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INTERSPECIFIC HYBRIDIZATION IN THE GENUS VIGNA

By

Nung Che Chen

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1980

ABSTRACT
INTERSPECIFIC HYBRIDIZATION IN THE GENUS XLEEé
BY
Nung Che Chen

Interspecific hybridization among four species of

Asian food legumes, namely, Vigna radiata (L.) Wilzcek

 

(mungbean), X. mungo (L.) Hepper (black gram), 2.

umbellata (Thunb.) Ohwi and Ohashi (rice bean) and

 

X. angularis (Wild.) Ohwi and Ohashi (adzuki bean) was
investigated. Pod-set and percentage of pods harvested
varied with interspecific cross combinations and cultivars
used. An increase in the number of F1 seeds was possible

in the cross, X. radiata x K. umbellata, through the use

 

of intraspecific hybrids as parents. An interspecific

hybrid of X. mungo x X. angularis was also accomplished

 

by the use of intraspecific hybrids as parents. Except

for the cross between X. umbellata and X. angularis, all

 

 

other reciprocal interspecific crosses were unsuccessful.
Hybrid seeds were obtained from the following crosses:

K. radiata x X. mungo, X. radiata x X. umbellata and

 

V. mungo x X. angularis. Hybrid plants were obtained

 

using embryo culture from the following crosses: X. radiata

 

 

x V. angularis, E. umbellata x X. angularis and X. angularis

 

 

x X. umbellata. A reciprocal hybrid of the interspecific

 

Nung Che Chen

cross between V. umbellata and V. angularis was successfully

 

 

made.

Growth and lethality of interspecific hybrid seedlings
were influenced by the genotypes of the parental species.
Parnetal genotypes also affected the fertility of inter-
specific hybrids which ranged from partially fertile to
completely sterile depending upon the interspecific
cross combinations.

Three chemicals, E-amino-n-caproic acid (EACA),
gentisic acid and L-lysine, were used to increase the
success of making interspecific crosses inthe genus
Vigna. The EACA as a foliar spray was the most effective
chemical in producing of interspecific hybrids between

V. radiata and V. umbellata. Daily foliar applications

 

of EACA to the plants of the maternal parent, V. radiata,

for 14 days prior to hybridization with V. umbellata

 

significantly increased the frequency of hybrid embryos

(22%) and the numbers of F seeds (from 2 to 9 times).

1
Concentrations of 100 to 1000 mg EACA/l were the most
effective. ,
Other techniques were also used to facilitate the

success of the interspecific cross, V. radiata x V.

umbellata. Number of F1 seeds was increased when pods

 

were allowed to mature with part of the pedicels were

severed 10-14 days after pollination and when they were

Nung Che Chen

cultured in vitro. The number of F1 seeds increased by

9 and 5 times for partial detached and detached pods,

 

respectively. Partial and complete removal of leaves
from the maternal plant 4 days after pollination also
increased the number of F1 seeds from 7- to 10—fold

as compared to the control. "Side-grafting” between two

species and the use of V. radiata var. sublobata as a

 

species bridge were effective in reducing embryo

abortion and increasing the numbers of F1 seeds by 11

and 45 times, respectively. Hybrid sterility was
circumvented by doubling the chromosome number.

Triploid plants (amphidiploid x V. radiata) were
successfully obtained by using embryo culture. Variation
in morphological characteristics and fertility was
observed in the progenies by selfing the backcross
progenies. Similar observations were made in the

progenies resulting from the second backcrossing.

A.»

 

We

ACKNOWLEDGMENTS

The author wishes to express his deep gratitude to his
former major professor, Dr. Larry R. Baker, for his guidance
and encouragement that made this study possible, and for his
valuable suggestions and critical review in the preparation
of this manuscript.

The author extends his sincere appreciation to his
major advisor, Dr. S. Honma, and also to the other members
of his committee, Drs. J.F. Fobes, J.F. Kelly, M.W. Adams
and P.S. Carlson, for their guidance, suggestions and
review of this manuscript.

Sincere appreciation is also expressed to Dr. w. Tai
for the use of his research facilities, and to Drs. J.C.
Moomaw and J.J. Riley, former Director and Associate
Director, respectively, The Asian Vegetable Research and
Development Center (Taiwan) for their support and encourage-
ment. A special thanks is extended to his colleague,

James Parrot, for his assistance with embryo culture
techniques.

Heartfelt gratitude is extended to his wife, Suman
for the many sacrifices she had to make while giving him
her unqualified support and also to his sons, Wayne and

hdike, for their belief in their father.

Guidance Committee:

Section I, II and III are segments of related thesis
research information condensed into formats suited and

intended for publication in Euphytica (Section I and III)

 

and in the Journal of the American Society for Horticultural

 

Science (Section II).

ii

.2 a.)

 

. . ..Z 2.;

TABLE OF CONTENTS

 

Page

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . i

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

LIST OF FIGURES . . . . . . . . . . . . . . . . . . vii

SECTION I

INFLUENCE OF PARENTAL CULTIVAR AND PARENTAL

HETEROZYGOSITY ON INTERSPECIFIC CROSSABILITY

AMONG FOUR SPECIES OF VIGNA . . . . . . . . 1
Summary. . . . . . . . . . . . . . . . . . 1
Introduction . . . . . . . . . . . . . . 3
Materials and Methods. . . . . . . . . . . . . 6
Results and Discussion . . . . . . . . . 10
General Discussion and Conclusion. . . . . . . 47
References . . . . . . . . . . . . . . . . . . 50

SECTION II

EFFECT OF E-AMINO-N-CAPROIC ACID ON INTER-

SPECIFIC HYBRIDIZATION IN THE GENUS VIGNA . . . . . 53
Abstract . . . . . . . . . . . . . . . . . . . 53
Introduction . . . . . . . . . . . . . . 55
Materials and Methods. . . . . . . . . . . . . 57
Results and Discussion . . . . . . . . . . . .' 60
Literature Cited . . . . . . . . . . . . . . . 79

SECTION III

TECHNIQUES IN CIRCUMVENTING CROSSABILITY

BARRIERS IN THE INTERSPECIFIC CROSS, VIGNA

RADIATA x V. UMBELLATA. . . . . . . . . . . 81
Summary. . . . . . . . . . . . . . . . . . . . 81
Introduction . . . . . . . . . . . . . . 82
Materials and Methods. . . . . . . . . . . . . 84
Results and Discussion . . . . . . . . . . . . 88
References . . . . . . . . . . . . . . . . . . 105

iii

Table

10.

ll.

12.

13.

LIST OF TABLES

SECTION I

Description of fourV Vi na species used as parents
for interspecific hy r1 1zation . .

Crossability for the interspecific cross of V.
radiata (M) and V. mungo (B).

Effects of parental cultivar on the growth and
fertility of interspecific hybrids of V. radiata

x V. mungo. . . . . . . . .

Comparison of the morphological characteristics
of V. radiata (M865), V. mungo (T-9) and their
interspec1f1c hybrid. . . . . . . . . . . . .

Crossability for the interspecific hybridization
of V. radiata (M) and V. umbellata (R). . . .

 

Comparison of the morphological characteristics
of V. radiata (Tainan #1), V. umbellata (HK) and
the1r 1nterspecific hybrid. . . . . . . . .

 

Effects of parental cultivar cross on growth and
fertility of the interspecific hybrid of V.

radiata x V. umbellata.

 

Crossability for the interspecific hybridization
of V. radiata (M) and V. angularis (A). . . .

 

Comparison of the morphological characteristics
ofV . radiata (Tainan #1),V . angularis (KS#ZlO)
and the1r 1nterspecific hybrid.

 

Crossability for the interspecific hybridization
of V. mungo (B) and V. umbellata (R).

 

Crossability for the interspecific hybridization
of V. mungo (B) and V. angularis (A).

 

Comparison of morphological characteristics of
V. mun o (NIZOS x T- 9) an ularis (KS#210)
and t e1r interspecific Hybr1§..

