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This is to certify that the

dissertation entitled

Cytogenetic Studies of the Dry Beans,
Species of the Genera Phaseolus and Vigna.

presented by

Gary Roy Bauchan

has been accepted towards fulfillment
of the requirements for

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CYTOGENETIC STUDIES OF THE DRY BEANS,
SPECIES OF THE GENERAL PHASEOLUS AND VIGNA (LEGUMINOSAE)

By

Gary R. Bauchan

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1982

.
Q

 

 

ABSTRACT

CYTOGENETIC STUDIES OF THE DRY BEANS,
SPECIES OF THE GENERA PHASEOLUS AND VIGNA (LEGUMINOSAE).

BY

GARY R. BAUCHAN

Most of the species in the genera Phaseolus and Xigna are diploid
(2n=2X=22) with only two exceptions, both of them being tetraploid
species (2n=4X=44). The basic chromosome number is X=ll. Most of
these species have small chromosomes with a homogeneous karyotype,
resulting in difficult karyotypic analysis. The discovery of polytene
chromosomes may prove to be helpful in attempts to identify the
individual chromosomes. Observations of meiosis in the pollen mother
cells has shown normal pairing at metaphase I, however, early bivalent
separation, which appear as univalents, have been observed. .Chromatin
bridges and quadrivalent associations in the hybrids indicate that
structural modifications, particularly inversions and translocations,
comprise the main evolutionary force for genome differentiation.

The cytology of Xigna radiata (L.) Wilczek,_2. umbellata, (Thunb.)
Ohwi & Ohashi, their hybrid, amphiploid, backcross progeny, and the
plants of subsequent generations were investigated to determine the
genome relationship among these plants for use in a breeding program
with the goal of incorporating beneficial characteristics of_K.

umbellata into V. radiata. Light microscope investigations included:

Gary R. Bauchan

root tip chromosome counts; the analysis of chromosome pairing during
all stages of microsporogenesis; and pollen stainability to estimate
plant fertility.

In this study the presence of secondary associations, multipolar
meiosis, the grouping of bivalents during meiosis, and the high basic
chromosome number has led to a re-evaluation of the ploidy levels in
these plants. Genome formulae have been assigned in accordance with
these findings under the assumption of genetic control of pairing with

dosage effects.

Copyright by
GARY R. BAUCHAN

1982

DEDICATION

This dissertation is dedicated to my late mother, Barbara, and to my

father, Roy G. Bauchan.

ii

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to Dr. William Tai
for his advice, encouragement, and friendship throughout the course of
this research. I would like to thank Dr. Wayne Adams, Dr. John Beaman,
Dr. Alfred Saettler, and Dr. Shigemi Honma for their advice and
willingness to read my dissertation.

I also wish to thank my fellow colleagues; Marcia Machado, N.C.
Chen, Jim Parrot, Joanne Whallon, John Blackson, Rich Rabeler, and
Chuck Elzinga, for their friendship and assistance offered throughout
the completion of this work.

A special note of thanks and appreciation is extended to my
sisters, Jenny, Nancy, Wendy, and Tracy; my grandparents, Mr. and Mrs.
Harry Witkowski and Mrs. Florence Bauchan; and my in-laws, Mr. and Mrs.
Walter Kloc, for their support and encouragement throughout the years.
Also I wish to thank the late Guy W. VanderWest for his help in
financing my college education.

Last but not least, a special thanks to my wife, Francine, for her

unfailing support, encouragement and assistance.

iii

TABLE OF CONTENTS

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . ii
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . iii
List of Tables . . . . . . . . . . . . . . . . . . . . . . . v

List of Figures . . . . . . . . . . . . . . . . . . . . . . vi

SECTION I. Cytogenetics of Phaseolus and Vigna

Abstract

Introduction . . . . . . . . . . . . . . . . . . . . . 1
Discussion . . . . . . . . . . . . . . . . . . . . . . 1
Conclusion . . . . . . . . . . . . . . . . . . . . . . 17
Appendix . . . . . . . . . . . . . . . . . . . . . . . 18

References . . . . . . . . . . . . . . . . . . . . . . 21

SECTION II. Cytology and Fertility of Vigna radiata and_x.
umbellata

 

Abstract

Introduction . . . . . . . . . . . . . . . . . . . . . 31
Materials and Methods . . . . . . . . . . . . . . . . . 33
Results . . . . . . . . . . . . . . . . . . . . . . . . 35
Discussion . . . . . . . . . . . . . . . . . . . . . . 41

References . . . . . . . . . . . . . . . . . . . . . . 50

iv

Table

LIST OF TABLES
SECTION I
Chromosome numbers of some economically important

species in the genera Phaseolus and Vigna.

Intergeneric, interspecific and intraspecific crosses
that have been performed.

Observations of meiosis and pollen fertility in the
hybrids.

SECTION II

Metaphase I chromosome associations in the amphiploid
(2n=44).

Meiotic chromosome behavior of 301 (Amphiploid x

.2. radiata backcross).

Pollen stainability of the amphiploid and BC1.

Chromosome number counts from self progeny of BC1.

13

15

38

39

4O

41

LIST OF FIGURES

SECTION I

Figure

1.
2.

10.
11.

Root tip cell in Vigna radiata (2n=22).
Metaphase I in V. umbellata showing secondary
associations of_two pairs of bivalents and co-
orientation of the other chromosomes.

Metaphase I in X: umbellata showing a Split metaphase
plate with 5/6 segregation of bivalents. .

Anaphase I in the interspecific hybrid V. radiata x
V.umbe11ata with 5/6 disjunction and II 1agg1ng
Ehromosomes. . . .
Metaphase I in the amphiploid showing 22 bivalents.
Metaphase I in the backcross progeny (Amphiploid x
.2. radiata) showing 11 bivalents and 11 univalents.

SECTION II

Diagram of a cross between Vigna radiata and V.
umbellata.

 

Root tip cell in the Amphiploid (2n=44).

Metaphase I in the Amphiploid showing 22 bivalents.
Root tip cell in BCl (2n=33).

Metaphase I in BCl showing 11 bivalents and 11
univalents. . . . . . . . . . . . . . . .
Metaphase I in BCl showing a Split plate with 5/6
segregation of bivalents and 11 univalents scattered

throughout the cell.

Anaphase I in BCl showing 11/11 segregation of
chromosomes and 11 lagging chromosomes.

Metaphase II in BCl showing 11 chromosomes per cell
with several fragments.

Quartet stage in 801 showing four "normal" cells
plus two microcells.

Root tip cell in 81 (2n=23).

Summary of the proposed genome formulae.

vi

34
36
36
36

36

36

36

36

36
36
44

 

 

SECTION I

Cytogenetics of Phaseolus and Vigna

ABSTRACT

CYTOGENETICS OF PHASEOLUS AND VIGNA
By

Gary R. Bauchan

Most of the species in the genera Phaseolus and Vigna are diploid
(2n=2X=22) with only two exceptions, both of them being tetraploid
species (2n=4X=44). The basic chromosome number is X=ll. Most of
these species have small chromosomes with a homogeneous karyotype,
resulting in difficult karyotypic analysis. Polytene chromosomes may
prove to be helpful in attempts to identify the individual chromosomes.
Observations of meiosis in the pollen mother cells have shown normal
pairing at metaphase I, however, early bivalent separation, which
appear as univalents, have been observed. Chromatin bridges and
quadrivalent associations in the hybrids indicate that structural
modifications, partiCularly inversions and translocations, comprise the
main evolutionary force for genome differentiation. Cytological
studies on these plants have been conducted which seem to indicate that
they may not be diploids but rather allotetraploids with a basic
chromosome number of X=6. The observation of secondary associations,
multipolar meiosis, the grouping of bivalents during meiosis, and the

high basic chromosome number has led to this proposed re-evaluation.

Introduction

 

Cytogenetic studies in Species of the genera Phaseolus and Vigna
are handicapped because these species have: 1) very small chromosomes,
2) homogeneous karyotypes (i.e., similar chromosomes within and between
species), and 3) the lack of knowledge concerning the driving forces of
evolution (i.e., chromosome structure modifications or chromosome num-
ber changes). It is difficult to obtain meiotic stages because usually
only one bud in an inflorescence is undergoing meiosis at any given
time. Therefore, to obtain a bud at a stage suitable for analysis of
the chromosome behavior is a major problem.

Most of the cytogenetic studies in these genera have been conduct-
ed in southeast Asia, probably due to their agricultural and economic
importance in this region. Many of the Vigna species are endemic to
India, perhaps this may be a catalyst for active cytogenetic research
in that region. In recent years, reports originating in the laborator—
ies in the western countries, including the United States, have in-
creased.

Discussion

Karyotype

The basic chromosome number of X=11 has been reported for all of
the species thus far examined of the genera Phaseolus and Vigna (Table
1). Most species are diploids with a somatic number of 2n=2X=22 (Fig-
ure 1). Dana (1964) was the first to report a Spontaneously occurring

allotetraploid, Vigna glabrescens with 2n=4X=44. A Similar chromosome

 

count has been reported by Frahm-Leliveld (1965b) for Vigna radiata

 

var. sublobatus, however, this plant may be the same Species that Dana

1

 

 

Table 1. Chromosome numbers of some economically important species in the genera Phaseolus

and Vigna.

Phaseolus

.3. acutifolius

.3. coccineus
fig. multiflorus

.E. lunatus

E, polystachys

_fl. vulgaris

P. vulgaris var.
aborigineus
= P. aborigineus

Mathew

11

11

11

11

22

22

24

22

22

22

22

References

Karpechenko, 1925; Marechel, 1969 &
1970; Shibata, 1962; Smartt, 1970;
Sarbhoy, 1978; Sinha & Roy, 1979;
Sarbhoy, 1980.

Karpechenko, 1925; Senn, 1938; Nagl,
1962; Thomas, 1964; Lamprecht, 1966;
Marechal, 1969 & 1970; Smartt, 1970;
Cionini & Avanzi, 1972; Mok & Mok,
1976; Joseph & Bouwkamp, 1978; Sinha &
Roy, 1979; Blackson & Tai, 1982.

Kleinman, 1923.

Karpechenko, 1925; Kawakami, 1930;
Senn, 1938; Berger et a1., 1958;
Dhaliwal et a1., 1962; Thomas, 1964;
Marechal, 1969 & 1970; Smartt, 1970;
Sinha & Roy 1979; Sarbhoy, 1980.

Allard & Allard, 1940; Dhaliwal et
31., 1962.

Karpechenko, 1925; Weinstein, 1926;
Katayama, 1928; Kawakami, 1930;
Malinowski, 1935; Senn, 1938; Takagi,
1938; Thomas, P., 1945; Berger et al.,
1958; Shibata, 1962; Moh, 1962;
Thomas, H., 1964; Marechal & Otoul,
1965; Yarnell, 1965; Lamprechet, 1966;
Nagl, 1969; Marechal, 1969 & 1970;
Smartt, 1970; Mok & Mok, 1976; Biswas
a Bhattacharya, 1976; 8hattacharya,
1978; Sarbhoy, 1978; Sinha & Roy,
1979; Sarbhoy, 1980; Blackson & Tai,
1982.

