might
form a 7-ring bivalent since they went through 5 mitotic

and 2 meiotic cycles of chromosome association and VVV under
unbalanced homologous pairing to form trivalents. Therefore,
no chromosome elimination would result in this first back- F—
cross. The conclusion reached is that the phenomenon of

chromosome elimination involving the genus Hordeum could be

alleviated by l) neutralizing the genome interaction to H.

 

vulgare by introducing single dosage of H. vulgare genomes P“
at first cross; 2) chemical disturbance of normal spindle V
function on Specific genomes; 3) maintain a wild cytoplasm
which would prevent cyto-nuclei harmony for a specific
genome (Figure 22)
In an attempt to have artificial control of directional
chromosome elimination, Sagar and Ramanis (1967, 1973) used
UV irradiation to block the chloroplast DNA elimination
mechanism and subsequently produced large numbers of bipar-
ental zygotes for genetic analysis in Chlamydomonas. Ponte-
corvo (1971) observed that X-ray or gamma irradiation of one
parent before somatic cell fusion of mouse and Chinese hamster
cell lines would arrest those chromosomes which were direc-

tionally lost under no treatment.

Karyotype analysis

 

Agropyron trachycaulum was quite distinct morpholo-

 

gically from both H. jubatum and H. vulgare. But in the

80

hybrid of AHV442 and AHV444, H. trachycaulum chromosomes

 

could not be distinguished from either H. jubatum or H.
vulgare chromosomes in either meiotic or mitotic chromosome
configurations. Their relationship can only be speculated

on the basis of the genome formula and meiotic chromosome

associations. Karyotypic analysis has been employed to typify

the relationship in terms of evolutionary speciation between
Species by karyotypic similarity. At the present stage,
karyotypic analysis was unable to define the relationships

of karyotypic similarity between H. trachycaulum, H. jubatum,

 

and H, vulgare. But it established the potentiality of
detecting chromosome differentiation leading to gene intro-
gression in comparison to subsequent hybrids.

Individually, the karyotype of H. jubatum has three
pairs of satellite chromosomes and two pairs of telocentric
chromosomes which are quite different karyotypically from H.
vulgare which has two pairs of satellite chromosomes and the
rest are median and submedian chromosomes. Compatibility
between genomes of H. jubatum and H. vulgare might depend on
their physical structure. Therefore, there is quite a gap
for these two genomes to match up physically in meiotic
synapsis. In fact, the asynaptic behavior between genomes
of H. vulgare (2X) and H. jubatum (4X) has been observed
(Murry, 1975). The karyotypic analysis is in accord with
the chromosome association as a compatible indicator between

genomes of H. vulgare and H. jubatum.

 

81

In the genus Hordeum, cultivated H. vulgare is not the
only species evolved to become incompatible with other wild
forms. For instance, H. jubatum was Significantly different

in karyotype to H. lechleri, H. californicum and H. pusillum.

 

AS a result, persistent 7-14 univalents were Observed in the
hybrid Of H. jubatum when it was crossed with any of the
three species. In addition, there was a significant number
of univalents among combinations of 7 Species in the genus
Hordeum (Rajhathy, 1961). Chromosome exchange, as well as
gene transgression, was limited in most of these hybrids.
Karyotypic variation, accompanied by uncompatibility within
the genus Hordeum, of heterogeneous genome origin, and, sub-
sequently, genome differentiation were assumed within the
genus Hordeum. Based on hybridization within the genus
Hordeum, H. jubatum has been described as a segmental allo-
tetraploid.

In addition, spontaneous hybrids between Hordeum Spp,

with other genera such as AgrOpyron, Elymus, Secale, and

 

Hgynaldia were well documented. On the other hand, a gene
pool overlap among genera could be reflected in their karyo-

typic ambiguity within the tribe Triticeae. In this study,

 

a plot of chromosome index range and frequency of H. jubatum

shared the co-center with those of H, trachycaulum and H.

 

vulgare (see Fig. 7). A Similar pattern was also found among
the hybrids of AHV444, AHV442, and VH—35.