Crossability for the interspecific hybridization
of V. umbellata (R) and V. angularis (A).

 

 

iv

Page

11

l4

18

20

22

24

26

29

33

36

4O

Table Page _

14. Comparison of the morphological characteristics
of V. umbellata (HK), V. angularis (Chien Shien)
and their interspecific hybridi. . . . . . . . . . 44

 

 

SECTION II

1. Effect of EACA treatments on the success of the
interspecific cross of V. radiata x V. umbellata . 61

 

2. Effect of EACA applied as post-pollination
treatments on the success of the interspecific
cross of V. radiata x V. umbellata . . . . . . . . 64

 

3. Effect of duration of treatment with 1000 mg/l
EACA on viable seed set of the interspecific
cross of V. radiata x V. umbellata . . . . . . . . 66

 

4. Response of two varieties of V. radiata to EACA
treatments by interspecific hybr1dizat1 on with
V. umbellata . . . . . . . . . . . . . . . . . 67

 

5. Relative effect of EACA and gentisic acid on the
success of the interspecific cross of V. radiata
x V.umbe11ata . . . . . . . . . . . . . . . . . . 70

 

6. Relative effect of EACA and L- lysine on the
success of the interspecific cross of V. radiata
x V. umbellata . . . . . . . . . . . . . . . . . . 72

 

7. Effects of EACA and L- lysine on deve10pment of
14- day- old pods, seeds and embryos produced from
the cross of V. radiata x V. umbellata . . . . 74

 

8. Effects of EACA, gentisic acid and L-lysine on
deve10pment of 12- day- old V. radiata x V.
umbellata embryos. . . . . . . . . . . . . . . . . 76

 

SECTION III

1. Effect of the partial detached and detached pods
on the success of the interspecific cross, V.
radiata x V. umbellata . . . . . . . . . . . . . . 9O

 

2. Effect of post-pollination time for partial
detached and detached pods on the success of the
interspecific cross, V. radiata x V. umbellata . . 92

 

Table , Page

3. Effect of defoliation on the success of the
interspecific cross, V. radiata x V. umbellata. . 93

 

4. Effect of time and degree of defoliation on the
success of the interspecific cross, V. radiata
x V. umbellata. . . . . . . . . . . . . . . .'. . 94

 

5. Effect of mixed pollen on facilitating inter-
specific hybridization of V. radiata and
V. umbellata. . . . . . . . . . . . . . . . . . . 97

 

6. Influence of interspecies grafting on overcoming
crossability barriers for the interspecific
cross of V. radiata x V. umbellata. . . . . . . . 98

 

7. Effect of V. radiata var. sublobata as a bridge
species to improve the crossability of V. radiata
and V. umbellata. . . . . . . . . . . . . . . . . 100

 

 

vi

LIST OF FIGURES

 

Figure Page
SECTION I
l. Interspecific hybrids of V. radiata x V. mungo
and V. radiata x V. umbellata. . . . . . . . . . . 15

Z. Interspecific hybrids of V. radiata x V.

 

 

 

angularis and V. mungo x V. anguIaris.-. . . . . . 30
3. Interspecific hybrids of V. umbellata x V.
angularis and the reciprocal hybrid. . . . . . . . 42

 

SECTION III

1. Comparative growth of pod, seed and embryo of
natural self of V. radiata and the interspecific
hybrid, V. radiata x V. umbellata. . . . . . . . . 89

 

2. Plants of interspecific hybrid V. radiata x V.
umbellata, amphidiploid, triploid and progenies
othhe triploid. . . . . . . . . . . . . . . .

 

102

vii

SECTION I
INFLUENCE OF PARENTAL CULTIVAR AND PARENTAL
HETEROZYGOSITY ON INTERSPECIFIC CROSSABILITY

AMONG FOUR SPECIES OF VIGNA

SUMMARY

Interspecific crossability among four species of

Vigna, namely, V. radiata (mungbean), V. munoo (black

__L

gram), V. umbellata (rice bean) and V. angularis (adzuki

bean), was investigated. Pod-set and percentages of pods

 

 

harvested varied with the combinations of two parental
cultivars of each species for most of the interspecific
hybrid crosses. The use of intraspecific hybrids as
parents was slightly superior to the use of cultivars.

A remarkable increase in viable seed production was found

for the interspecific cross, V. radiata x V. umbellata,

 

by using intraspecific hybrid as parents. Furthermore,
a successful interspecific hybrid of V. mungo x V.

angularis was accomplished through the use of intra-

 

specific hybrid parents. Reciprocal cross differences
were common in all the interspecific combinations.

Except for the cross between V. umbellata and V. angularis,

 

 

all other reciprocal interspecific crosses were unsuccessful.
Viable seeds were obtained from the interspecific
crosses of V. radiata x V. mungo, V. radiata x V.

umbellata and V. mungo x V. angularis. Hybrid plants

 

 

were obtained from cultured embryos for the interspecific

crosses of V. radiata x V. angularis, V. umbellata

 

 

x V. angularis and V. angularis x V. umbellata. A

 

 

 

successful reciprocal hybrid of the interspecific cross

[\J

between V. umbellata and V. angularis is reported.

 

 

Growth and lethality of the interspecific hybrid
seedlings were influenced by the genotypes of both
parental species. Parental genotypes also affected the
fertility of interspecific hybrids. Complete hybrid
sterility was found in the interspecific crosses of

V. radiata x V. umbellata, V. radiata x V. angularis

 

 

and V. mungo x V. angularis, while reduced fertility

 

was observed for the interspecific hybrids of V. radiata

_. mungo, V. umbellata x V. angularis and V. angularis

 

 

 

V
x V. umbellata.

 

INTRODUCTION

Interspecific hybridization is a useful tool in
plant breeding to create new genetic variation. Interest
in interspecific hybridization has gained impetus as
improved hybridization techniques increased the
possibilities of successful crosses (Sanchez-Monge G
Garcia-Olmedo, 1977).

The four oriental species of food legumes, Vigna
radiata (L.) Wilzcek (mungbean), V. mgngg (L.) Hepper
(black gram), V. umbellata (Thunb.) Ohwi and Ohashi

 

(rice bean) and V. angularis (Wild.) Ohwi and Ohashi

 

(adzuki bean), are important pulse crops in parts of
Asia and Africa (Rachie 8 Roberts, 1974). These species
are distinguished from other food legumes by their Asian
origin and their yellow flowers and were recently

separated from Phaseolus by Verdicourt (1970).

 

Varying degrees of success in interspecific hybridi-
zation of Vigna have been reported (Dana, 1966, 1967; De G
Krishnan, 1966; Al-Yasiri 8 Coyne, 1966; Sawa, 1973;
Biswas 8 Dana, 1975; Chen et a1., 1977; Chowdhury 8
Chowdhury, 1977; Ahn G Hartmann, 1977, 1978a, 1978b).

The degree of success was influenced by the parental
cultivars used and the parental heterozygosity. Honma
and Heeckt (1958, 1959), using different cultivars and

intraspecific hybrids, were successful in obtaining the

interspecific hybrids of V. vulgaris and V. lunatus and

V. coccineus x V. lunatus. Mok et a1., (1978) reported

 

that the genotypes of the parents appear to be essential
in generating interspecific hybrids. By using different
parental genotypes, they were able to obtain the hybrids

of V. vulgaris x V. lunatus, V. vulgaris x V. acutifolius

 

and V. acutifolius x V. vulgaris through the aid of

 

embryo culture. The rate of growth and final size of these
hybrid embryos seemed to be influenced by the genotypes of
both parents. In other studies, Taira et a1., (1978)

reported that the Triticum turgidum (wheat) and Secale

 

cereale (rye) parental genotypes exerted a definite
effect on the frequency of normal embryo formation and
seed-set of wheat-rye crosses. Parental genotypes do
play an important role in the success of interspecific
hybridization.