Burkart & Brucher, 1953; Blackson &
Tai, 1982.

Table 1. (continued)

Vigna Geeetoghyge Segrophzge

V, aconitifolia 11
:15. aconitifolius

 

V, angularis 11

25 glabrescens 22

: V. radiata

— glabra
V

. mungo 11
= fl. mun 90

.V. radiata 11
P. aureus

.fl. radiata

.1. radiata var.
sublobatus
:-E. sublobatus

V. umbellata 11
:.fl. calcaratus
:-P. ricciardianus

 

‘1. unguiculata 11
_V. catjang
V, cylindrica
_V. sesquipedalis
. sinensis

 

|<

22

22

44

22

24

22

24

22

22

24

References

Tschechow 6 Kartaschow, 1932;
Marechal, 1969 6 1970; Biswas 6 Dana,
1976a; Sinha 6 Roy, 1979; Sarbhoy,
1980.

Marechal, 1970; Joseph 6 Bouwkamp,
1978; Sinha 6 Roy, 1979.

Dana, 1964, 1965a, b, 6 1966d;
Krishnan 6 De, 1965, 1968, 6 1970;
Swindell et al., 1973; Sarbhoy, 1980.

Karpechenko, 1925; Simmonds, 1954; Sen
6 Chheda, 1958; Dana, 1966b; De 6
Krishnan, 1966a 6 b; Bir 6 Sidhu,
1967; Krishnan 6 De, 1968; Biswas 6
Dana, 19758; Sinha 6 Roy, 1979;
Sarbhoy, 1980.

Rau, 1929

Karpechenko, 1925; Kawakami, 1930;
Frahm-Leliveld, 1953 6 1957; Simmonds,
1954; Shibata, 1962; Krishnan 6 De,
1965; Dana, 1966a, b, 6 G; De 6
Krishnan, 1966b; Bit 6 Sidhu, 1967;
Krishnan 6 De, 1968; Marechal, 1969 6
1970; Kaul, 1970; Biswas 6 Dana, 1975;
Josepha 6 Bouwkamp, 1978; Sinha 6 Roy,
1979; Sarbhoy, 1980.

Karpechenko, 1925; Rau, 1929.

Frahm-Leliveld, 1965b.

Janaki-Ammal, 1945; FrahmcLeliveld,
1953 6 1957; Shibata, 1962; Dana,
1966c 6 d; Singh 6 Roy, 1970; Biswas 6
Dana, 19758; Joseph 6 Bouwkamp, 1978;
Sinha 6 Roy, 1979; Sarbhoy, 1980.

Karpechenko, 1925; Tschechow 6
Kartaschowa, 1932; Senn, 1938;
Saunders, 1960; Sen 6 Bhowal, 19603 6
b; Shibata, 1962; Paris, 1964;
Frahm-Liliveld, 1965; Kodama, 1967;
Marechal, 1969 6 1970.

Rau, 1929; Kawakami, 1930; Berger et
81., 1958; Floresca et a1., 1960;
Miege, 1962.

Figure

Figure

Figure

Figure

Figure

Figure

N

9.)

U1

Root tip cell in Vigna radiata (2n=22). (2000x)

Metaphase I in_!. umbellata showing secondary associations

of two pairs of bivalents and co-orientation of the other
chromosomes. (700x)

Metaphase I in_V. umbellata showing a split metaphase plate
with 5/6 segregation of bivalents. (750x)

Anaphase I in the interspecific hybrid_V. radiata x V.
umbellata with 5/6 disjunction and 11 lagging chromosomes.

650x

Metaphase I in the amphiploid showing 22 bivalents.
(lOOOX)

Metaphase I in the backcross progeny (amphiploid x V.

radiata) showing 11 bivalents and 11 univalents. (550x)

 

6
(1964) studied. Faris (1964) surveyed l92 cultivars and strains of
Vigna unguiculata from 42 countries and found that all had 2n=2X=22.
Previous reports listed this species as having 2n=2X=24 (Rau, 1929;
Kawakami, 1930; Berger et a1., 1958; Floresca et a1., 1960; and Miege,
1962). hromosome counts of 2n=2X=24 have also been reported for
Phaseolus coccineus (Kleinmann, 1923) and Vigng_mgggg (Rau, 1929), but
chromosome counts reported prior to 1930 may be incorrect.

Chromosome size differences have been reported among different
chromosomes of the same chromosome complement as well as the total
chromatin length of the haploid set in different species. However, the
small size of all the chromosomes may make these measurements less
significant than in other species, with larger chromosomes.

Phaseolus vulgaris has the greatest total chromosome length of the
haploid set, 42.26 pm, and £3 lunatus the least, 35.2 pm. The greatest
length in Vigna species is found in X. radiata, 37.4 pm, and the
smallest in_V. umbellata, 28.4 pm (Sarbhoy, 1980). Different
cytological pretreatments or measurements at different stages of
meiosis can lead to variations in chromosome length, thus the
difference between the longest and the shortest complement may not be
statistically significant.

The range of individual chromosome size ranges from 0.17 pm to 3.1
pm for Phaseolus and from 0.64 pm to 3.7 um for Vigna (Sen and Bhowal,
1960; Joseph and Bouwkamp, 1977; Sinha and Roy, 1979; and Sarbhoy,

1980).

 

7
Mok and Mok (1976) observed two pairs of SAT-chromosomes in

Phaseolus vulgaris and P. coccineus, while Bhattacharya (1978) and

 

Sarbhoy (1977) observed only one pair in P. vulgaris. Sinha and Roy
(1979) reported that Vigna radiata, and V. radiata var. sublobatus have
one pair of chromosomes with a satellite. Sen and Bhowal (1960) have

seen two pairs of SAT-chromosomes in Vigna catjang, V. sesquipedalis,

 

 

and V, sinensis (V. unguiculata).

Sarbhoy (1977) suggested that Phaseolus vulgaris possesses a

 

symmetrical karyotype and the other Phaseolus species have an
asymmetrical karyotype. In Vigna species, 2. radiata is the only
species with a symmetrical karyotype. A symmetrical karyotype is one
in which the chromosomes are all approximately the same size, with
median or submedian centromeres. A change in the position of a
centromere or a change in the relative length of a chromosome arm due
to chromosome aberrations can convert a karyotype from a symmetrical to
an asymmetrical karyotype (Stebbins, 1971). Chromosome aberrations
such as inversions, translocations, duplications, and deletions have
been detected in both Phaseolus and Vigna Species (Dana, 1966a 6 d; De
and Krishnan, 1966b; Honma, 1968; Biswas 6 Dana, 1975, 1976a; and
Sarbhoy, 1977).

Since the small chromosome size makes karyotypic analysis of
mitotic metaphase chromosomes very difficult, other techniques (i.e.,
pachytene analysis, Giemsa banding, and fluorescent banding techniques)

have been used.

 

8
Pachytene chromosomes have been studied extensively in corn
because the different sizes and shapes of the heteropycnotic knobs have
diagnostic value in karyotype analysis. Krishnan and De (1965 and
1970) and De and Krishnan (1966a) used pachytene chromosomes to

karyotype Vigna radiata, V. mungo and V: glabrescens. They observed

 

"knobs" of varying lengths near the centromere. The difficulty in
distinguishing between individual chromosomes at the pachytene stage,
makes the results drawn from this technique inconclusive.

Giemsa and fluorescent banding techniques are rather new in
cytogenetic studies. In most plant species it is difficult to obtain a
good banding pattern with any degree of consistency. However, banding

patterns have been used to produce a karyotype of Phaseolus vulgaris

 

and P. coccineus. All the chromosomes Showed bands around the
centromere and terminal bands in one or both arms. The number of bands
varied from two to five bands per chromosome (Schweizer, 1976;
Bhattacharya, 1978; and Mok and Mok, 1976).

Nagl (1974) has written a review paper on the polytene chromosomes
found in suspensor cells of Phaseolus. The suspensor consists of a
basal cell plus several other cells which connect the embryo proper to
the embryo sac. Polytene chromosomes have been found in B: coccineus
and P. vulgaris. These polytene chromosomes are Similar to those found
in dipteran insects. The chromosomes show distinct banding patterns if
the plants are subjected to cold temperatures, unfavorable light
periods, inhibitions of RNA synthesis by actinomycin D, and exposure to
high concentrations of calcium ions, thus making it possible to

identify individual chromosomes and possibly the loci of individual

9

genes. Puffs, the site of RNA synthesis, also have been observed in
the polytene chromosomes (Nagl, 1969). These chromosomes are about 30
times longer and 15 times larger in diameter than metaphase chromosomes
found in root tips of the same plants.
Meiosis

Early reports indicated that meiosis in the pollen mother cells
was normal in most Phaseolus (Weinstein, 1926; Dhaliwal et a1., 1962;
Sarbhoy, 1977; and Sinha and Roy, 1979b) and Vigna species (Sen and
Chheda, 1958; Sen and Bhowal, 1960 a 6 b; Krishnana and De, 1965;
Frahm—Levliveld, 1965a 6 b; De and Krishnan, 1966b; Dana, 1966a, b, 6
c; Biswas and Dana, 1976a; Sarbhoy, 1977; and Sinha and Roy, 1979).
The 22 chromosomes form 11 bivalents at diakinesis and metaphase I.
I Segregation of the chromosomes was reported to be normal in subsequent
stages, resulting in pollen grains with 11 chromosomes each.

Marechal (1971) observed the meiosis of Phaseolus coccineus,_P.

 

formosus, P. obvallatus,_£. polyanthus, and P. vulgaris and noted the
nuber of chiasmata that formed between the homologous chromosomes. He
then made a series of crosses between these species and observed the
number of chiasmata that were formed. From this study he concluded
that P: formosus, P. obvallatus, and P. polyanthus are similar to P.
coccineus but were distinct enough to reclassify them as subSpecieS of
£3 coccineus i.e., P. coccineus subsp. formosus, etc.

Irregular meiotic behavior has been occasionally reported. The

presence of univalents has been observed in P. acutifolius, P.

 

coccineus, P. lunatus, P. vulgaris, Vigna aconitifolia, V. radiata, and

 

 

V. umbellata (Sarbhoy, 1977; Sinha and Roy, 1979; and Machado et a1.,

 

10
1982). The univalents may be attributed to 1) failure of the
chromosomes to pair at zygotene, 2) early separation of bivalents at
metaphase I, or 3) inversion heterozygosity (Sarbhoy, 1977).

Lack of homology has been the most important factor to cause
univalent formation during meiosis I in most plant species. However,
lack of pairing also can be caused by genetic factors as reported in
corn (Beadle, 1930), rice (Ramanujan and Parthsarthy, 1935), wheat
(Feldman, 1966), pea (Gattschalk and Baquar, 1971), and other plants.

Early separation of bivalents at metaphase I has been observed in

Phaseolus coccineus, P. lunatus, P, vulgaris, Vigna radiata, and V.