The genome of H, trachycaulum has been speculated to

 

play the role of a buffer or balancer between genomes of

 

82

H. jubatum and H. vulgare in the hybrid series of AHV444,
AHV442 mix octOploid and AHV-Bl. Dosage effect of a Specific
,genome has been well documented for the genome interaction
link with the phonomenon of chromosome elimination in certain
interSpecific and intergeneric hybrids. Phenotypically,

AgrOpyron characteristics are still contained in all hybrids

 

in Spite of a combined genome of H. jubatum and H. vulgare. F“

This might Show that H. trachycaulum has a wider range of

 

chromosome index than either H. jubatum or H. vulgare in

addition hybrids are maintained in H. trachycaulum cytOplasm.

 

 

Regarding evolution, the genus Agropyron has a broader germ-

 

plasm which has been used in replenishing the gene pool of
cultivated wheat. Cauderon (1962) reported allopolyploidy,
segmental allopolypoidy, and complex allOpolyploidy among

eight species in the genus AgrOpyron. In addition, spon-

 

taneous and artificial hybrids occurred frequently between

AgrOpyron spp. with other genera such as Triticum, AegilOpS,

 

Secale, Haynaldia, Hordeum, and ElyHuS. These hybrids are

 

 

also unique in maintaining AgrOpyron cytOplasm. In a pre-

 

vious study it was illustrated that most hybrid series were

derived from an Agropyron cytOplasm hybrid. Considering the

 

cytoplasmic effect, karyotypic similarity of entries involved
in hybridization is just one factor in a complex evolutionary

system.

SUMMARY

Comparative karyotype studies among AHV442, AHV444
and VH-35 were unable to detect chromosome exchange by
traditional cytological techniques due to the similarity of

the original karyotypes Of H. trachycaulum, Hordeum jubatum

 

and Hordeum vulgare.

 

H. vulgare genome was expressed morpholOgically in
hybrids and in the first backcross in prOportion to the
genome dosage involved. The factors of chromosome elimina-
tion facautated in the H. vulgare genome by the dosage ratio

to the combined genome of H. tracHycaulum and H. jubatum.

 

The chromosome elimination was circumvented by introducing
a single dosage of H. vulgare genome at beginning and then
doubling the dosage by colchicine before adding more dosage
in subsequent backcrosses to H. vulgare.

The potential of gene transfer from germplasm of

either H. trachycaulum or H. jubatum into H. vulgare was

 

discussed. Results have typified the concept of reconstruction
and replenishing barley germplasm for improving its environ-
mental stress, disease and insect resistance through hybri-

dization with its wild relatives.

83

 

w
Ill-“1'91.“ .

APPENDIX

APPENDIX

Chromosome Association in Hybrids Involving Agropyron

 

trachycaulum, Hordeum jgbatum and Hordeum vulgare

 

R. H. Huang,2 R. P. Steidl and W. Tai
Department of CrOp and Soil Sciences and
Department of Botany and Plant Pathology

Michigan State University, East Lansing, Michigan 48824

ABSTRACT

A natural cross between Agropyron trachygaulum (Link),

 

Malte (4X), and Hordeum jubatum L. (4X) collected in Alaska

 

was duplicated by making reciprocal crosses in the greenhouse.
Comparison of the progeny of these crosses with the natural

hybrid showed that AgrOpyron trachycaulum was the female in

 

the natural hybrid. The natural hybrid was doubled by col-
chicine and crossed with diploid and tetraploid Hordeum
vulgare L. emend. Lam.

The cytology of the natural hybrid, synthetic hybrids,

amphiploid, and hybrids of the amphiploid with Hordeum vulgare

 

was studied and genome formulas for the above were suggested.
The results suggest that the chromosome association of Hordeum

vulgare with its wild relatives could be enhanced with

 

lMichigan Agricultural Experiment Station Journal
Article No. 8453.

2The work of the first author was done in partial ful—
fillment for the Ph.D. degree at Michigan State University.

84

 

85
appropriate parental combination which contributes to genome

balance. The impact of Agropyron and Hordeum jubatum genomes

 

 

on Hordeum vulgare was discussed.