The causes of failures of interspecific crosses in
food legumes are not fully understood. In some cases,
the pollen tubes are unable to penetrate the stigma
and style (Chowdhury G Chowdhury, 1977). In other cases,
fertilization occurs, but embryo abortion takes place
during embryogenesis (Honma, 1956; Al-Yasiri 8 Coyne,
1966; Ahn G Hartmann, 1977, 1978a, 1978b). The failure
of interspecific hybridization due to embryo degeneration

is common in interspecific crosses of food legumes (Ahn

8 Hartmann, 1977; Chen et a1., 1977). Moreover,
interspecific hybrids obtained are often completely
sterile or only partially fertile, while relatively

few are fertile. This investigation reports the

effects of parental cultivars on interspecific
crossability, describes the interspecific hybrid plants
derived from four Vigna species, and compares the use

of homozygous with heterozygous parents for interspecific

crossability.

MATERIALS AND METHODS

Plant materials. Two parental cultivars each of
V. radiata (Tainan #1, M865), V. mungo (T-9, N1208),

V. umbellata (HK, 8-91) and V. angularis (Chien Shien,

 

 

KS#210) were used for hybridization (Table 1). In general,
the two selected parents of each species were diverse in
their origins and phenotypes. All possible combinations,
including reciprocals, were made between four species.
Intraspecific hybrids were produced by crossing the two
cultivars of each Species to obtain heterozygous parents

for the interspecific crosses.

Cultural conditions. Plants were grown under glass-
house conditions. During winter, temperatures were
adjusted to 260C (day) and 180C (night) with a photoperiod
of 14 hr light supplied by high intensity metal-halide
lamps (10-12 klx). Two plants were grown in a 20 cm pot
containing "Peat Lite Mix". A total of eight plants of
each parental cultivar were used for the female. All

parental cultivars including intraspecific F were

1
planted at the same time. Several plantings were made
of the male parents to assure adequate pollen. Plants
were fertilized with 3 g/pot of Peter's fertilizer

(ZON-ZOP-ZOK) biweekly. A short-day photOperiod was

used for V. umbellata to induce flowering.

 

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Crossing methods. Flowers used as female parents

 

were emasculated the day before anthesis and pollinated
immediately. The hybridization technique used was

similar to that described by Boling et a1., (1961).

For the same interspecific cross, pollinations were made
on both parental cultivars over the same period of

time so that comparisons could be made between parental
cultivars for crossability. A minimum of 100 pollinations
were attempted in all possible 60 cross-combinations.
Self-pollinated pods were removed daily from the plants

to eliminate competition.

Data collection and analysis. Data were taken on

 

the number of flowers pollinated, number of pods set and
number of mature pods harvested. Flowers which abscised
within 24 hr were excluded on the basis of mechanical
damage. Therefore, the number hybridization included
those flowers which opened normally the day after
pollination. Pod-setting was recorded on the fourth

day after pollination.

Mature pods were harvested individually and the
number of pods and seeds recorded. The 'hybrid' seeds
were germinated in petri dishes to determine their
viability. Germinating seedlings were transplanted to
Jiffy-7 pots. The lethality of seedlings was recorded.

Morphological characteristics of the 'hybrid' plants were

.—v

.a.

.-

‘o

observed and compared with the parental species. Evidence
of hybridity was determined from their morphological
features. Genetic markers for germination habit,

epicotyl color, petiole length of the primary leaves

and lobing of leaflets were used as indicators of
hybridity. Pollen viability was estimated from the
percentage of plump pollen grains that stained darkly

and uniformly from an Iz-KI solution (Johansen, 1940).

A minimum of 400 pollen grains were counted from each

of five flowers per 'hybrid' plant.

Embryo culture was attempted on interspecific
crosses which did not produce viable seeds from cross-
pollinations. Immature embryos from 14 to 18 day old
pods were excised and cultured on an artificial medium
described by Linsmaier and Skoog (1965). The resulting
seedlings were transplanted to Jiffy-7 pots and gradually
acclimatized to glasshouse conditions.

The effects of parental cultivar on crossability
was tested by chi-square for percent pod-set, percent
pods harvested, number of viable hybrid seeds, reciprocal
cross difference, and homozygous versus heterozygous

parents.

RESULTS AND DISCUSSION

Crossability of V. radiata and V. mungo. Inter-

 

specific hybridization was successful only when V.
radiata was used as the female parent. The overall
percentage for pods harvested was 85 and 75% for
V. radiata x V. mungo and V. mungg x V. radiata,
respectively (Table 2). Percent pods harvested was
generally high which suggested fertilization was
successful in order to obtain pod-set between these
two species. The chi-square test for reciprocal cross
difference in percent pods harvested was not significant
(Table 2).

In the V. radiata x V. mgngg crosses, differences
in percent pods harvested were highly significant
among the crosses and between the two cultivars of V.
radiata. The best combination was Tainan #1 x T-9 which
produced the highest percentage of pods harvested (92%).
The percentage of pods harvested was higher on cultivar
Tainan #1 than on M865; i.e., 91 and 62%, respectively
(Table 2). There was no significant difference between
the cultivars of the pollen parent for the number of
pods harvested as well as between the use of cultivars
and an intraspecific hybrid as parents for the species
cross. The set-pods developed normally to maturity.

Two kinds of hybrid seeds were obtained; viz., partially

10

11

Table 2. Crossability for the interspecific cross of V. radiata (M)
and V. mungo (B). ‘

 

Parents1 No. flowers Pods harvested Total seeds Viable seeds

 

 

 

 

 

 

 

 

 

 

 

 

Female Male crossed (%) obtained %) No./
attempt
Ml B1 125 92.0 949 87.7 6.7
M1 B2 140 90.7 .617 7 .l 3.
Mean 133 91.0 783 84.3 5.
MZ Bl 136 55.9 793 88.0 S.
MZ B2 145 67.6 968 73.6 4
Mean 141 61.7 881 80.0
(MlxMZ) (B2xBl) 146 91.1 1132 80.7 6.9
Bl M1 ‘ 243 60.1 431 0 0
Bl M2 203 52.1 228
Mean 223 56.5 330
B2 Ml 148 82.4 546
BZ M2 156 85.9 568
Mean 152 84.2 557
(BZxBl) (MlxMZ) 203 82.3 664 0 0
Comparison % pods harvested Comparison % pods harvested
Within M x B **2 Within B x M **
between M * between B *
between B ns between M ns
cultivars vs Fls ns cultivars vs Fls ns

M x B vs B x M
cultivar x cultivar ns

leFl ns

 

IM1 = Tainan #1, M2 . M865, Bl = r-9, 132 = NI 2.08.

zSignificant at 5% (*) or 1% (**); ns = not significant by chi-square
test .

12

shrivelled and ruptured seeds. The partially shrivelled
seeds were found to be viable by germination; while
the ruptured seeds were inviable. There were no
significant differences in number of viable seeds per
attempt among the different combinations of parental
cultivars.

Number of pods harvested varied significantly
according to the parental cultivars when V. mungo
was used as the female parent. The cultivar NI208
was superior to T-9 for crossability as indicated
by the higher percentages of pods harvested and numbers
of seeds produced (Table 2). Early pod development
appeared normal, but later aborted. Two types of
seeds, shrivelled and empty, were obtained in the
crosses of V. mgngg x V. radiata which were all
inviable.

The relatively high number of pods harvested
would suggest that apparently there are no barriers
in crossing of these two species for the parental
cultivars used. However, there were barriers to seed
development which produced inviable and viable seeds
for the cross of V. radiata x V. mungg, but completely
inviable seeds in the reciprocal cross, V. mgngg x
V. radiata. Attempts to culture immature hybrid embryos

of V. mungo x V. radiata failed to produce any seedlings.

13

The lS-day-old hybrid embryos were of adequate size
(3.4 mm), but 10 of 21 cultured embryos formed only
callus, and all others failed to grow. The reciprocal
difference in crossability of V. radiata and V. mungg
suggests interaction between genic and cytOplasmic
factors (Stebbins, 1958). This interaction may be the
cause of hybrid embryo degeneration when V. mungg is

used as the female parent (Ahn 5 Hartmann, 1977).