 

umbellata (Sarbhoy, 1977; and Machado et a1., 1982). Short
chromosomes, such as those found in Phaseolus and Vigna, have a lower
frequency of chiasma formation (Darlington, 1930 and 1937; and Kostoff,
1940), and thus have a tendency to separate early (Machado et al.,
1982). These separated bivalents appear as univalents which orient
themselves opposite each other in the equatorial region. These paired
univalents behave normally and do not affect normal segregation
(Sarbhoy, 1977).

Inversion heterozygosity is known to cause univalent formation in
various Species of Citrus (Raghuranshi, 1962a 6 b). Chromatin bridges
with their accompanying fragments suggests the presence of inversion
heterozygotes. Chromatin bridges have been observed in Phaseolus

acutifolius, P. coccineus, P. vulgaris, Vigna mungo, and V. radiata

 

 

(Sarbhoy, 1978 and Sinha and Roy, 1979).
Dana (1964) reported that the Spontaneously occurring
allotetraploid 2n=2X=44 shows regular meiosis with 22 bivalents at

metaphase I. Dana (1964 and 1966d), Krishnan and De (1968 and 1970),

 

11
and Biswas and Dana (1976b) have used Vigna glabrescens in many of

their crossing programs. When 23 glabrescens was used as the female

 

parent with Vig23_umbellata as the male parent, Dana (1964) reported
that the offspring was triploid with the majority of the metaphase
plates possessing 11 bivalents and 11 univalents. Other meiotic
abnormalities included failure of the chromosomes to line up on the
metaphase plate, early movement of the bivalents at metaphase I,
laggards at anaphase I, and unequal distribution of chromosomes during

the second division. When Dana (1966d) studied the pachytene stage of

 

this hybrid, he discovered inversion loops, duplications, deletions,
and heteromorphic regions. Dana (1965a) crossed V. glabrescens with

Vigna umbellata. Biswas and Dana (1976b) treated this hybrid with

 

colchicine to produce a plant with 2n=66. These plants Showed meiotic

irregularities such as multivalents, laggards, and unequal segregation

 

of chromosomes. In 72% of the cells observed, 33/33 segregation
occurred and autosyndetic pairing was observed. Krishnan and De (1968)

used Vigna mungo as the female parent and_V. glabrescens as the male

 

 

parent. The resulting hybrid had 33 chromosomes with the majority of
the cells containing 12 bivalents and 9 univalents. Quadrivalents,
trivalents, early separation of the bivalents, two to six Spindles
during anaphase II, and one to five micronuclei in the tetrads also
were observed. Krishnan and De (1970) completed a pachytene analysis
of the allotetraploid and produced a karyotype of 19 of the 22 pairs of

chromosomes. They suggested that the chromosomes of X: glabrescens are

 

similar to some of the chromosomes of Vigna radiata and V. mungo.

These authors have called this Spontaneous tetraploid an allotetraploid

12

with the different genomes being derived from V: radiata and X, EEEES;
however, the genomic relationships between these two species have not
been well established.

A number of hybrids have been produced (Table 2), but not all
Fls produced viable seeds, due to the presence of isolating
mechanisms. These isolating mechanisms include: 1) hybrid inviability
or weakness, 2) developmental hybrid sterility, 3) segregational hybrid
sterility, and 4) F2 breakdown (Stebbins, 1971b). Through the use of
embryo culture, some of these barriers have been overcome.

Only a few of the hybrids have been studied cytogenetically (Table
3). Some of the meiotic abnormalities that have been observed include:
1) failure of the chromosomes to align themselves on the metaphase
plates, 2) multivalent formation, 3) early bivalent separation, 4)
chromatin bridges with fragments, 5) laggards, 6) univalents, 7)
multiple spindles, and 8) micronuclei formation. See Table 3 for
specific information on each hybrid studied.

Some of these hybrids have been treated with colchicine to produce
amphiploids. If the hybrids were infertile, the amphiploids proved to
be more fertile as shown by the percentage of viable pollen (Table 3).
Complete fertility was not restored due to abnormal meiosis.
Multivalents, unequal separation of chromosomes, and micronuclei were
observed in the amphiploids.

In one of the crosses, Phaseolus vulgaris x‘P. ritensis, the

 

amphiploid was produced and then backcrossed to P. ritensis, yielding a
triploid. This triploid was then backcrossed producing a trisomic of

2n=23. The trisomics were selfed and produced a small number of

 

13

Table 2. Intergeneric, interspecific, and intraspecific crosses that have been performed.

Phaseolus

Interspecific

'E. acutifolius x.fi. coccineus - Al-Yasiri 6 Coyne, 1966.

.3. acutifolius xifl. vulgaris - Mok et a1., 1978.

_E. coccineus x_fl. acutifolius - Al-Yasiri 6 Coyne, 1966.

.3. coccineus x‘fl. lunatus - Honma 6 Heeckt, 1958; Wolfenbarger 6 Sleesman, 1961.

P, coccineus x.fl. ulgaris - Honma 6 Heeckt, 1952; Al-Yasiri 6 Coyne, 1966; Smartt 6 Hag,
1972; lmbrahim 6 Coyne, 1975.

*P. lunatus x.fl. polystachzus - Lorz, 1952; Dhaliwal et a1., 1962; LeMarchand et a1., 1976.

.E. lunatus xqfl. ritensis - LeMarchand et al., 1976.

_E. ritensis x P. lunatus - LeMarchand et al., 1976.

.5. vulgaris x_fi. acutifolius - Honma, 1956; Al-Yasiri 6 Coyne, 1966; Mok et al., 1978.

*P. vulgaris x.fi. coccineus - Lamprecht, 1941; Tschermak-Seysenegg, 1942; Gates, 1951;
Wall 6 York, 1957; Bemis 6 Kedar, 1961; Al-Yasiri 6 Coyne,
1966; Marechal, 1971.

P. vulgaris x.fl. dumosus - Jaarerslag, 1957.

*P. vulgaris x‘fi. filiformis - Marechal 6 Baudoin, 1978.

_E. vulgaris x13. lunatus - Honma 6 Heeckt, 1959; Al-Yasiri 6 Coyne, 1966; Mok et a1., 1978.

.5. vulgaris x.£. ritensis - Break 6 Kooistra, 1975.

Intreepecific

f3. vulgaris x_fi. vulgaris - Honma, 1958.

*Crosses that have been studied cytogenetically.

 

 

14

Table 2. (continued)

Vigna
Interepecific
V, angularis XIX. mungo - A1 Yasiri 6 Coyne, 1966.

1<
O

aggularis x.l° umbellata - A1 Yasiri 6 Coyne, 1966; Chen, 1980; Parrot, 1981.

glabrescens x.!. mango - Parrot, 1981.
glabrescens x‘V. radiata - Parrot, 1981.

P< b< r<
O O O

glabrescens XIV. umbellata - Dana, 1964 6 1965a; Biswas 6 Dana, 1976b; Parrot, 1981.

|<
O

mungg x_V. angularis - Chen, 1980.
mungo x.x. glabrescens - Krishnan 6 De, 1968.

2.:
|<
O

|<

munqg x V. radiata - Luyeye, 1975.

a:
|<
O

mungo x.!. umbellata - A1«Yasiri 6 Coyne, 1966; Biswas 6 Dana, 1975a.

|<
O

radiata x V, angularis - Ahn 6 Hartman, 1978a; Chen, 1980.

2.:
|<
O

radiata x.l. glabrescens - Dana, 1965b; Parrot, 1981.

#
|<
O

radiata x.V. mungo - Sen 6 Ghosh, 1960; Bhag-Singh et a1., 1964; Chavan et 31., 1965;
Dana, 1966b; De 6 Krishnan, 1966b; Luyeye, 1975; Ahn 6 Hartman,
* 1977; Chen, 1980.

V; radiata x.1. umbellata - Dana, 1966c; Sawa, 1974; Evans, 1975; Ahn 6 Hartman, 1977;
Chen, 1980; Parrot, 1981; Machado et a1., 1982.

fll. radiata x V: angularis - Evans, 1975; Ahn 6 Hartman, 1978b; Chen, 1980.

|<
O

umbellata XIX. mungo - Evans, 1975.
{1. umbellata x V. radiata - Evans, 1975.

Intrsspecific

_V. unguiculata x_1. unguiculata - Premseker 6 Raman, 1972.

Intergeneric
Phaseolus vulgaris x Vigna mungo - Strand, 1943; Al-Yasiri 6 Coyne, 1966.
Phaseolus vulgaris x Vigna umbellata - Al-Yasiri 6 Coyne, 1966.

Vigna aconitifolius x Phaseolus trilobus - Chavan et a1., 1965; Biswas 6 Dana, 1976a.

 

Vigna angularis x Phaseolus acutifolius - Al-Yasiri 6 Coyne, 1966.
Vigna aggularis x Phaseolus vulgaris - Al-Yasiri 6 Coyne, 1966.

Vigna muggo x Phaseolus vulgaris - Wolfenbarger 6 Cleesman, 1961; Al-Yasiri 6 Coyne, 1966.

 

*Vigna radiata x Macroptilium lathyroides - Biswas 6 Dana, 1975b.

 

 

Vigna radiata x Phaseolus trilobata - Dana, 1966a.

 

*Cross that have been studied cytogenetically.

 

15

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16

semi-sterile trisomics, plus fertile normal diploid plants (Braak and
Kooistra, 1975). A Similar situation occurred when the amphiploid of
Vigga_radiata x‘V, umbellata was backcrossed to V: radiata. The
resulting plants, when selfing occurred, produced a small number of
plants that were trisomic (Bauchan and Tai, 1982).

Mok and Mok (1977) treated shoot tips with 0.1% colchicine for two
days, six hours each day. Along with the expected autotetraploids,
they discovered two monosomics, 2n=21. The use of aneuploids, such as
2n+1=23 or 2n-1=21, can prove to be useful in genetic studies and/or in
a breeding program.

Shoot tips of different varieties of Phaseolus vulgaris (Biswas

and Bhattacharya, 1976), Vigna mungo (Sen and Chheda, 1958), and V.

 

unguiculata (Sen and Bhowal, 1960b) were treated with 0.25% colchicine

 

for two days, six hours each day, to produce autotetraploids. Numerous
examples of meiotic abnormalities were observed: multivalents, early
separation of bivalents, laggards, nondisjunction, three or more
spindles at anaphase II, unequal segregation of the chromosomes,
formation of micronuclei, and polySpory.

Kaul (1970) treated Vigna radiata with 0.01, 0.025, and 0.05%, 2,2

 

dichlorOprOpionic acid (DPA). The treated plants showed 50% pollen
sterility at 0.01% DPA and 100% pollen sterility at 0.025% and 0.05%
DPA. Thirty-eight percent of the cells showed a normal meiosis with 11
bivalents. Multivalents, early separation of bivalents, bivalent
associations (both Side-to-side and end-to-end), laggards, unequal
segregation of chromosomes into 10/12 groupings, chromatin bridges, and

polyspory were observed.