 

Agropyron is one of the most variable genera in the

 

Gramineae. H. trachycaulum is a tetraploid with a chromosome

 

number 2n=28 (Myers, 1947). The genome formula for H. trachy-
caulum (2n=28) was given as SSXX in which "S" denotes the J
genome of H. Spicatum (PurSh) Scrib and Smith and "X" is a
genome of unknown origin. (Dewey, 1968, 1969). Boyle and

Holmgren (1954) suggested that H. jubatum and H. trachycaulum

 

 

(Link) Malte share a common genome as AACC for H. jubatum

and AABB for H. trachycaulum. This conclusion has been sup-

 

ported by Bowden (1960) and Gross (1960). Gross's studies
produced two different partially fertile octOploid plants
(2n=56) by colchicine treatment of sterile tetraploids H

Agrohordeum macounii. A Similar hybrid was reported by

 

Mitchell and Hodgson (1965) and classified as H ngohordeum

 

pilosilemma. Recent cytological studies on the genome of H.

 

jubatum with other related Species Show that H. jubatum is a
segmental allotetraploid. (Wagenaar, 1959, 1960; Rajhathy
et al., 1964; Starks and Tai, 1974).

Starks and Tai (1974) also suggested that the chromo-
some association in H. jubatum is genetically controlled with
a dosage effect: when there is a single dose from each genome
of H. jubatum, homoeologous pairing occurs. However, if each
genome appears twice, homologous pairing prevails. They sug-

gested the genome formula AAA'A' for H. jubatum.

86
Riley and Kempanna (1963), Feldman (1966) Observed

genetic control of chromosome pairing in Triticum aestivum.

 

They suggested the genes promoting dissociation between
homoeologous chromosomes act at the time of premeiotic
somatic chromosome pairing.

The genus Agropyron has been well documented as a

 

source for desirable agronomic traits in cultivated wheat.
(Elliott, 1957; Rillo et al., 1970; Sears, 1973; Sinigovets, E-

1974; Knott et al., 1977). Species of ngopyron have hardly

 

been thought of as useful germplasm for improving H. vulgare.

The intergeneric hybrid between wild species of AgrOpyron

 

 

and Hordeum can be Obtained spontaneously and synthetically.
Hybridization between distant Species and genera have
intrigued plant breeders as a way to broaden and replenish
the germplasm of economic plants. The factors involved in
using wild relatives to improve cultivated barley were evalu-
ated by Steidl (1976), but the attempts at using some wild
Species within the genus Hordeum to improve cultivated barley
have had unexpected results. For example, crosses between
H. bulbosum L. (2X) and H. vulgare (2X) abruptly produced
haploid H. vulgare due to a phenomenon of chromosome elimi-
nation (Kao and Kasha, 1969; Kash and Sadasivaiah, 1971;
Subrahmanyam and Kasha, 1973; and Ho and Kasha, 1975).

Transfer of disease resistance from H. leporinum Link to cul-

 

tivated barley was reported by Hamilton et al. (1955) and
from H. bulbosum by Schooler (1964). The first and second

backcrosses of [H. jubatum X H. compressum (6X)] to H. vulgare

 

produced weak and sterile H. vulgare type plants due to

87

H. jubatum cytOplasm (Steidl, 1976). The phenomenon of chro-
mosome elimination is reported in the hybridization between
H. vulgare (2X) and Secale (2X) (Fedak, 1977).

The purpose of this study is to further elucidate the

genomic nature of H. trachycaulum, H. jubatum and H. vulgare

 

by comparative studies among natural, synthetic and their
amphidiploid cross with H. vulgare. Attempts were also made

to use H. jubatum and H. trachycaulum as sources of germplasm

 

in barley breeding programs.

The stocks of AgrOpyron trachygaulum, Hordeum jubatum
and the Spontaneous hybrid of these two Species used in this
study were collected in Alaska with the help of Drs. W. W.
Mitchell and R. Taylor. The amphiploid was obtained from
natural hybrid with colchicine treatment by Dr. R. P. Steidl.