Growth and morphology of V. radiata x V. mungo

 

hybrids. The hybrid plants differed according to the
parental cultivars used. Hybrid lethality was high when
the cross combinations involved N1208 of V. 33359. The
F1 plants of Tainan #1 x N1208 and M865 x N1208 grew
slowly and poorly with small crinkled leaves (Table 3,
Fig. 1-a,b,c). Hybrid plants of Tainan #1 x NI208 died
at various stages of growth development and never reached
the flowering stage. The hybrids from the cross of
Tainan #1 x T-9 grew normally at early stages but
suddenly lost their vigor before or upon flowering

and eventually died. The root systems of Tainan #1 x T-9
plants were observed to grow slowly and eventually succumb
to root rot (Fig. 1-d). The hybrid of M865 x T-9 was

the superior among the five cross combinations. The
plants displayed hybrid vigor in the seedling stage

which extended throughout their life cycle. Hybrid plants

14

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Figure l.

15

Interspecific hybrids of V. radiata x V} mungg and

V} radiata x V. umbellata. 1-a: Hybrid plants of
Tainan #1 x T-9 (left), Tainan #1 x N1208 (middle)

and (Tainan #1 x M865) x (N1208 x T-9). 1-b: Hybrid
plants of Tainan #1 x T-9 (left), M865 x N1208
(middle) and M865 x T-9. l-c: Hybrid plants of

M865 x T-9 (left) and M865 x N1208. 1-d: Root
systems, tOp: Tainan #1 (left), Tainan #1 x T-9
(middle) and T-9; bottom: M865 (left), M865 x T-9
(middle) and T-9. l-e: Seedlings of V. radiata
(Tainan #1, left), V. umbellata (HK, right) and their
interspecific hybrid (middle) showing the germination
habits. 1-f: Plants of V. radiata (Tainan #1, left),
V. umbellata (HK, right) and their interspecific hybrid
(middle).

\ ‘4

 

 

 

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17

derived from two heterozygous intraspecific hybrids of
two parental species; e.g., (Tainan #1 x M865) x (NI208 x
T-9), had a range of vigor from weak like the plants of
Tainan #1 x N1208 and M865 x NI208 to vigorous like the
plants of M865 x T-9.

The hybrid plants resembled the female parent for
certain characters; the male for other characters; or
else was intermediate in expression (Table 4). The
morphological characteristics similar to male parent or
intermediate between two parental species were the most
important traits to use as markers to verify hybridity.
In the cross of V. radiata x V. mungo, the characters of
ovoid seeds, seed L/W ratio and concave hilum cushion
liked the male parent; whereas the seed color and seed
weight were intermediate.

The F1 plants were partially fertile and flowered
profusely and continuously, and had a low pod-set.

The infrequent mature pods harvested from self-pollinated
flowers usually contained one or two fully developed
seeds. Many pods aborted prematurely with only empty or
ruptured seeds. Pollen stainability ranged from 17 to
47% depending upon the combination of parental cultivars
(Table 3). The hybrid plants of M865 x T-9 had the
highest percentage of stainable pollen grains with an
average of 47% compared to over 90% for the two parental

species (Table 4). The stained pollen grains were

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18

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19

relatively plump and uniform, whereas the unstained

pollen grains were smaller and variable in size.

Crossability of V. radiata and V. umbellata. The

 

cross between V. radiata and V. umbellata was only

 

successful when V. radiata was used as the seed parent.
The pod-set was high (85% average) and few viable hybrid

seeds were obtained (Table 5). When V. umbellata was

 

used as the female, pod-set was only 21% and no viable
seed was produced. The reciprocal cross differences for
both pod-set and number of viable seeds were highly
significant. P

No differences in pod-set between the parental
cultivars was found when V. radiata was used as the
female parent. However, there were significant differences
in numbers of viable seeds obtained for this inter-
specific cross (Table 5). Pods developed normally to
maturity. The mature pods contained seeds which appeared
from very shrivelled to partially filled. Upon
germination only 1 to 11% of the total seeds harvested
were viable. The percentages of viable seeds
depended upon the hybrid combinations. The combination
of M865 x HK produced the most viable seeds per 100
attempts, 48, while Tainan #1 x S-9l produced only 8
viable seeds. Though no differences in viable seed

numbers were found between the interspecific crosses

20

Table 5. Crossability for the interspecific hybridization of
V. radiata (M) and V. umbellata (R).

 

 

 

 

 

 

 

 

Parents1 No. flowers Pod-set Total seeds Viable seeds
Female ‘Male crossed (%) obtained (%) No./100
attempts
Ml R1 154 89.6 878 6.7 38.3
Ml R2 160 90.0 996 1.3 8.1
Mean 157 89.8 937 3.8 22.9
M2 R1 142 74.5 699 9.7 47.9
M2 R2 155 85.2 538 7.8 27.1
IMean 149 79.9 619 8.9 37.5
(MlxMZ) (R2le) 210 92.9 1483 11.1 78.6
Comparison % pod set Viable seeds Comparison %
per 100 attempts pod-set
Within.M x R nszfi ** Within R.x M’ **
between M ns ns between R **
between R ns ** between M ns
cultivars vs F15 ns. ** cultivars vs
F 5 ns
1

M x R vs R x M
cultivar x cultivar ** **

** ink
F1 x F1

 

lMi = Tainan #1, M2 = M865, R1 = HK, R2 = s-91.

2(**) significant at 1%; ns = not significant by chi-square test.

21

for the two V. radiata cultivars, the difference

between the two V. umbellata cultivars as the pollen

 

parents was highly significant. The combination of
intraspecific hybrids of the two species significantly
increased the number of viable seeds, 79 per 100
attempts as compared to 30 for the parental cultivars.

In the cross of V. umbellata x V. radiata, pod-set

 

differences were highly significant among the four
combinations of parental cultivars. No effect of two
V. radiata pollinators on pod-set was observed, but
the differences in pod-set were highly significant

between two V. umbellata cultivars. The pod-set on

 

HK (32%) was higher than that on S-91 (11%). Pods on

V. umbellata usually abscised within a week of pollina-

 

tion. The use of heterozygous parents did not improve
the low pod-set and embryo abortion.

The reciprocal cross differences in crossability
suggest that cytoplasmic factors play an important
role in the unidirectional success of this interspecific

CI‘OSS .

Growth and morphology of V. radiata x V. umbellata

 

hybrids. The germination habit of hybrid seedlings was
intermediate between the parental species which were

epigeal and hypogeal for V. radiata and V. umbellata,

 

respectively (Table 6, Fig. l-e). The cotyledon

22

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page: guzopu

Acananu zxa
AEEV mama thsflhm

 

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o mé H E. m.m .+. 5%. 155 £33 389::

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23

positions of the hybrid seedlings were at the soil
surface, and the average length of hypocotyl was 24 mm
compared to 65 mm for the V. radiata parent.

The hybrid seedlings showed poor vigor and the
initial growth was slow. Abnormal seedlings with three
or four primary leaves were noted. Growth of the hybrid
plants was vigorous once the seedling stage was past
(Fig. l-f). Hybrid plant growth usually exceeded the
parental species and was characterized by a thicker
stem, larger leaves and more branches (Table 6).

The hybrid plants were also intermediate for
expression of petiole length of the primary leaves and

flower color (Table 6). The V. umbellata characters

 

of indeterminate growth and simple raceme were dominant
in the F1 plants. The indeterminate growth habit of
the hybrid plants produced continuously juvenile

shoots and behaved as a perennial with no signs of
senescence.

The F1 plants flowered profusely and continuously
within the same racemes for a prolonged period and set
few pods during cool temperature period, but abscised
shortly thereafter. Stainable pollen grains were from
1.2 to 2.0% depending upon the hybrid combination of
parental cultivars (Table 7). The unstained pollen

grains were variable in size, while the stained pollen

24

Table 7. Effects of parental cultivar cross on growth and
fertility of the interspecific hybrid of V. radiata
x V. umbellata.