 

 

 

17

Goswami (1980) exposed Vignaflmungg_seeds to a 35Kr dosage of
x-rays. He studied the meiosis of these plants and observed the lack
of chromosome pairing at diakinesis and metaphase I. Chromatin
bridges, lack of cytokinesis at the end of telophase I, and the
formation of micronuclei and microcells at the quartet stage were some
of the irregularities that were observed. Ojomo and Chheda (1971)
exposed Vigna_unguiculata seeds to x-rays; they observed chromatin
bridges, sticky chromosomes, fragments and/or laggards in these
plants.

Conclusions

 

1.) Except for one Spontaneous allotetraploid, all studied
species of Phaseolus and Vigna are diploids with 2n=2X¥22, with the
basic chromosome number being X=1l.

2.) There is no evidence to support the idea that changes in
chromosome number contribute to genome differentiation.

3.) Most of these Species have small chromosomes with homogeneous
karyotypes. Therefore, karyotyping may not be as useful for cytotaxo-
nomic studies as in other species.

4.) Meiosis is normal with the formation of 11 bivalents at meta-
phase I for the diploid species, however, early bivalent separation,
which appear as univalents, do occur. The allotetraploid species also
have normal meiosis with 22 bivalents forming at metaphase I.

5.) Chromatin bridges and quadrivalent associations in the hy-
brids, although found at a low frequency, have led authors to believe
that structural modifications, particularly inversions and
translocations, comprise the main evolutionary force for genome

differentiation.

 

APPENDIX

 

 

 

 

 

 

 

Appendix

Cytological studies in the genera Phaseolus and Vigna have been
conducted at Michigan State University. These studies have yielded
different results and interpretations than those reviewed above. The
presence of one to five bivalents in association with each other per

metaphase plate have been observed in Vigna angularis, V: mungo, Xx

 

radiata, V: umbellata, Phaseolus coccineus, and P. vulgaris var.

 

aborigineus (Blackson and Tai, 1982; Hussein, personal communication;

 

and Machado et a1., 1982) (Figure 2). In cells with less than five
bivalents in association, most of the remaining chromosomes were
co-oriented in a manner suggestive of an interrelationship.

According to Rieger et a1. (1976), this attraction of bivalents,
known as secondary association, occurs if they are related by genetic
and evolutionary factors. There is general agreement that the
occurrence of secondary associations indicates homology between the
chromosomes involved (Lawrence, 1931; Meurman, 1933; and Therman,
1951).

Along with the secondary associations, Split Spindles showing
five/six segregation of bivalents at metaphase I and multipolar meiosis
have been observed for all of the species described (Figure 3).
Multipolar meiosis is a Spontaneous or induced spindle apparatus
abnormality resulting in genome separation during meiosis (Li 6 Tu,
1974; Thompson, 1962; Tai, 1970). Tai (1970) prOposed that multipolar
meiosis occurs via genome specific spindle organizer, cell organelles
which govern chromosome migration and cytokinesis, which provide an

18

 

 

19
evolutionary mechanism for haploidization in higher plant polyploids.
The Spontaneous appearance of multipolar meiosis in the Phaseolus and
Vigna species mentioned suggests that these species may be comprised of
two genomes.

In the Phaseolus and Vigna species studied; the existence of
secondary associations, multipolar meiosis, the grouping of bivalents
into groups of five and six, and the high basic chromosome number
(X=11) suggests that these species may be allotetraploids functioning
at the diploid level. These species would consist of two genomes, one
containing six and the other five chromosomes. Six would most likely
be the basic chromosome number and five the derivative number arising
due to a loss of a pair of chromosomes from the other genome.

Additional evidence to support this theory has been collected by

studying the cytology of the hybrid between Vigna radiata and V.

 

umbellata, (Bauchan and Tai, 1982; and Machado et a1. 1982).

Machado et a1. (1982) observed up to nine bivalents in the hybrid,
suggesting allosyndetic pairing, while the presence of multivalents,
suggested autosyndetic pairing. This ambivalence suggests that there
must be more than one genome in each parent and the presence of some
homology between the genomes of_V; radiata and V, umbellata (Figure 4).

Observations of meiosis in the amphiploid by Bauchan and Tai
(1982) showed normal bivalent pairing (Figure 5). The absence of
multivalents in both the parental species and the amphiploid may be the
result of genetic control of pairing with dosage effects; i.e., a

single dose allows homoeologous pairing, whereas a double dose promotes

 

 

 

 

 

20

homologous pairing (Riley and Chapman, 1958; Feldman, 1966; Driscoll,
1972; and Starks and Tai, 1974). These observations further support
the theory that the parental species are allotetraploids.

Split metaphase plates resulting in the 12/10 segregation of
bivalents were also observed in the amphiploid. This adds evidence to
the hypothesis that these species consist of two genomes, one
containing six chromosomes and the other five.

The meiosis of the backcross progeny (amphiploid x.V. radiata) was
very irregular (Bauchan and Tai, 1982). A majority of the cells
observed contained 11 bivalents and 11 univalents (Figure 5).

Although the evidence thus far seems to suggest that the species
contained in the genera Phaseolus and Vigna_are allotetraploids with
the basic number of X=6, only seven Species have been studied
critically. Much more research needs to be done on the cytology of

these Species and their hybrids in order to learn their relationships.

REFERENCES

 

 

References

 

1. Ahn, C. S. and R. W. Hartman. 1977. Interspecific hybridizations
among four Species of the genus Vigna. .12; The First
International Mungbean Symposium. University of the
Phillippines, Los Banos. pp. 240-246.

2. . 1978 a. Interspecific hybridization between Mung bean
(Vigna radiata) and Adzuki bean (V. angularis). Journal of
the American Society for Horticultural Science. 103:3-6.

 

 

3. . 1978 b. Interspecific hybridization between Rice bean
(Vigna umbellata) and Adzuki bean (X. angularis). Journal of
the American Society for Horticultural Science. 103:3-6.

 

4. Allard, H. A. and H. F. Allard. 1940. The wild bean Phaseolus
pglystachyus, Journal of the Washington Academy of Science
30:335-337.

 

5. Al-Yasiri, S. A. and D. P. Coyne. 1966. Interspecific
hybridization in the genus Phaseolus. CrOp Science 6:59-60.

6. Bauchan, G. R. and W. Tai. 1982. Cytology and Fertility of Vigna
radiata x X, umbellata. CrOp Science. Submitted.

7. Beadle, G. W. 1930. Genetical and cytological studies on
Mendelian asynapsis in maize. Memoir. Cornell Agricultural
Experiment Station No. 129.

8. Bemis, S. W. P. and N. Kedar. 1961. Inheritance of morphological
abnormalities in seedlings of two species of Phaseolus.
Heredity 52:171-178.

9. Berger, C. A., E. R. Witkus, and R. M. McMahan. 1958. Cytotaxo-
nomic studies in the Leguminosae. Bulletin of the Torrey
Botanical Club 85:405-414.

10. Bhag-Singh, A., A. N. Khanna, and S. M. Valdya. 1964. Crossabil-
ity studies in the genus Phaseolus. Journal of Irari PG.
School 2:47-50.

11. Bhattachatya, S. 1978. Giemsa banding patterns of chromosomes in
Phaseolus vulgaris. Cytologia 43:581-588.

 

12. Bir, S. S. and S. Sidhu. 1967. Cytological observations on the
North Indian members of the family Leguminosae. Nucleus
10:47-63.

21

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

22

Blackson, J. H. and W. Tai. 1982. Cytological and statistical
analysis of secondary associations in Phaseolus Species
(Leguminosae). Journal of Heredity. SuEmitted.

Biswas, A. K. and N. K. Bhattacharya. 1876. Induced polyploidy in
Legumes. III. Phaseolus vulgaris. Cytologia 41:105-110.

 

Biswas, M. R. and 3. Dana. 1975 a. Black gram x Rice bean cross.
Cytologia 40:787-795.

1975 b. Phaseolus aureus x.P; lathyroides cross.

 

 

 

NucIeus 18:81-85.

1976 a. Phaseolus aconitifolius x P. trilobus.

 

 

Indian Journal of7Genetics afid Plant Breedifig 36:125-131.

1976 b.A110polyploid of tetraploid Phaseolus Spp. x

 

_E. calcaratus cross. Biologia Plantarum 18:

 

Braak, J. P. and E. Kooistra. 1975. A successful cross between
Phaseolus vulgaris and P. ritensis with the aid of embryo
culture. Euphyt1ca 24: 669-

 

Burkart, A. and H. Brucher. 1953. Phaseolus aborigineus, die
mutmabliche andine Stammform der7Rulturbohne. Zuchter
23:65-72.

 

Chavan, V. M., G. D. Patil, and D. G. Bhapkar. 1965. Improvement
of cultivated Phaseolus species - need for interspecific
hybridization. Ind1an Journal of Genetics 26A:152-154.

Chen, N. C. 1980. Interspecific hybridization in the genus Vigna.
Ph.D. Dissertation. Michigan State University.

Cionini, P. G. and S. Avanzi. 1972. Pattern of binding of
tritiated actinomycin D to Phaseolus coccineus polytene
chromosomes. I. Nucleolus organizing chromosomes.
Experimental Cell Research 75:154-158.

 

Dana, S. 1964. Interspecific cross between tetraploid Phaseolus
Spp. and P. ricciardianus. Nucleus 7:1-10.

 

1965 a. Hybrid between tetraploid Phaseolus spp. and

 

 

_P. calcaratus.. Biologia Plantarum 7:7-12.

1965 b. Phaseolus aureus x tetraploid Phaseolus Spp.

cross. Revista De B1oIog1a 5: 109- 114.

 

1966 a. Species cross between Phaseolus aureus and P.

 

 

trilobus. Cytologia 31: 176- 187.

1966 b. The cross between Phaseolus aureus and P.

 

 

mungo. Genetica 37: 259- 274.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

23

1966 c. Cross between Phaseolus aureus and P.

 

 

ricciardianus. Genetica Iberica 18:141-156.

 

1966 d. Chromosome differentation in tetraploid

 

Phaseolus Species and P. ricciardiannus. Nucleus 9:97-101.

 

Darlington, C. D. 1930. Chromosome studies in Fritillaria III.
Cytologia 2:37-55.

 

1937. Recent advances in cytology. Ed. 2. J. A.

 

Churchill Ltd., London.

De, D. N. and R. Krishnan. 1966 a. Studies on pachytene and
somatic chromosomes of Phaseolus mungo. Genetica 37;581-587.

 

1966 b. Cytological studies of the hybrid Phaseolus

 

aureus x-P. mungo. Genetica 37:588-600.

Dhaliwal, A., L. H. Pollard, and A. P. Lorz. 1962. Cytological
behavior of an F1 Species cross (Phaseolus luntus L. var.
Fordhook X._P. polystachyus L.). Cytologia 27:369-374.