Diploid H. vulgare "Larker" (CI 10648), "Coho" (CI 13852)
and one tetraploid H. vulgare from Dr. A. B. Schooler were used
to cross with the amphiploid. The plants were grown in a
_greenhouse at 18-20°C. Spikes used as the female were emascu—
lated and covered with aluminum foil one or two days before
pollination. Fresh pollen was shed onto the stigma by tapping
the designated male Spike. Pollination was repeated on two
consecutive days. Embryo culture was employed to obtain the
hybrid in crosses between the amphiploid and H. vulgare.
Immature seeds were sterilized for five minutes with commercial
Clorox diluted with an equal part Of distilled water. The
embryos were dissected from the underdeveloped seeds and

transferred to Norstog media (1973).

 

88

For cytological studies, root tips were collected after
the plant had been subjected to cold treatment (0-2°C) over-
night.

Farmer's solution was used for fixation of the root tip
and Spike. The aceto-carmine smear technique was used for

cytological slide preparation.

RESULTS AND DISCUSSION EF
The crosses and hybrids obtained are Shown in Table 1.
The morphology of the hybrids was generally intermediate

between the parents. H. tracHycaulum when used as female has

 

 

a relatively higher seed set than H. jubatum in reciprocal
crosses. Fertility was partially restored in the amphiploid
which was subsequently used as the female to cross with H.
vulgare.

The meiotic chromosome association of plants was observed
in the pollen mother cells and is summarized in Table 2.

Root tip chromosome counts showed that the natural
hybrid had 2n=28; amphiploid, 2n=56; amphiploid crossed with
diploid H, vulgare, 2n=35 (Figure l); and crossed with tetra-
ploid H. vulgare had 2n=42 (Figure 2).

Metaphase I was the main stage used for interpretation
of chromosome association. Meiotic chromosome behavior of
the synthetic hybrid of reciprocal crosses was similar to that
of the Spontaneous hybrid. Generally, they all had a minimum
of seven univalents, an average of less than seven bivalents,
and a few trivalents. Various numbers of micronuclei were

Observed in quartets. The pollen grains were of different

89

omomm: wusuaso omunEm «

 

 

 

 

Adah» HouCH3V

 

 

Immucmv INHucmy I
.3 om men 3133 x 933-33033H3353 mum Hs> .3
Ammucmv Immucmy I
.3 3 3m N33 3 amvuouonunos3 mum 33> .3
Amequv
“maucmw I Immucmv
.3 am an mum Hs> .3 3133 x advueuoamunmeH
.onoov
Saucmv I Immucmv
43 m mm mum H3> .3 «333 x a<vuouoadunde<
Immucmv Iomncmv
.3 ma em mummas> .3 ~133 x 331-330H3H332<
.mmncmv I Ammucmv I
H mm ma Edasmomnomuu .4 Bowman“ .m
immucmv I Ammuewv
an an om Ssumnsn .3 suasmoscomuu .4
mucmHm wanna: pom mommm omuocflaaom mommouu
mo .02 Hmuoa mmxfiom .Oz

 

 

acuumnuouuQS3 cu omcumuno mucmam new mommm mo nmnssz

dH magma

 

3333 333333> .m x 333 x 333 - 3303333323 «3

Axmv mumm~s> .m 3 Ann 3 adv I ofloaofinofim 3.

 

90

 

 

3 33.3 33.33 33.3 n 3332 333u333

333 3 3-3 33-33 33-3 "33333 .3333>3<
3 33.3 33.33 33.3 n 3332 333nc33

33 3 3-3 33-3 33-3 ”33333 .333>3<
33.3 33.3 33.33 33.3 n 333: 333-333

33 3-3 3-3 33-33 3-3 "33333 3333 x 333-3303333323
33.3 33.3 33.3 33.33 3 333: 333-333