 

1

 

 

 

Interspecific Seedling Leaf size (LxW, cm) Pollen

hybrid pedigree height Terminal leaflet Lateral leaflet stain-
(cm) ability (%)

Tainan #1 x I-IK 9.3 21.9 x 15.8 20.6 x 13.8 1.8

Tainan #1 x S-91 10.8 22.4 x 15.9 20.8 x 14.4 2.0

M865 x HK 10.4 18.0 x 12.2 16.2 x 11.3 1.5

M865 x S-91 7.9 19.1 x 14.5 18.2 x 13.4 1.2

(Tainan #1 x M865)

x (S—91 x HK) 10.2 17.7 x 13.5 16.8 x 12.0 1.3

1

Planted on June 10; seedling height was measured at 3 weeks
after planting.

25

grains were somewhat larger than the parental pollen.
This was also observed by other workers (Ahn G Hartmann,
1977; Machado et a1., unpublished). The sterility

of the hybrid plants might be caused by the lack of
chromosomal homology and resultant meiotic irregularities
(Dana, 1967; Ahn & Hartmann, 1977; Machado et a1.,

unpublished).

Crossability of V. radiata and V. angularis. Neither

 

the parental cultivars of V. radiata nor V. angularis

 

affected pod-set when V. radiata was used as the female
parent (Table 8). The percentages of pod-set noted on
the fourth day after pollination were high; viz., 77%
for the four combinations of parental cultivars and 85%
for the interspecific cross between the two intraspecific
hybrids. However, the pods only grew to about 3-5 cm
in length and finally abscised some 1 to 3 weeks after
cross-pollination. A few pods were retained longer on
the plants but embryo abortion eventually occurred. The
empty pods contained very small, shrivelled, nonviable
seeds. No differences in pod-set and seed deve10pment
were observed between the use of parental cultivar and
intraspecific hybrid as the parents. The 15-day-old
hybrid embryos appeared small, averaging 1.2 mm in length.
There were significant differences for pod-set
between the parental cultivars of seed parent in the

crosses of V. angularis x V. radiata. However, pod-set

 

26

Table 8. Crossability for the interspecific hybridization of
V. radiata (M) and V. angplar-is (A).

 

 

 

 

 

 

 

 

 

 

 

 

7
Parents1 No. flowers Pod-set Pod abscision“
Female Phle' crossed (%) (days)
ND. A1 130 80.8 8.6
Ml A2 206 79.1 13.4
Mean 168 79.8 11.0
MZ A1 133 72.2 5.2
M2 A2 115 76.5 8.2
Mean 124 74.2 6.7
(MlxMZ) (A2xAl) 214 84.6 9.8
A1 M1 106 62.3 11.8
A1 M2 118 » 68.6 15.6
Mean 112 66.1 13.7
A2 ND 125 24.0 11.0
A2 M2 131 35.1 10.2
Mean 128 29.7 10.7
(AZxAl) (MlxMZ) 147 57.8 10.8
Comparison % pgd-set Comparison % pgd-set
Within M x A ns Within A x M **
between.M ns between A **
between A ns between.M ns
cultivars vs Fls ns cultivars vs Fls ns
M'x A.vs A.x M Mix A.vs A.x M
cultivar x cultivars ** Fl x F1 *
1

M1 = Tainan #1, M2 = M865, A1 = Chien Shien, A2 = KS#210.
2Mean of days to pod abscision after pollination.
3Significant at 5% (*) or 1% (**); ns = not significant by chi-square

test.

27

was equally affected by the two pollen parent

V. radiata cultivars. Pod-set on the two parental
cultivars was not significantly different from the

° intraspecific hybrids. The pods stopped growing

about 10-15 days after cross-pollination, then either
abscised or the embryos aborted leaving empty pods

on the plants.

1 Significant reciprocal differences in pod-set
were found within parental cultivars and intraspecific
hybrids used as parents in this interspecific cross.
These results were not similar to findings of Ahn and
Hartmann (1978a), perhaps due to cultivar differences.
In this study, pod-set on V. radiata was higher than that

on V. angularis. Pod-set on V. radiata and V. angularis

 

averaged 77 and 47% respectively, where cultivars were
used as parents, whereas, the use of intraspecific
hybrids as parents increased pod-set to 85 and 58%,

respectively (Table 8).

Growth and morphology of V. radiata x V. angularis
hybrids. As mentioned previously, the reciprocal crosses

of V. radiata x V. angularis did not produce viable seed.

 

The hybrid embryos of V. radiata x V. angularis were very

 

small, but appeared normal. Embryo culture of 20
embryos from 14-18 day old pods produced 15 seedlings

of which only 3 plants reached flowering. The other 12

28

plants died during early seedling stages. Two of the
hybrid plants were cultured from the cross of Tainan #1 x
KS#210, the other one from the cross of (Tainan #1 x
M865) x (KS#210 x Chien Shien).

The plants derived by embryo culture were definitely
hybrid (Table 9, Fig. 2-a,b,c). They were intermediate
between their two parents for petiole length and shape
of primary leaves. However, the hybrids resembled the

V. angularis pollen parent for color of epicotyl, lobing

 

of leaflet margins, and simple compact racemes.

The Tainan #1 x KS#210 hybrid plants grew vigorously
with thicker stems, larger leaves and many branches,
compared to the parental cultivars. However, abnormal
growth occurred after the plants reached the 8 to 10
trifoliate leaf stage. The plants formed crinkled
leaves and gradually ceased vegetative growth. The
hybrids rarely flowered as flower buds aborted prior to
anthesis. Slightly distorted plant growth and development
was noted for the hybrid plant derived from the cross
of (Tainan #1 x M865) x (KS#210 x Chien Shien). This
difference might be accounted for by the parental
heterozygosity of this interspecific hybrid pedigree.

No pod-set was observed on any of these hybrid
plants. The flowers contained small numbers of pollen

grains with 0.6 - 9.8% of stainable pollen grains as

29

 

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Figure 2.

30

Interspecific hybrids of V. radiata x‘V} angplaris
and V. @239. x V. angplaris. Z-a: Hybrid plants

of V} radiata (Tainan #1) x‘V} angplaris (KS#210).
Z-b: Terminal leaflets of V} radiata (Tainan #1,
left), V} angplaris (KS#210, right) and their inter-
specific hybrid (middle). 2-c: Interspecific hybrid
plants of V. radiata x V. angplaris: Tainan #1 x
KS#210 (left) and (Tainan #1 x M865) x (KS#210 x
Chien Shien). 29d: Plants of V} mppgg_(N1208 x T-9,
left), V} angplaris (KS#210 x Chien Shien, right) and
their interspecific hybrid (middle). 2-e: Terminal
and lateral leaflets of V} mppgg (N1208 x T-9, left),
;V. angglaris (KS#210 x Chien Shien, right) and their
interspecific hybrid.(midd1e). 2-f3 Mature plants of
V. M82 (N1208 x T-9, left), V. anglaris (KS#210 x
Chien Shien, right) and their interspecific hybrid
(middle).

31

 

 

 

 

 

 

 

 

 

 

 

 

32

compared to over 90% for the parental species (Table 9).
The sizes of unstained pollen grains were highly
variable. Chromosomal irregularity and a low frequency
of pairing for this species cross have been reported

(Ahn G Hartmann, 1978a).

Crossability of V. mungo and V. umbellata. When

 

V. mppgp was used as the female, significant differences
in pod-set among the different combinations of

parental cultivars were determined (Table 10). Regardless
of the pollen parent cultivar, T-9 set significantly

more pods than NI208. The averages for pod-set on

T-9 and N1208 were 82% and 53%, respectively. There

were no differences between the V. umbellata cultivars

 

when used as the pollen parent. Pod-set was not affected
by the use of F1 parental species for the cross. The
hybrid pods developed normally and reached maturity.
About 43 to 71% of the pods were harvested depending
upon the combinations of parental species used for the
cross. The mature pods contained two kinds of seeds;
viz., small, undeveloped and normal full-sized seeds
which were slightly shrivelled. The slightly shrivelled
seeds appeared normal when imbibed with water, but
failed to germinate with highly distorted cotyledons

and poorly developed embryo axes.