 

 

Evans, A. M. 1975. Species hybridization in the genus Vigna.
IITA Tropical Grain Legume Bulletin. Ibadan, Nigeria. pp.
1-5.

Faris, D. G. 1964. The chromosome number of Vigna sinesis (L.)
Savi. Canadian Journal of Genetics and Cytology 6:255-258.

 

Feldman, M. 1966. The effect of chromosome SB, 5D, and SA on
chromosome pairing in Triticum aestivum. Proceedings of the
Academy of Science, USA. 55:1447-1453.

 

Floresca, E. T., J. M. Capinpin, and J. V. Pancho. 1960. A
cytogenic study of Bush Sitao and its parental types.
Phillippine Agriculturist 44:290-298.

Frahm-Leliveld, J. A. 1953. Some chromosome numbers in tropical
leguminous plants. Euphytica 2:46-48.

1957. Observations cytologiques sur quelques
Legumineuses trOpices et subtrOpicales. Rev. Cytol. et. Biol.
Veg. 18:273-287.

1965 a. Cytological data on some wild tropical Vigna

 

species and cultivars from cowpea and asparagus bean.
Euphytica 14:251-270.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

24

1965 b. Cytological observations in the genus

Phaseolus. Acta Botanica Neerlandica 14:159-162.

Gates, R. R. 1951. Epigeal germination in the Leguminosae.
Botanical Gazette 113:515-517.

Gattschalk, W. and S. R. Baquar. 1971. The effect of mutated
genes on the germ cell formation in Pisum. Pakistan Journal
of Science, Indian Research 14:223-227.

Goswami, L. C. 1980. Cytogenetics and breeding of x-ray induced
chlorophyll mutants in Vigna mungo. Genetica Agraria

34:299-312.

Honma, S. 1956. A bean interspecific hybrid. Journal of Heredity
47:217-220.

1968. Inversion in the chromosomes of Phaseolus

vulgaris. Cytologia 33:78-81.

Honma, S. and O. Heeckt. 1958. Bean interspecific hybrid
involving Phaseolus coccineus x P. lunatus. American Society
for Horticultural Science 72:360-364.

 

1959. Interspecific hybrid between Phaseolus vulgaris

and_P. lunatus. Journal of Heredity 50:233-237.

1952. Genetic transfer of hypogeal character of
Phaseolus coccineus to other species of Phaseolus. XVI
International Horticultural Congress. Brussels, Belgium. pp.
145-153.

Ibrahim, A. M. and D. P. Coyne. 1975. Overcoming unilateral
cross-ability in the interspecific cross Phaseolus coccineus x
P. vulgaris. Annual Report of Bean Improvement Cooperative
18:32.

Jaaverslag, T. O. 1957. Directic vande Tuinbouw en het
Tuin-bouwonderwijs's. Grravenhagen, The Netherlands.

Janaki-Ammal, E. K. 1945. Chromosome number count for the genus
Phaseolus ricciardianus. In} Darlington, C. D. and E. K.
Janaki-Ammal. 1945. Chromosome atlas of cultivated plants.
George Allen and Unwin Ltd., London.

 

Joseph, L. S. and J. C. Bouwkamp. 1978. Karyomorphology of
several species of Phaseolus and Vigna. Cytologia
43:595—600.

Karpechenko, G. D. 1925. Chromosome number c0unts. Bulletin of
Applied Plant Breeding 14:143.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

25

Katayama, Y. 1928. The chromosome number in Phaseolus and Allium
and an observation on the size of stomata in different species
of Trillium. Nogaku Kwaiho (Journal of the Scientific and
Agricultural Society of Japan) 303:52-54.

Kaul, C. L. 1970 . Investigations into the cause of sterility.
IV. Gametocide - induced male sterile Phaseolus aureus.
Genetica 41:316-320.

 

Kawakami, J. 1930. Chromosome numbers in Leguminosae. The
Botanical Magazine (Tokyo) 44:319-328.

Kleinmann, H. 1932. Uber Kern-und Zellteilungenium Cambium. Bot.
Arch. 4:113-147.

Kodama, A. 1967. Cytological studies on root nodules of some
species in Leguminosae II. The Botanical Magazine (Tokyo)
80:92-99.

Kostoff, D. 1940. Fertility and chromosome length. Journal of-
Heredity 31:33-34.

Krishnan, R. and D. N. De. 1965. Studies on pachytene and somatic
chromosomes of Phaseolus aureus. Nucleus 8:7-16.

 

1968. Cytological studies in Phaseolus II. .P. mungo
x tetraploid Phaseolus Spp. and the amphiploid. Indian
Journal of Genetics and Plant Breeding 28:23-29.

 

1970. Pachytene chromosomes and the origin of a

 

tetraploid species of Phaseolus. Cytologia 35:34-35.
Kumar, L. S. S. and A. Abrahm. 1942. Current Science 11:112.

Lamprecht, H. 1941. Die artgreze zwischen Phaseolus vulgaris und
.3. multiflorus. Heredity 27:151-175.

 

 

1966. Die Entsthung der Arten und hoheren Kategorien.

 

Experimenteller Nachweis des Ablants der Evolution. Wein, New
York.

Lawrence, W. J. C. 1931. The secondary associations of chromo-
somes. Cytologia 2:352-384.

LeMarchand, G., R. Marechal, and J. C. Bandet. 1976. Observation
sur quelques hybrides dan le genre Phaseolus. III. Extrait du

Bulletin des Recherches Agronomiques de Gembloux.
11:183-200.

 

 

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

26

Li, H. W. and D. 8. Tu. 1947. Studies on the chromosomal
aberrations of the amphiploid, Triticum timOpheevi and
Aegilops bicoris. Bot. Bull. Acad. Sincia. 1:173-186.

 

 

Lorz, A. P. 1952. An interspecific cross involving the lima bean,
Phaseolus lunatus. Science 115:702-703.

 

Luyeye, L. 1975. Distinction entre Vigna radiata et 2, mungo.
Extrait du Bulletin des Recherches Agrononiques de Gembloux
10:372-373.

Machado, M., W. Tai, and L. Baker. 1982. Cytogenetic analysis of
the interspecific hybrid, Vigna radiata x V, umbellata.
Journal of Heredity. (In press).

Malinowski, E. 1935. Studies on hybrid vigor in Phaseolus
vulgaris. Zietschr. Indukt. Abstamm U. Verrerbungslehre
70:96-124.

Marechal, R. 1969. Donnees cytologiques sur les especes de la
sous-tribu des Papilionaceae-Phaseoleae-Phaseolineae. Bul.
Mard. Bot. Nat. Belg. 39:125-165.

1970. Donnees cytologiques sur les especes de la
sous-tribu des Papilionaceae-Phaseoleae-Phaseolineae. Deuxime
serie. Bul. Jard. Bot. Nat. Belg. 40:307-348.

1971. Observations sur quelques hybrides dans 1e genre
Phaseolus. Extrait du Bulletin des Recherches Agronomiques de
Gembloux 6:461-489.

Marechal, R. and J. B. Bandoin. 1978. Observations sur quelques
hybrides dans 1e genre Phaseolus. IV. Extrait du Bulletin des
Recherches Agronomiques de Gembloux 13:233-240.

Marechal, R. and Otoul, E. 1965. Contribution a Petrude
cytotaxinomique des Papilionaceae-Phaseolus-Phaseolineae. I.
Bul. Jard. Bot. (Bruxelles) 35:73-84.

Meurman, O. 1933. Chromosome morphology, somatic doubling and
secondary association in Acer plantanoides. Heredita
18:145-173.

 

Miege, J. 1962. Quatrieme liste de nembers chromosomiques
d'especes d'Afrique Occidentale. Rev. Cytol. et Biol. Veg.
3-4:149-164.

Moh, C. C. 1962. The effects of p-dichlorobenzene pretreatment
and temperatures on the chromosome morphology of Phaseolus
vulgaris. Genetics 31:971-972.

Mok, D. W. S. and M. C. Mok. 1976. A modified Giemsa technique
for identifying bean chromosomes. Journal of Heredity
67:187-188.

 

 

 

 

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

27

. 1977. Mbnosomics in common bean, Phaseolus vulgaris.
Theoretical and Applied Genetics 49: 145- 149.

 

Mok, D. W. S. , M. C. Mok, and A. Rabakoarihonta. 1978.
Interspecific hybridization of Phaseolus vulgaris, with P.
lunatus, and P. acutifotius. Theoretical afiHIApplied Genetics

52:209-215. '-

 

 

Nagl, W. 1962. Uber end0polyploide, restitutionskernbildung und
kernstrukturen im suspensor von angiospermen und einer
gymnosperme. Osterreich, Bot. Zeitscher. 109:431-494.

. 1969. Puffing of polytene chromosomes in a plant
(Phaseolus vulgaris). Naturwissenschaften 56:221-222.

 

. 1974. The Phaseolus suspensor and its polytene
chromosomes. Z. PfIanzeanysiol. 73:1-44.

Ojomo, O. A. and-H. R. Chheda. 1971. Mitotic events and other

disorders induced in cowpea, Vi na ungui ulata by ionizing
radiation. Radiation Botany 5-38I.

Parrot, J. F. 1981. Recovery of interspecific Vigna hybrids via
embryo culture. M.S. Thesis. Michigan State University.

Premseker, S. and V. S. Raman. 1972. A genetic analysis of the
progenies of the hybrid Vigna sinensis and V. sesquipedalis-
Madras Agricultural Journal 59:

 

Raghuranshi, S. S. 1962 a. Cytogenetical studies in the genus
Citrus,_§. assamensis. Caryologia 15:143-149.

1962 b. Cytogenetical studies in the genus Citrus.
IV. Evolution in the genus Citrus. Caryologia 15:150.

Ramanujan and Parthsarthy. 1935. An asynaptic mutant in rice.
Proceedings of the Indian Academy of Science, Bangalore.

Rau, N. S. 1929. Further contributions to the cytology of some
cr0p plants of South India. Journal of the Indian Botanical
Society 8:201-206.

Rieger, R. A., A. Michaelis, and M. M. Green. 1976. Glossary of
Genetics and Cytogenetics. Springer-Verlag, New York.

Sarbhoy, R. K. 1977. Cytological studies in the genus Phaseolus.
III. Evolution in the genus. Cytologia 42:401-413.

. 1978. Cytogenetical studies in the genus Phaseolus.
I 6 II. Somatic and meiotic studies in 15 species 0
Phaseolus. Cytologia 43:161-180.

1980. Karyological studies in the genus Phaseolus.

——c'_1'yto ogia 45:363-373.

 

 

 

 

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

28

Saunders, A. R. 1960. Inheritance in the cowpea (Vigna sinensis)
III. Mutations and Linkages, South African JournaI of
Agricultural Science 3:327-347.

 

Sawa, M. 1974. On the interSpecific hybridization between the
Adzuki bean, Phaseolus angularis and the green gram, P.
radiatus. II. Japanese JournaI of Breeding 24:282-286.