33 3-3 3-3 3-3 33-3 "33333 333 x 333-3333333333
3 33.3 33.3 33.33 H 3332 333-333

33 3 3-3 33-3 33-3 "33333 333 x 333-3333333333
3 33.3 33.3 33.33 " 3332 333u333

33 3 3-3 3-3 33-3 ”33333 333 x sac-3333332

3 3 3.33 3 u 3332 333u333 I

333 3 3 33 3 "33333 2333333 .3

3 3 3.33 3 u 3332 333-333 I

003 o o «A o uwmcmm Edasmowzomnu .4

33333 >3 333 33 3 333333

MO .02 COHHMHUOmmm OEOmOEOHSU

 

 

mucmam HO :oflumHOOmmd OEOmOEOHnU one

€.N OHQMB

 

(l)
(2)
(3)

(4)

91

Figure 1A

Somatic metaphase in AHV442 with 2n=35 (X1360)

Somatic metaphase in AHV444 with 2n=42 (X880)

Metaphase l in AHV442 with 131 + 811 (3 ring by arrow) +
2 III (X915)

Metaphase 1 in AHV444 with 131 + 1311 (7 ring by arrow) +

1 III (X915)

b..___ _. _-.

93

Sizes; none stained with Iz-KI. The anthers were shrunken
and the plants were completely sterile.
The amphiploid from the Spontaneous hybrid had very
few multivalents but the number of univalents was substantially
decreased. The average number of bivalents was 25 with a range

from 22 to 28.

The progenies from the amphiploid as female crossed

r
with the diploid or the tetraploid H. vulgare are coded as
AHV442 and AHV444, respectively. The chromosome number Of
2n=35 is expected in AHV442, likewise 2n=42 was obtained in
AHV444. Univalents averaged 6.53 in AHV442 and 7.09 in AHV444. L1

 

Bivalents in AHV442 averaged 13.93 and 15.92 in AHV444. Only

a few multivalents were observed in these hybrids. Morpholo-
gically, these hybrids have significant H. vulgare character-
istics such as a prominent auricle and spike style. Some
agronomic characteristics of the wild species such as pubescent
leaves and mildew resistance were perceivable in these hybrids.

With a common genome between H. trachycaulum and H.

 

jubatum, the chromosome association in the hybrid should
average 7 bivalents, a minimum of 14 univalents, and no multi-
valents. However, in the combined data of reciprocal crosses,
Boyle and Holmgren (1954) reported the presence Of multi-
valents in 23.55 percent of the cells observed. It is hard

to explain the presence of multivalents if both parents of

the amphiploid are allOpolyplOids. We Observed trivalent
associations in the synthetic hybrids from reciprocal crosses.

In the synthetic hybrids trivalents occurred in 22.22 percent

94

of the cells when the female parent was H. trachycaulum.

 

When H. jubatum was the female parent, the frequency increased
to 28.72 percent.

Since both parents were tetraploids, the hybrid received
two genomes from each parent. The two genomes from H. Egg-
chycaulum are non-homologous with each other, but the chromo-
somes from H. jubatum are homoeologous with each other. Thus,
in the hybrid a single set of chromosomes from each of the
two genomes of H, jubatum is present and the chromosomes Should

pair autosyndetically, A-A. With one genome of H. trachycaulum

 

in common with one of the two genomes of H. jubatum, any tri-
valent occurring in the hybrid should have a chromosome

contributed by H. trachycaulum and two chromosomes contributed

 

by H. jubatum, A-A'-A.

In the synthetic hybrids, 6-7 chromosomes often behaved
in a manner quite different from the rest of the chromosomes
and were Often excluded from the main group. We have tenta-
tively concluded that these are the chromosomes belonging to

the B genome from.H. trachycaulum. Separation and grouping

 

of chromosomes based on genome originality was suggested by
Huskins and Chouinard (1950), Thompson (1962), Bammi (1965),
Tai (1970), and Rizzoni et a1. (1974). It has been demon-
strated that some homolOgy exists among the two genomes from

H. jubatum and one genome from H, trachycaulum. The chromo-

 

somes of the fourth group, the B genome, were comparatively

separated and excluded from the main group.