In reciprocal crosses where V. umbellata was used

 

Table 10. Crossability for the interspecific hybridization of

LV. mungo (B) and V. umbellata (R).

 

 

 

 

 

 

 

 

 

 

,
Parentsl No. flowers Pod-set Pod abscision“
Female Male crossed (%) (days)
Bl R1 202 79.7 8.0
B1 R2 165 83.6 8.4
Mean 184 81.5 8.2
B2 R1 166 49.4 7.2
B2 R2 181 56.4 6.6
Mean 174 52.9 6.9
(B2xBl) (R2le) 231 87.9 6.3
R1 Bl 152 48.7 5.5
R1 B2 132 36.4 4.3
Mean 142 43.0 4.9
R2 B1 118 33.1 4.0
R2 B2 124 58.9 4.4
Nkfiul 121 46.3 4.2
(R2le) (B2xBl) 177 45.8 4.9
Comparison % Pod-set Comparison % Pod-set
Within B x R **3 Within R.x B *
between B * between R ns
between R ns between B ns
cultivars vs F 5 ns cultivars vs F15 ns
B x R.vs R x B B x R vs R x B
cultivar x cultivar * F1 x Fl **

 

 

#31 = T-9, BZ = N1208, R1 = HK, R2 = s-91.
2Mean of days to pod abscision after pollination.

3Significant at 5% (*) or 1% (**); ns = not significant by chi-square

test .

34

as the seed parent, the pod-set was significantly
affected by the hybrid combination of parental cultivars
(Table 10). However, there were no significant

differences for pod-set between V. umbellata cultivars,

 

V. mppgp cultivars and intraspecific hybrids as the
parents. Reduced pod growth was followed by abscision
within one week of pollination. Reciprocal differences
for both pod-set and subsequent pod growth was observed
as pod-set was higher and pod retention longer when

V. mungo was used as the seed parent than V. umbellata.

 

Embryo culture of hybrid embryos from the cross of

V. mungo x V. umbellata was attempted. Thirty-eight

 

embryos taken from 15 to 19 day-old pods of (N1208 x T-9)
x (S-91 x HK) were cultured. Normal seedlings were
obtained from 6 embryos, 22 formed callus and/or etiolated
shoots with deformed leaves, and 10 did not grow. The
hybrid embryos appeared normal in shape and size (4 mm
in length). However, all 6 plantlets died during early
seedling stages. Hybrids obtained through embryo culture
have been reported (Ahn 8 Hartmann, 1977; Biswas 8
Dana, 1975). In one case, the plants flowered but
were completely sterile (Biswas 8 Dana, 1975).

Other attempts to make this interspecific cross

have been reported (Chowdhury 8 Chowdhury, 1977). When

 

V. umbellata was used as the female, the pollen tubes

35

did not penetrate the stigma, while in the reciprocal
cross the embryos aborted due to early degeneration

of the endosperm.

Crossability of V. mungo and V. angularis. There

 

was a significant difference in pod-set for the cross

of V. mungo x V. angplaris depending on the cultivar

 

used as the pollen parent. Regardless of the V. mppgg
cultivar, pod-set on Chien Shien and KS#210 was 49 and
73%, respectively (Table 11). The difference in pod-set
between the two V. mppgp cultivars used as seed parent
was not significant. The use of intraspecific hybrids

as parents significantly increased the number of pods
that reached maturity without a significant increase

in pod-set. The mature pods were generally empty as only
28 partially filled and shrivelled seeds were obtained.
Of these seeds, 7 germinated but 6 seedlings died soon
after germination. They were observed to have distorted
cotyledons and poor root development. Only one seedling
survived and flowered. This was the first successful
interspecific hybrid plant obtained from seed. The use
of heterozygous parental species did successfully enhance
the crossability of this species cross. On the other
hand, cultures of 8 embryos (2.5 mm in length) from

14-18 day-old pods produced 3 seedlings which died at

the early seedling stages. The root systems were poorly

36

Table 11. Crossability fOr the interspecific hybridization of
V. mungo (B) and V. angularis (A).

 

 

 

 

 

 

 

 

 

Parents1 No. flowers Pod-set Pods harvested Pod abscision2
Female Mare crossed (%) (%) (days)
B1 A1 118 52.5 35.6 8.8
B1 A2 186 78.5 53.8 9.1

Mean 152 64.4 46.7 9.0
B2 A1 135 45.9 33.3 9
BZ A2 123 64.4 42.3 8.2

Mean 129 54.3 37.6 8.1
(B2xBl) (AZxAl) 164 84.2 63.4 9 3
A1 B1 112 36.6 7
A1 BZ 108 21.3 .8

Mean 110 29 . 1 . 3
A2 B1 124 12.9 4.8
.A2 B2 120 8.3 5.

Mean 122 10.7 4.
(AZxAl) (B2xBl) 138 36.2 0 5.8
Comparison % Pod-set % Pod Comparison % Pod-set

3 harvested
Within B x.A * ns Within A.x B **
between B ns ns between A **
between A * ns between B ns
cultivars vs Fls ns * cultivars vs Fls *
B x.A vs.A x B
cultivar x cultivar ** **
** **
F1 x F1

 

131 = T-9, 132 = N1208, A1 = Chien Shien, A2 = KS#210.

2Mean of days to pod abscision after pollination.
3Significant at 5% (*) or 1% (**); ns = not significant by chi-square
test.

37

developed as was previously reported to be a primary
cause of seedling mortality (Ahn 8 Hartmann, 1977).
Successful hybrid plants might possibly be attained
by either modifying the culture medium, particularly
the sucrose content, as reported by Honma (1955) to
encourage better growth of the root system or by
grafting onto parental rootstocks as described by
McLean (1946).

When V. angularis was used as the female, there

 

was a highly significant difference in pod-set between

the two cultivars of V. angularis. Pod-set on Chien

 

Shien and KS#210 were 29 and 11%, respectively (Table 11).
The differences in pod-set between the two V. mppgp
cultivars used as male parents was not significant.
Moreover, a significant increase in pod-set was
obtained by using intraspecific hybrids as parents; viz.,
36% compared to 19% for parental cultivars. Unfortunately,
all pods abscised within 10 days after pollination.
Reciprocal differences in crossability were clearly
shown in crosses between these two species. When
cultivars were used as parents for hybridization, pod-set

on V. mungo and V. angularis were 62 and 19%, respectively;

 

whereas pod-set increased to 84 and 36%, respectively,
when intraspecific hybrids were used as parents (Table 11).
Pod abortion was delayed by using V. mungo as the female

parent.

Growth and morphology of V. mungo x V. angularis
hybrid. The single plant obtained from the cross of
(N1208 x T-9) x (KS#210 x Chien Shien) grew slowly
during the early seedling stage, but became increasingly
vigorous as it became more established (Fig. 2-d,f).

An examination of morphological characteristics
strongly suggested that the plant was a species hybrid
(Fig. 2-e). It was intermediate between the two species
parents for germination habit, petiole length and shape
of primary leaves, and growth habit (Table 12). The
hybrid resembled the V. angularis parent in color of
epicotyl, lobing of leaflet margins, and simple compact

racemes .

Crossability of V. umbellata and V. angularis. In

 

the cross of V. umbellata x V. angularis, no significant

 

 

difference for pod-set was found between the two cultivars

of V. umbellata or between the use of cultivars and

 

intraspecific hybrids as female parents (Table 13).
Pod-set was significantly influenced by the cultivar

of V. angularis used as pollen parent. The pollen

 

parent KS#210 produced a higher pod-set (29%) than
Chien Shien (45%). The pods developed slowly and
abscised l to 2 weeks after pollination.

The reciprocal cross using V. angularis as the

 

female showed no differences in pod-set between the

 

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40

Table 13. Crossability for the interspecific hybridization of
X. umbellata (R) and y_. anglaris (A).