Schweizer, D. 1976. Giemsa and fluorochrome banding of polytene
chromosomes in Phaseolus vulgaris and P. coccineus. In: K.
Jones and P. E. Brandham (Eds.). CurrEht Cfiromosome'RESearch.
Elserier/North - Holland Biochemical Press, The Netherlands.
pp. 51-56.

 

Sen, N. K. and J. G. Bhowal. 1960 a. Cytotaxonomic studies on
Vigna. Cytologia 25:195-207.

1960 b. Colchicine-induced tetraploids of six
varieties of Vigna sinesis. Indian Journal of Agricultural
Science 30:149-16I.

Sen, N. K. and H. R. Chheda. 1958. Colchicine induced tetraploids
of five varieties of black gram. Indian Journal of Genetics
and Plant Breeding 18:238-248.

Sen, N. K. and A. K. Ghosh. 1960. Interspecific hybridization
between Phaseolus aureus Roxb. (green gram) and P. mun 0
(black gram). Bulletin of the Botanical Society—of Bengal
14:1-4.

 

Senn, H. A. 1938. Chromosome number relationships in the
Leguminoisae. Bibliographia Genetica 12:175-345.

Shibata, I. 1962. Estudio citologicos de plant as colombianas
silvestres y cultivadas. (Cytological studies on some wild
and cultivated plants of Colombia). Nogyo Daigaku (Journal of
Agricultural Science, Tokyo) 8:49-62.

Simmonds, N. W. 1954. Chromosome behavior in some trOpical
plants. Heredity 8:139-145.

Singh, A. and R. P. Roy. 1970. Karyological studies in
Trigonella, Indigotera, and Phaseolus. The Nucleus 13:41-54.

 

Sinha, S. S. N. and H. Roy. 1979 a. Cytological studies in the
genus Phaseolus. I. Mitotic analysis of fourteen species.

Cytologia 44:191-199.

1979 b. Cytological studies in the genus Phaseolus
II. Meiosis analysis of Sixteen Species. Cytologia
44:201-209.

Smartt, J. 1970. Interspecific hybridizations between cultivated

American Species of the genus Phaseolus. Euphytica
19:480-489.

 

 

 

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

29

Smartt, J. and N. Hag. 1972. The behavior of Phaseolus coccineus
as seed parent in interspecific crosses with P, vulgaris.
Annual Report of Bean Improvement COOperative 15:88-91.

 

Stebbins, G. L. 1971 a. Chromosomal Evolution in Higher Plants.
Addison-Wesley Publishing Co., Reading, Mass.

. 1971 b. Processes of Organic Evolution. 2nd ed.
Prentice-Hall Inc., Englewood Cliffs, New Jersey.

Strand, A. B. 1943. Species crosses in the genus Phaseolus.
Proceedings of the American Society for Horticultural Science
42:569-573.

Swindell, R. E., E. E. Watt, and G. M. Evans. 1973. A natural
tetraploid mungbean of suspected amphidiploid origin. The
Journal of Heredity 64:107.

Tai, W. 1970. Multipolar meiosis in diploid crested wheat-grass,
AgrOpyron cristatum. American Journal of Botany
57:1160-1169.

 

Takagi, N. 1938. A list of chromosome numbers in some ornamental
plants. Bulletin of Miyazaki College Agricultural Forestry
10:83-87.

Therman, E. 1951. Somatic and secondary pairing in Ornithogalum.
Heredity 5:253-269.

 

Thomas, H. 1964. Investigations into the inter-relationships of
Phaseolus vulgaris_£. and P. coccineus. Genetica 35:59-74.

 

Thomas, P. T. 1945. Unpublished counts.

Thompson, M. M. 1962. Cytogenetics of Rubus III. Meiotic
instability in some higher polyploids. American Journal of
Botany 49:575-582.

 

Tschechow, T. and N. Kartaschowa. 1932. Cytologia 3:221.

Tschermak-Seysenegg, E. V. 1942. Uber basterden Zwischen Fisole
(Phaseolus vulgaris) and feuerbohne (P. multiflorus) and ihre
eventuelle praktische verwertbarkeit. Zuckter 14:153-164.

 

 

Wall, R. J. and T. L. York. 1957. Inheritance of seedling
cotyledon position in Phaseolus Species. Journal of Heredity
48:71-74.

Weinstein, A. 1926. Cytological studies on Phaseolus vulgaris.
American Journal of Botany 13:248-263.

 

 

130.

131.

30

Wolfenbarger, D. and J. P. Sleesman. 1961. Resistance to the
potato leafhopper in lima bean lines, interspecific Phaseolus
crosses, Phaseolus Spp., the cowpea, and the bonavist bean.
Journal of Economic Entomology 54:1077-1079.

 

Yarnell, S. H. 1965. Cytogenetics of the Vegetable Crops. IV.
Legumes. The Botanical Review 31:247-328.

 

 

 

SECTION II

Cytology and Fertility of Vigna radiata and _V. umbellata

 

 

ABSTRACT

CYTOLOGY AND FERTILITY OF VIGNA RADIATA AND E. UMBELLATA

 

By

Gary R. Bauchan

The cytology of Vigna_radiata (L.) Wilczek,_V. umbellata (Thunb.)
Ohwi 6 Ohashi, their hybrid, amphiploid, backcross progeny, and the
plants of subsequent generations was investigated to determine the
genome relationship among these plants for use in a breeding program
with the ultimate goal of incorporating beneficial characteristics of
_Z. wmbellata into 1. radiata. Light microsc0pe investigations
included: root tip chromosome counts; the analysis of chromosome
pairing during all stages of microsporogenesis; and pollen stainability
to estimate plant fertility.

In this study the presence of secondary associations, multipolar
meiosis, the grouping of bivalents during meiosis, and the high basic
chromosome number has led to a re-evaluation of ploidy levels in these
plants. Genome formulae have been assigned in accordance with these
findings under the assumption of genetic control of pairing with dosage

effects.

 

Introduction

 

The food legumes are an important component for increasing the
protein portion of primarily vegetarian diets. AS a group, the food
legumes include Species adapted to a broad range of conditions.
However, since most of the cultivated food legumes are self pollinated,
the selection pressures imposed upon them have created individual
species with relatively narrow gene bases. The Leguminosae as a whole,
is endowed with a richness of genetic diversity. Increased genetic
recomination within this extensive gene pool would significantly
increase the genetic base of the agricultural legumes. One method of
accomplishing this exchange is through the use of wide hybridization,
to transfer beneficial genes from the wild Species to the cultivated
Species (Adams, personal communication).

Vigna radiata (L.) Wilczek (mungbean) is an important food legume

 

crop in Asia and Africa. In the 1972-73 Asian Vegetable Research and
Development Center (AVRDC) Annual Report (1974) it is stated that
mungbeans are an excellent source of protein, but its yield potential
is low and it is susceptible to many insect and disease pests. ‘V.
umbellata (Thunb.) Ohwi 6 Ohashi (rice bean) is a species closely
related to V. radiata and is reported to be highly resistant to bean

fly (Melanagromyze phaseoli), Cercospora leaf spot, powdery mildew and

 

 

root diseases (AVRDC, 1975). A goal of this project is to incorporate
these characteristics of V. umbellata into V. radiata.

Stebbins (1950) and Grant (1975) discussed in detail the pre-
zygotic and post-zygotic isolating mechanisms that prevent the
achievement of a cross between two species which would allow the

31

 

32

transfer of desirable traits. There are methods for overcoming these
barriers, one of which is to produce the F1 hybrid, double the
chromosome number to produce an amphiploid, and backcross the
amphiploid to the cultivated parent with the eventual goal of producing
trisomics (2n+l). These trisomics can then be used for genetic studies
to assign genes to particular chromosomes. They also can be used for
genetic recombination through crossing-over of homologOus,
homoeologous, or secondarily associated chromosomes; or by the breakage
of the chromosomes using mutagens to facilitate translocation of a
piece or pieces of chromosomes from one species into another (Kimber
and Sears, 1980).

Limited success has been reported in achieving the interspecific
cross Vigna radiata x V. umbellata (Dana, 1966; Evans, 1975; AVRDC,
1975; Ahn and Hartman, 1977; and Chen et a1., 1977). In all cases the
F1 hybrid was highly sterile. Meiotic studies of this cross (Dana,
1966; Ahn and Hartman, 1977; and Machado et a1., 1982) showed many
cytological irregularities with incomplete bivalent pairing.

The objectives of the present study were: 1) to determine the
cytological relationships between V. radiata and V3 umbellata, their
hybrids, amphiploids, backcross progeny and the plants of the
subsequent generations; 2) to determine the relationship between

cytology and fertility in these plants.

 

 

33

Materials and Methods

 

All original plant material was obtained from the AVRDC. The
cultivars used in the breeding program were V. radiata PI377276 and
AVRDC accession number 2013; and X: umbellata AVRDC accession number
4065. Hybridizations and recovery of the hybrids via embryo culture
were accomplished as reported by Chen (1980) and Parrot (1981). Among
the sterile hybrids grown at the AVRDC, one plant was found to have
some fertility. This plant was sent to our laboratory at Michigan
State Unviersity for cytological analysis. Preliminary studies of this
plant indicated that chromosomal doubling of the original F1 hybrid
had occurred. The F1 was successfully backcrossed t°.K° radiata by
the use of embryo culture. These backcrossed plants grew Slowly but
flowered profusely and produced a few self-pollinated seeds which upon
germination produced mature plants morphologically similar to X:
radiata. The petigree is diagramed in Figure 1.

All plants were greenhouse grown. Four plants of the amphiploid,
three BCl plants and four 81 plants were studied cytologically.

Germinated seedlings and roots from stem cuttings were used to
provide root tip material for chromosome counts. Roots were harvested
and placed into Carnoy's fixative of ethanol, chloroform and glacial
acetic acid (623:1, v/v/v) for 24 hours. The roots were hydrolyzed for
15 minutes in IN HCl at 60°C, placed in Feulgen's stain for 30 minutes,
and squashed in propiono-carmine.

Meiotic observations were made on pollen mother cells (PMC'S).

The flower buds were fixed in Carnoy's fixative for 24 hours,

transferred to 70% ethanol and stored in a refrigerator. PMC'S were

34

Vigna radiata (PI377276) x Vigna umbellata (AVRDC #4065)
2n=22) (2n=22)

 

 

 

F1 Hybrid
(2n=22)

doubling of
chromosomes

Amphiploid x Vigna radiata (AVRDC #2013)
(2n=44) (2n=22)

 

 

BCl
(2n=33)

selfing

 

Sl
(2n=22 or 23)

Figure 1. Diagram of a cross between Vigna radiata and V. umbellata.

 

35

squashed in propiono-carmine. All cytological interpretations were
based upon observations from temporary slides.

These plants were extremely difficult to work with owing to their
small chromosomes (1-4 pm in length) (Sen and Bhowal, 1960; Joseph and
Bauwkamp, 1978; Sarbhoy, 1977; and Sinha and Roy, 1979), homogeneous
karyotype, and the fact that only one bud per inflorescence is
undergoing meiosis at any one time (Bauchan and Tai, 1982).