 

95

The chromosome association Observed in the amphiploid
showed a mean of 2.36 univalents, 25.63 bivalents, 0.71 tri-
valents, and 0.06 quadrivalents. Since few multivalents
occurred in the amphiploid, obviously there was no pairing
between homoeologous genomes from H. jubatum. However, the
frequency of trivalents and quadrivalents was lower than
expected with a combined total of 0.76 multivalents per cell.

The dosage theory of Starks and Tai (1974) is further
confirmed by the chromosome behavior in the amphiploid.

The lack of multivalents in our amphiploid can be explained

on the same basis. We suggest that the gene or genes con-

 

trolling chromosome pairing in the amphiploid also promotes
dissociation between less like chromosomes (the homoeologous
chromosomes) and that genes affect pairing among the one

genome, A', from H. trachycaulum and the two genomes, AA,

 

from H. jubatum. In the hybrid, AAA'B, allosyndetic pairing
of H. jubatum chromosomes would occur to form about seven
bivalents (AA') with a maximum of seven trivalents (AAA').
The chromosome number doubled in the amphiploid; however, the
number of bivalents Observed in the hybrid did not become the
number of quadrivalents in the amphiploid. The relationship

between the one genome of H. trachycaulum in common with one

 

genome of H. jubatum is not as close as a regular homologous

set.
Therefore, preferential compatibility among genomes
existed. We suggest changing the genome formula of the

plants to: H. trachycaulum, A3A3BB; H. jubatum, AlAlAZAz;

 

96

the hybrid, A1A2A3B; and the amphiploid, A1A1A2A2A3A3BB.

The amphiploid has been successfully crossed with
diploid and tetraploid cultivated barley, H. vulgare.
The hybrid between the amphiploid and the diploid H. vulgare
is expected to be a pentaploid with 2n=35 chromosomes; coded
AHV442.

By the formula, the genome constitution of AHV442
plant would be as A1A2A3BV, V is the designation for the
H. vulgare genome. It has the same genome constitution as

the natural hybrid in addition to an extra V genome from H.

vulgare. Therefore, the association should be seven bivalents

 

of allosyndetic pairing between A1A2 or 1-3 trivalents from
A1A2A3 plus at least 14 univalents each from the B and V
genomes.

By Observation, it was shown that there was an average
of 6.53 univalents and 13.98 bivalents (Figure 3). Multi-
valents were very rare. There has been no documentation Of
the fact that H. vulgare genome would associate with genomes
of its wild relatives. Murry (1975) observed no chromosome
association in hybrids between H. jubatum and H. vulgare.
Therefore, seven univalents in this hybrid have been specu-
lated to be from the V genome rather than the B. Consequently,
fourteen bivalents should come from allosyndetic pairing of
the A genome with B under the influence Of the V genome. The
hybrid derived from the cross between the amphiploid with
tetraploid H. vulgare had a genome constitution of A1A2A3BVV

with a chromosome number 2n=42. The univalents averaged

97
7.92, the bivalents 15.92 and the trivalents increased from
0.16 in AHV442 to 0.86 in AHV444 (Figure 4). Under micro—
SCOpiC Observation, seven large ring bivalents apparently
from VV genomes could be seen that were different from other
genomes which prevailed in a form of a rod bivalent. The
seven univalents probably belonged to the B genome as an
association pattern of A1A2A3B genomes in a natural hybrid
without the influence of two dosages of the V genome. A
concomitant increase of H. vulgare characters in the plant
was observed comparing two doses of V in AHV444 with only
one dose of V in AHV442. Subrahmanyam and Kasha (1973 and
Ho and Kasha (1975) reported that rate and degree of chromo-
some elimination was dependent on a well-defined ratio of
genomes in the hybrid of H. vulgare X H. bulbosum. In addition,
they demonstrated that the process was genetically controlled
and is essential to H. vulgare genomes ratio to other genomes.
Manipulation on the cell level had the advantage of reducing
the gene load in single cells rather than in the whole plant.
Tissue culture plus the apprOpriate treatment might genener-

ate the plant pOpulation with a variable chromosome make-up.

ACKNOWLEDGMENTS
The authors wish to thank Joanne Whallon and Dr. Carter

Harrison for their help in manuscript preparation.

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