 

 

 

 

 

 

 

 

 

Parents1 No. flowers Pod-set Pod abscisionZ Shrivelled seeds
Female Male crossed (%) (days) (no . / attempt)
R1 A1 126 27.8 5.6
R1 A2 118 36.4 9.3

Mean 122 32.0 7.5
R2 Ad 148 31.1 5.0
R2 .AZ 194 53.6 6.6

.Mean 171 43.9 5.8 0
(R2le) (A2xAl) 188 52.1 10.5 0
Al R1 138 65.2 24.3 1.
Al R2 116 74.1 21.7 1.8

iMean 127 69.3 23.0 1.7
A2 R1 166 63.9 22.1 2.6
A2 R2 104 75.0 21.4 3.1

Mean 135 68.2 21.8 2.8
(AaxAl) (RZXRl) 146 84.9 23.5 3.3
nggarison % Pod-set Comparison_ % Pod-set

. . *3 . .

Within R x A Within A x R ns
between R ns between A ns
between A * between R ns
cultivars vs F13 ns cultivars vs Fls ns

R x A.vs A x R R x A.vs A.x R
cultivar x cultivar ** F1 x F1 **

 

1R1 = HK, R2 = 5-91, A1 = Chien Shien, A2 = KS#210.

2Mean of days to pod abscision after pollination.
3Significant at 5% (*) or 1% (**), ns = not significant by chi-square
test.

41

parental cultivars of both species, nor differences
between cultivars compared to heterozygous hybrids as

parents (Table 13). Pods on X. angularis developed

 

-,

normally up to 3 weeks, then aborted.

Pod-sets were significantly higher for K. angularis

 

x X. umbellata than the reciprocal. Crosses using

 

parental cultivars of X. umbellata and y. angularis

 

 

resulted in 39 and 69% pod-set, respectively; whereas,
52 and 85%, respectively, were achieved by using
intraspecific hybrids as parents. No viable seeds were
obtained for this cross. From the culture of 20 embryos
(1.3 mm in length), 3 hybrid plants were obtained. Two

of these hybrid plants were from the cross of X. umbellata

 

x X. angularis and the other from its reciprocal cross.

 

Thus, interspecific hybrids were successfully secured

for the reciprocal crosses of X. umbellata x X. angularis

 

(Fig. 3-f).

Growth and morphology of V. umbellata x V. angularis

 

hybrid. The two plants derived by embryo culture from
the cross of HK x Chien Shien grew normally (Fig. 3-a).
Epicotyl color, shape of primary leaves, seed size and
seed weight of the hybrid plants were intermediate
between the two parental species (Table 14). The hybrid

plants resembled the female parent, X. umbellata (HK), for

 

indeterminate growth habit, elongated racemes and concave

Figure 3.

Interspecific hybrids of X. umbellata x !. anglaris
and the reciprocal hybrid. 3-a: Plants of X.
umbellata (HK, left), )1. angularis (Chien Shien, right),
and their interspecific hybrid (middle). 3-b: Leaves
of X. umbellata (HK, left), X. angularis (Chien Shien,
right) and their interspecific hybrid (middle). 3-c:
Hybrid plants of X. umbellata x X. angularis, showing
fruiting (left) and continuous vegetative growth (right).
3-d: F2 seedlings of X. umbellata x y_. anglaris,
showing the variation of primary leaf shapes and their
parents, X. umbellata (left) and X. angularis (right).
3-e: Terminal leaflets of X. umbellata x E. angularis
F2 plants, showing the variation of leaf shapes with
their parents, X. umbellata (top left two) and K.
angularis (top right two). 3-f: Reciprocal inter-
specific hybrid plant, X. anglaris x X. umbellata.

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

44

 

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hilum cushion. The pollen parent, X. angularis (Chien

 

Shien), character of lobed leaflet was dominant in
the hybrid to the entire leaflet margin of the female,

Z. umbellata (HK). The presence of lobed leaflets

 

in the hybrid plants clearly indicated a hybrid (Fig.
3-b).

The hybrid plants expressed indeterminate growth
by producing juvenile shoots even after flowering
(Fig. 3-c). They flowered profusely and produced
viable seeds, though the fertility was less than the
parental species (Fig. 3-c). The pollen stainability
of the hybrids was 77% compared to 95 and 93% for HK
and Chien Shien, respectively (Table 14). The length
of pod and number of seeds per pod for the F1 plants
was less than the parental species due to the low fertility.

Ahn and Hartmann (1978) reported that the hybrid

of X. umbellata x X. angularis had regular meiosis

 

with 11 bivalents and a high degree of chromosome
homology. In the current study, F2 seeds from F1 plants
germinated normally. From 60 F2 plants, a continuous
variation was observed for epicotyl color and shape

of primary leaves (Fig. 3-d,e). Combination of parental
types was observed; e.g., the purple epicotyl of X.

umbellata with the cordate primary leaves of K. angularis

 

 

and vice versa were found in the population (Fig. 3-d).

46

The reciprocal crossability of this hybrid and the
relatively high fertility of the F1 and F2 generations
indicated that the only isolating barrier to this
species cross was embryo abortion which can be overcome

by embryo culture. Accordingly, K. umbellata and K.

 

angularis are probably closely related and genetic

exchange between them may be feasible.

GENERAL DISCUSSION AND CONCLUSIONS

Stebbins (1958) reviewed several mechanisms
responsible for the isolation of species including
hybrid inviability, lack of vigor and sterility.

These general mechanisms might operate between the

four species of Xigna in this study. Five interspecific
hybrids were successfully made from the six possible
crosses of four species. In general, pod-set occurred
in all of the interspecific crosses. Embryo abortion

took place in the crosses of X. radiata x X. angularis,

 

E. mungo x X. umbellata and K. umbellata x K. angularis.

 

 

 

The remaining interspecific crosses produced viable hybrid
seeds with varying degrees of success.

The degree in crossability based on the use of two
cultivars per species, and for cultivars versus
intraspecific hybrid parents, suggested that parental
genotypes and heterozygous parental genotypes might
be important factors in successful interspecific crosses.
Growth and development of hybrid seedlings was also
influenced by the parental genotypes of both parents.

As a result, the lethality and vigor of hybrid seedlings
could be improved and recovered by judicious selection
of parental genotypes. In this study, the interspecific

hybrid of X. mungo x X. angularis and the reciprocal hybrid,

 

X. umbellata x X. angularis, were produced through the use

 

 

47

48

of proper parental genotypes.

Reciprocal difference in crossability for pod-set,
embryo abortion and viable seed were found for all six
interspecific crosses. Except for the cross between

X. umbellata and X. angularis, no other reciprocal

 

 

interspecific crosses were successful. The unilateral
success of most crosses suggested that cytoplasmic
factors may be involved in the isolating mechanisms
between these species (Ahn G Hartmann, 1977).

In the present study, viable hybrid seeds were only
obtained for the interspecific crosses of X. radiata x

X. mungo, X. radiata x X. umbellata and Z. mungo x X.

 

angularis. Hybrid seedlings of these species crosses
can also be obtained through embryo culture. Hybrid
seedlings obtained through embryo culture grew normally

and reached flowering for the following F crosses:

l
X. radiata x X. angularis, K. umbellata x X. angularis

 

 

 

and X. angularis x K. umbellata.

 

 

The hybrid plants of X. radiata x X. mungo and

X. umbellata x X. angularis were partially fertile,

 

 

while the hybrids of X. radiata x K. umbellata, X. radiata

 

x X. angularis and K. mungo x X. angularis were sterile.

 

 

Cytological studies have shown poor chromosome pairing
probably due to lack of homology (Sawa, 1973; Biswas 6
Dana, 1975; Ahn & Hartmann, 1977; Machado et al.,

unpublished).

49

Ahn and Hartmann (1977) postulated the evolutionary
relationships of these four species based on crossability

and divided them into two subgroups; viz., X. radiata with

 

 

K. mungg and X. umbellata with K. angularis. Electro-
phoretic survey of isozyme patterns of these four Xigna
species made no suggested genetic relationship between
these four species due to the large number of variant
bands (Bassiri 8 Adams, 1978). Based on the cultivars
used in this study, the degree of crossability for these
four Xigna species might be divided into four groups.
The cross of X. radiata x X. mungg yielded most Fl seeds
which suggested these two species are closely related.