To estimate fertility, pollen grains from mature anthers were
stained with cotton blue. Only those grains of normal shape staining
blue were counted as stainable and hence assumed viable.

All observations were made using a Zeiss Photoscope II
phase-contrast microscope with a Planapo 63X, N.A.=1.4 objective.
Photomicrographs were taken with an attached Linhof 4X5 box camera

using Kodak Contrast Process Ortho film.

Results

Amphiploid of V. radiata x V. umbellata

 

Root tip chromosomes of this plant were 2n=44 (Figure 2). Meiosis
was mostly regular with 184 of the 248 metaphase I cells observed
containing 22 bivalents (Figure 3). Early separation of one or two
bivalents occurred in 19% and 7% of the cells, respectively.
Occasionally (4%), a Split metaphase plate was discovered with one half
containing 12 bivalents grouped together and the other half containing
10 bivalents grouped together (Table 1). Normal distribution of the
chromosomes occurred at all of the remaining stages of meiosis.

However, only 44% of the 3500 pollen grains counted proved to be

36

Axommfiv .AmNHCNV Hm cH HHoo nHu Boom

Axmmev
.mHHmoouoHE oau msHa mHHmo :HmEuocz meow wcHsozm Hum CH mwmum umuumso

Axonwv
.mucoEwmuw Hmuo>om :uHB HHoo use meOmoEouzo HH wcH30:m Hum cH HH mwmnamumz

Axomwv .moBOmoEouno
wcwamH HH new moEOmoEouco mo :oHummmummm HH\HH wcHBOAm Hum :H H mumsmmc<

AxoomHv .amHum>o cm mmumoHenH Bouu< .HHmo ecu usozwnounu monogamom
mucon>Hn Ho coHumwowwom ©\m :qu oumHm uHHam m wcHaosm Hum :H H ommsmmumz

Axomav .ucon>Hn m Cu ucon>Hce am we coHumHUOmmm cm
oumoHocH msouw< .mucon>H:= HH mam mucon>Hn HH wcHsonm Hum GH H ommnamuo:

AxommHv .Amm1cmv Hom :H HHoo aHu uoom

Axmaev .msemam>an AN mangoes eaoHaHnaa< was an H meanness:

AxooHHv .Aqeucmv eHoHawsme< one cH HHoo 6H6 Boom

 

 

.oH

mustm

muanm

mustm

mustm

mpstm

ousmHm
musmHm
oustm

mustm

 

37

 

 

 

 

38
stainable (Table 3).

Table 1. Metaphase I chromosome associations in the amphiploid

 

 

(2n=44).
Chromosome Associations Cells
No. of
_I .3; Cells Percent
0 22 184 74
2 21 48 19
4 20 16 7
Total 248 100

 

Backcross Progeny (BC1), Amphiploid x V. radiata

 

Mitotic chromosome counts of the backcross progeny showed that BCl
has 33 chromosomes (Figure 4). A summary of the meiotic behavior is
shown in Table 2. Meiosis was highly irregular. 221 PMC'S were
observed at metaphase I and most (99%) of the cells contained 11
bivalents and 11 univalents (Figure 5). Periodically one to three
bivalents were found to have a univalent loosely associated (Figure 5).
This association varied from close proximity to wide separation with
only a thin strand joining the chromatin. Side-to-side, side-to-end,
and end-to-end associations were also noted. A few of the cells (12%)
showed a split spindle with one part containing six bivalents and the
other containing five bivalents with the 11 univalents scattered
throughout the cell (Figure 6). Early separation of a bivalent was

observed in 2% of the cells.

39

 

cop so
mm: mm:

N, a.

an SN
HCOQH 0m mdfimu

b0

.02

 

sauce
*.m-oooaoaz
6nouooHoecoaoH:
Hosaoz

 

muouuasa

.w up n oCHon CQEEoo uses on» .PHnn scum mmwua> goose: 02H

.uouumao non mHHmo use; cmzu once mchucou..

.P OCHon coeeoo umos on» Law: our scum moHua> unguaac use HodosceeoHe do genes: 05—:

 

co. m assoc
HHI. H: ON
.. P we
cos A assoc P. P we
«H1. H11 m mm m n, cos .NN _SSOS
Sr P e Ha a e_ co. Fe sauce H11 mu: o. A,
as m P. «N N as mmH Hm F. as AFN _F PP
acmouma assoc demo Suwanee Adamo dame Seances assoc Haas scouts; assoc .HH: 1H1
be use do use Lo use do mcoHuwHoomm<
.oz muummomm .oz mucmsoaum .oz anemone; .oz oeomoeouzu
mmnmmmmmmmm SH meanness: Himmmmmmmm 41mmmmmmmmm

 

.HmmOuoxomn muwHome .N.x oHoHQHcme<v Hum do uoH>mcon oeomoeouco pooHoz

.N oHamH

40

Anaphase I displayed the normal bipolar distribution of
chromosomes with 11 univalents remaining at the metaphase plate in all
of the observed cells (Figure 7). These univalents invariably
underwent precocious centromere division, but generally migrated to the
poles and were included in the telophase I nuclei.

The second division of meiosis was difficult to obtain in this
material probably due to the very short length of these stages. Of the
cells observed at metaphase II, 11 chromosomes plus several fragments
could be found in each cell (Figure 8). The distribution of
chromosomes during anaphase II varied widely and did not Show any
consistent pattern.

The quartet stage reflected the irregular behavior noted in the
previous stages. Of the 84 quartets observed, 69% contained
micronuclei or consisted of more than four cells; 31% of the quartets
appeared normal at this stage. Many (57%) of the quartets consisted of
four "normal" Sized cells and one to several smaller cells (Figure 9).
Of the 1800 mature pollen grains counted only 15% were stainable (Table

3).

Table 3. Pollen stainability of the Amphiploid and BC1.

 

 

No. Stainable Percent Stainable
Plant Pollen Counted Pollen
Amphiploid 1554 3500 44

BCl 274 1800 15

 

41
Progeny from Selfing of BCl
Only mitotic studies were made on these plants. From the four
plants studied, 79 root tip cells were counted with 70% containing 23

chromosomes (Figure 10), and 30% containing 22 (Table 4).

Table 4. Chromosome number counts from self progeny of BC1.

 

Chromosome No. of
Number Cells Percent
22 24 30
23 2.5. _79
Total 79 100

 

Discussion

Cytological studies of the parents, 1. radiata and X: umbellata,
were reported by Machado et a1. (1982), who stated that micro-
Sporogenesis was mostly normal. At metaphase I, 11 bivalents could be
found, with one to five of the bivalents found in association with each
other. According to Rieger et a1. (1976), this attraction of
bivalents, known as secondary association, occurs if they are related
by genetic and evolutionary factors. There is general agreement that
the occurrence of secondary association indicates homology between the
chromosomes involved (Lawrence, 1931; Meurman, 1933; and Therman,
1951). Hussein (personal communication) found secondary associations

in other closely related species such as X: angularis and V: mungo.

42

Similar findings were also reported in Phaseolus coccineus, P.

 

vulgaris, and P. vulgaris var. aborigineus (Blackson and Tai, 1982).

 

Along with the secondary associations, split Spindles showing
five/six segregation of bivalents at metaphase I and multipolar meiosis
have been described for both parental species (Machado et a1., 1982).
Multipolar meiosis is a Spontaneous or induced spindle apparatus
abnormality resulting in genome separation during meiosis (Li and Tu,
1947; Thompson, 1962; and Tai, 1970). Tai (1970) prOposed that
multipolar meiosis occurs via genome specific spindle organizers, cell
organelles which govern chromosome migration and cytokinesis, which
provide an evolutionary mechansim for haploidization in higher plant
polyploids. The spontaneous appearance of multipolar meiosis in Vigna
Species may be the first indication that these plants may contain more
than a Single genome.

The existence of a high basic chromosome number, especially uneven
ones such as 11 found in Vigna, suggests that the diploid genome is
possibly comprised of two genomes differing by structural modifications
(Stebbins, 1950 and Grant, 1963). The basic chromosome number may be
either five or Six with one of the two being an aneuploid number
(either a gain or a loss of a chromosome). The existence of secondary
associations, multipolar meiosis, the grouping of bivalents into groups
of five and six, plus the high basic chromosome number suggests that
the parental Species used in this breeding program are perhaps segment-
ed allotetraploids consisting of two genomes of five and Six, respec-

tively. Thus the following genome formula VrVrV'rV' for X:

I‘

radiata and VUVUV'UV'u for V: umbellata (V=6 chromosomes and

43
V'=5 chromosomes) are suggested (Figure 11).

The subscripts r and u are used to designate the origin of the
genomes from V, radiata and V, umbellata. Since only 11 bivalents were
observed in the parental Species V and V' are not homologous. The
presence of secondary associations, suggests some interrelationships
between these genomes.

The question exists, which is the basic number and which is the
derivative number, five or six. The basic number could be six with
five being the derivative number, five arising due to the loss of a
pair of chromosomes from the other genome. This loss would not have
severe genetic consequences.

Machado et al. (1982) reported the meiotic irregularity of the
F1 hybrid between_V. radiata and V, umbellata. A maxium of nine
loosely paired bivalents was noted as well as a few trivalents and
quadrivalents at metaphase I. Multipolar meiosis was also observed
with the bivalents segregating from the univalents. During the latter
stages of meiosis, fragmentation of the univalents occured forming
microcells at the quartet stage. In the parents, the homologous
chromosomes and spindle organizers allow a normal meiosis; in this
interspecific hybrid, non-homology of the chromosomes and spindle
organizers lead to fractionation of the chromosome complement.

Due to the presence of up to nine bivalents in the hybrid,
allosyndetic pairing, pairing of the chromosomes from different
gametes, is suggested. It is believed, therefore, that there must be
some relationship between the genomes of V: radiata and_V. umbellata.

However, the rare occurrence of multivalents also suggests some

44

Vigna radiata** x Vigna umbellata**
I I I l
vrvrV rV r VuvuV uV u

 

 

F1 Hybrid*
Vrv ' rVuV ' U
2n=4X-22

doubling of

 

 

 

chromosomes
Amphiploid** x Vigna radiata**
vrer'rv'rvuvuv'uv'u
2n=8X=44
BC1*
VrVrV'rV'rVuV'u
2n=6X=33
selfing
Sl*
VrV'r + 1 (either Vu or V'u chromosomes)
2n=22 or 23

Figure 11. Summary of the prOposed genome formulae.

45

autosyndetic pairing in addition to allosyndetic pairing. It is
possible that there is some homology among all four genomes in the two

’ I 1
parental spec1es. Thus the genome formula VrV rVuV can be

L1
assigned to these hybrids.

Earlier workers have suggested that V. radiata and V. umbellata
are diploids with the genome formula of AA and BB or AA and A'A',
respectively. The F1 hybrid then would be AB or AA'. If the genome
formula for the hybrid was AB then only univalents could occur at
metaphase I in the hybrid. The maximum chromosome association that
could occur would be 11 bivalents as a result of allosyndetic pairing
between the A and A' genomes. However, the occurrence of multivalents
indicated autosyndetic pairing, thus substantiating the hypothesis that
there are more than one homo- or homoeologous genome occuring in each
parent.

Pollen viability of the F1 hybrid was very low (1.5%) and the
plants were sterile, probably due to the irregular meiosis. However,
Stebbins (1958) pointed out that although meiotic irregularities exist,
genetic causes may also account for sterility. Since the purpose of
making the cross was to recombine characteristics of the parents,
fertility (or lack thereof) is of prime concern.

The doubling of the chromosomes of the F1 hybrid restored
partial fertility, as Shown in the normal bivalent pairing (22 II) at
metaphase I. The absence of multivalents in both the parental species

and the amphiploid further substantiates the theory that the parental

Species are allotetraploids.

46

Two factors may explain the absence of multivalents in these
plants; low chiasma frequency of the chromosomes in Vigna because of
their small Size (Kostoff, 1940), and the genetic control of pairing.
Chromosome pairing is governed by a gene or genes with the type of
pairing resulting from a dosage effect; i.e. a single dose allows
homoeologous pairing whereas a double dose promotes homologous pairing
(Riley and Chapman, 1958; Feldman, 1966; Driscoll, 1972; and Starks and
Tai, 1974).

Loosely paired bivalents were observed in both parental species
suggesting the presence of a double dose of the gene(s) controlling
pairing. In the F1 hybrid, a Single dose from each parent is
present, thus allowing auto- and allosyndetic homoeologous pairing to
occur as determined by the presence of bivalents and multivalents,
respectively. The dosage of the gene(s) controlling pairing was
doubled in the amphiploid, therefore no multivalents occurred and
secondary associations were absent.

Split Spindles occurred in 4% of the cells observed in the
amphiploid, resulting in the 12/10 segregation of bivalents at
metaphase I. This reflects the grouping of chromosomes of the same
genome; groups of Six and five in the parental Species and groups of 12
and 10 after doubling in the amphiploid. This suggests that the
parental species consist of two genomes, one containing six and the
other five chromosomes.

Early separation of bivalents appearing as univalents at metaphase
I may be caused by the low frequency of chiasma found in these small

chromosomes. Succeeding stages of meiosis Show that these aligned

47
univalents behave normally and do not affect the segregation of chromo-
somes. These observations suggest that the partially homologous Vr’
V'r, Vu, and V'u genomes paired autosyndetically as bivalents and
were doubled in their genomic constitution. Therefore, the genomic
formula for the amphiploid is VrVrV'rV'rVuVuV'uV'u
(Figure 11).

The partial sterility of this amphiploid as seen in the reduced
viability of the mature pollen (44%) may be caused by various kinds of
physiological imbalances in Spite of nearly regular meiotic behavior
(Stebbins, 1971). This plant does have some of the desired
characteristics such as disease and insect resistance (AVRDC, 1975).
Due to its poor pollen production and sterility, this amphiploid has
limited utilization in agriculture. Therefore, the backcross of the
amphiploid to V. radiata was performed.

The backcross progeny (BC1) proved to be very weak plants with low
fertility. Meiosis was very irregular, with a majority of the cells
containing 11 bivalents and 11 univalents. The absence of multivalents
at metaphase I would suggest homology between only two of the genomes.
Because of the double dosage of the gene(s) controlling pairing from
the V: radiata genomes, no secondary associations between the V.
umbellata genomes could occur. The dosage effect also prevents the
pairing 0f.!: umbellata with V. radiata chromosomes. Therefore, it can
be assumed that the univalents are of V. umbellata origin. However,
the observation of loose associations suggests partial homology between
all of the genomes present. This partial homology could be the result

of genetically controlled homoeologous pairing. The genome formula

48
may be expressed as VrVrV'rV'rVuV'u (Figure 11).

Multipolar meiosis occurred in Bcl as in the others. At metaphase
I, split Spindles were observed, the bivalents segregating into groups
of five and six, thus further substantiating the earlier hypothesis
that the two genomes in the parental Species contained Six and five
chromosomes, respectively. THe remaining 11 univalents were scattered
throughout the cell. During anaphase I these univalents remained at
the metaphase plate and eventually became fragmented due to precocious
centromere division. These fragments will either be lost in the
cytOplasm or become incorporated into the telophase nuclei.

During the second meiotic division these fragments become
scattered and either formed micronuclei in a quartet of four cells or
microcells during the final stages of meiosis. A high percentage of
abnormal quartets were observed in these plants. Even the "normal"
looking quartets may contain a portion of these fragments, accounting
for the low percentage of viable pollen.

Despite chromosome abnormalities and possible genic imbalances, a
few seed from BCl were obtained through natural selfing. Upon
germination of the seeds and analysis of the root tip cells, it was
determined that these plants are trisomics (2n+1) with 2n=22 or 23.
Due to the inability to distinguish between the different chromosomes
of the original parents, the origin of the Single additional chromosome
was not discernable. It can be assumed that during the process of
selfing, the progeny received its entire genome from V. radiata with

the V. umbellata chromosomes being eliminated by multipolar meiosis.

49

Possibly only a fragment or a whole chromosome remains from the Vu or
V'u genome, depending quite possibly upon the degree of homology
between the V: umbellata genomes and the genomes of_V. radiata. THis
is the optimum Situation for incorporating beneficial genes into the
cultivated variety. In addition, the morphological characteristics of
the plants resemble the V. radiata parent. Thus, the genome formula
could be VrV'r + one Vu or V'u chromosome (Figure 11).

Seed that was produced by the BC1 plants was sent to the AVRDC
with these purposes in mind: 1) screening of the plants for beneficial
characteristics, 2) mutigenesis to induce translocations, and 3)

potential use in crOp improvement.

REFERENCES

References

 

l.

10.

11.

12.

13.

14.

Ahn, C. S. and R. W. Hartman. 1977. Interspecific hybridizations
among four species of the genus Vigna. _£3: The First
International Mungbean Symposium. University of the
Phillippines, Los Banos. pp. 240-246.

.Asian Vegetable Research and Development Center (AVRDC). 1974.

Annual Report for 1972-73. Shanhua, Tiawan.
1975. Annual Report for 1974. Shanhua, Tiawan.
Bauchan, G. R. and W. Tai. 1982. Cytogenetics of Phaseolus and
Vigna. In: Advances in the Grain Legumes. ed. D. Wallace.
(In Press)
Blackson, J. H. and W. Tai. 1982. Cytological and statistical
analysis of secondary associations in Phaseolus species

(Leguminosae). Journal of Heredity. Submitted.

Chen, N. C. 1980. Interspecific hybridization in the genus Vigna.
Ph.D. Dissertation. Michigan State University.

, J. F. Parrot, T. Jocobs, L. R. Baker, and P. S.

 

Carlson. 1977. Interspecific hybridization of food legumes
by unconventional methods of plant breeding. In: Proceedings
of the First International Mungbean Symposium,_Dniversity of
the Phillip[ines, Los Banos.

Dana, S. 1966 a. Species cross between Phaseolus aureus and P.
ricciardianus. Genetica Iberica 18:141-156.

 

 

Driscoll, C. L. 1972. Genetic suppression of homoeologous
chromosomes pairing in hexaploid wheat. Canadian Journal of
Genetics and Cytology 14:39-42.

Evans, A. M. 1975. Species hybridization in the genus Vigna. IITA
Tropical Grain Legume Bulletin. Ibadan, Nigeria.

Feldman, M. 1966. The effect of chromosomes SB, 5D, and SA on
chromosomal pairing in Triticum aestivum. Proceedings of the
National Academy of Science, USA. 55:1447-1453.

 

Grant, V. 1963. The Origin of Adaptions. Columbia Unversity
Press, New York.

1975. Genetics of Flowering Plants. Columbia
University Press, New York.

Joseph, L. 8. nd J. C. Bouwkamp. 1978. Karyomorphology of several
species of Phaseolus and Vigna. Cytologia 43:595-600.

50

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

51
Kimber, G. and E. R. Sears. 1980. Uses of wheat aneuploids. “In:
Polyploidy, Biological Relevance. ed. W. H. Lewis, Plenum

Press, New York.

Kostoff, D. 1940. Fertility and chromosome length. Journal of
Heridity 31:33-34.

Lawrence, W. J. C. 1931. The secondary association of
chromosomes. Cytologia 2: 352-384.

Li, H. W. and D. 8. Tu. 1947. Sutdies on the chromosomal

aberration of the amphiploid, Triticum timgpheevi and Aegilops

 

bicoris. Bot. Bull. Acad. Sinica. 1:173-186.

Machado, M., W. Tai, and L. Baker. 1982. Cytogenetic analysis of
the interspecific hybrid, Vigna radiata x Vigna umbellata.
Journal of Heredity. (In Press)

Meurman, O. 1933. Chromosome morphology, somatic doubling and
secondary association in Acer plantanoides L. Hereditas
18:145-173.

 

National Research Council. 1972. Committee on Genetic
Vulnerability of Major CrOps. National Academy of Sciences,
Washington, D.C.

Parrot, J. F. 1981. Recovery of interspecific Vigna hybrids via
embryo culture. M.S. Thesis. Michigan State Unviersity.

Rieger, R. A., A. Michaelis, and M. M. Green. 1976. Glossary of
Genetics and Cytogenetics. Springer-Verlag, New York.

Riley, R. and V. Chapman. 1958. Genetic control of the
cytologically diploid behavior of hexaploid wheat. Nature
182:1244-1246.

Sarbhoy, R. K. 1977. Cytological studies in the genus Phaseolus.
III. Evolution in the genus. Cytologia 42:401-413.

Sen, N. K. and J. G. Bhowal. 1960. Cytotaxonomic studies on
Vigna. Cytologia 25:195-207.

Sinha, S. S. N. and H. Roy. 1979. Cytological studies in the

genus Phaseolus. I. Mitotic analysis of fourteen Species.
Cytologia 44:191-199.

Starks, G. J. and W. Tai. 1974. Genome analysis of Hordeum
jabatum and H. compressum. Canadian Journal of Genetics and
Cytology 16:663-668.

Stebbins, G. L. 1950. Variation and Evolution in Plants.
Columbia University Press, New York.

 

30.

31.

32.

33.

34.

52

1958. Hybrid inviability, weakness and sterility of

 

interspecific hybrids. Advances in Genetics 9:147-215.

1971. Chromosomal Evolution in Higher Plants.

 

Addison-Wesley Pub. Co., Boston.

Tai, W. 1970. Multipolar meiosis in diploid creasted wheatgrass,
Agropyron cristatum. American Journal of Botany
57:1160-1169.

 

Therman, E. 1951. Somatic and secondary pairing on Ornithogalum.
Heredity 5:253-269.

 

Thompson, M. M. 1962. Cytogenetics of Rubus. III. Meiotic
instability in some higher polyploids. American Journal of
Botany 49:575582.

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