Few F1 seeds of X. radiata x X. umbellata and X. mungo

 

x K. angularis were obtained which suggested these two

 

pair of species are slightly related. Interspecific Fl

plants of X. radiata x v. angularis and X. umbellata x

 

 

K. angularis could only be obtained by embryo culture

indicating these two pairs of species are remotely related.

 

Neither seeds nor embryos of X. mungo x y. umbellata were
obtained as these two species are probably more

distantly related.

REFERENCES

Ahn, C.S. and R.W. Hartmann. 1977. Interspecific
hybridization among four species of the genus
Vi na. In: Proc. First Intl. Mungbean Symposium,
A51an Vegetable Research and Development Center,
Shanhua, Taiwan. p. 240-246.

Ahn, C.S. and R.W. Hartmann. 1978a. Interspecific
hybridization between mungbean (Vigna radiata (L.
Wilczek) and adzuki bean (Vigna angularis (Willd.
Ohwi and Ohashi). J. Amer. Soc. Hort. Sci. 103:

 

 

MVk—J

Ahn, C.S. and R.W. Hartmann. 1978b. Interspecific
hybridization between rice bean (Vi na umbellata
(Thunb.) Ohwi and Ohashi) and adzu 1 ean (V.
angularis (Willd.) Ohwi and Ohashi). J. AmEr. Soc.
Hort. Sci. 103: 435-438.

 

 

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

 

Bassiri, A. and M.W. Adams. 1978. An electrophoretic
survey of seedling isozymes in several Phaseolus
species. Euphytica 27: 447-459.

 

Biswas, M.R. and S. Dana. 1975. Black gram x rice
bean cross. Cytologia 40: 787-795.

Boling, M., D.A. Sander and R.S. Matlock. 1961.
Mungbean hybridization techniques. Agron. J. 53:
54-55.

Chen, N.C., J.F. Parrot, T. Jacobs, L.R. Baker and
P.S. Carlson. 1977. Interspecific hybridization
of food legumes by unconventional methods of plant
breeding. In: Proc. First Intl. Mungbean Symposium.
Asian Vegetable Research and Development Center,
Shanhua, Taiwan. p. 247-252.

Chowdhury, R.K. and J.B. Chowdhury. 1977. Intergeneric
hybridization between Vigna mungg (L.) Hepper and
Phaseolus calcaratus Roxb. Indian J. Agric. Sci.

' - 21.

 

 

Dana, S. 1966. The cross between Phaseolus aureus

Roxb. and E. mungo L. Genetica. 37: 259-2 '.

50

51

Dana, S. 1967. Hybrid from the cross Phaseolus
aureus Roxb. x P. calcaratus Roxb. J: Cytology and

Gene. 2: 92-97.‘

De, D.N. and R. Krishnan. 1966. Cytological studies
of the hybrid Phaseolus aureus x P. mungo.
Genetica 37: 588-600.

 

 

 

Honma, S. 1955. A technique for artificial culturing
of bean embryos. Proc. Amer. Soc. Hort. Sci. 65:
405-408.

Honma, S. 1956. A bean interspecific hybrid. J.
Hered. 47: 217-220.

Honma, S. and O. Heeckt. 1958. Bean interspecific
hybrid involving Phaseolus coccineus x P. lunatus.
Proc. Amer. Soc. Hort. SET. 72: 360-364.

 

Honma, S. and 0. Heeckt. 1959. Interspecific hybrid
between Phaseolus vulgaris and P. lunatus. J. Hered.
50: 233-237.

 

Johansen, D.A. 1940. Plant Microtechnique. McGraw-
Hill Book Co., Inc., New York.

Linsmaier, E.M. and F. Skoog. 1965. Organic growth
factor requirements of tobacco tissue cultures.
Physiol. Plant. 18: 100-127.

McLean, S.W. 1946. Interspecific crosses involving
Datura ceratocaula obtained by embryo dissection.
Amer. J. Bot. 33: 630-638.

 

Mok, D.W.S., M.C. NOR and A. Rabakoarihanta. 1978.
Interspecific hybridization of Phaseolus vulgaris
with P. lunatus and P. acutifolius. Theor. Appl.

GenetT 52: 209-215. -

 

 

Rachie, K.0. and L.M. Roberts. 1974. Grain legumes
of the lowland tropics. Adv. Agron. 26: 1-132.

Sanchez-Monge, E. and F. Garcia-Olmedo (ed.) 1977.
Interspecific hybridization in plant breeding. Proc.
8th EUCARPIA Congress, Madrid, Spain.

52

Sawa, M. 1973. On the interspecific hybridization
between the adzuki bean, Phaseolus angularis (Willd.)
W.F. Wight and the green gram, Phaseolus radiatus L.
I. Crossing between a cultivar of the green gram and
a semi wild relative of adzuki bean, in endemic name
"Bakaso". Japan. J. Breed. 23: 61-66.

 

 

Stebbins, G.L. 1958. The inviability, weakness, and
sterility of interspecific hybrids. Adv. Genet.
9: 147-215.

Taira, T., T. Lelley and E.N. Larter. 1978. Influence
of parental rye on the development of embryos and
endosperm of wheat-rye hybrids. Can. J. Bot. 56:
386-390.

Verdicourt, B. 1970. Studies in the Leguminosae-
Papilionoideae for "Flora of Tropical East Africa."
IV. Kew Bul. 24: 507-569.

SECTION II

EFFECT OF E-AMINO-N-CAPROIC ACID ON INTERSPECIFIC
HYBRIDIZATION IN THE GENUS VIGNA

ABSTRACT

The possibility of using chemicals to circumvent
crossability barriers in interspecific hybridization was
studied. Three chemicals, E-amino-n-caproic acid (EACA),
gentisic acid and L-lysine were tested for their effect
on interspecific crosses in the genus Vigna. The EACA
as a foliar spray was the most effective chemical studied.
Daily foliar applications of EACA to the plants of the

maternal parent, Vigna radiata (mungbean) for 14 days

 

 

before cross-pollination with V. umbellata (rice bean)
significantly increased the numbers of viable hybrid
seeds. Concentrations of EACA less than 100 mg/l appeared
ineffective, whereas concentrations over 2500 mg/l were
phytotoxic. Concentrations of 100 to 1000 mg/l were the
most effective. The overall numbers of viable seeds
obtained were increased from 2- to 9-fold by EACA treat-
ment depending upon the experiment number. Different
cultivars of maternal Vigna species responded differently
to EACA concentrations. Additionally, 1000 mg/l EACA
applied as a post-pollination treatment increased numbers
of viable seed similar to pre-pollination treatments.
Treatment by either EACA or gentisic acid significantly
enhanced the deve10pment of normal hybrid seeds produced

for the V. radiata x V. umbellata cross. Applications

 

of equal concentration EACA and L-lysine were as effective

53

54

as EACA applied alone for producing F1 seeds of this
interspecific cross. Appropriate chemical treatments
increased the frequencies of normally appeared hybrid
embryos by 15 to 22% relative to the control. The success
with which the embryos could be cultured in 13333 was
improved accordingly. The proposed use of EACA for
interspecific hybridization involving selected cultivars

of V. radiata and V. umbellata reduced crossability

 

barriers and greatly facilitated the movement of germ-

plasm between these two Vigna species.

INTRODUCTION

Interspecific hybridization has played an important
role in the evolution of flowering plants in nature (5).
In plant breeding, interspecific hybridization has been
used when a desired character can not be identified in
the gene pool of a given species, but is present in a
second species. The Leguminosae species are endowed with
a richness of genetic diversity unparalleled by other
economic plants; e.g., N-fixation, protein quality and
quantity, oil content. The relatively narrow gene base
(17, 19, 20) of some individual species, however, has
limited varietal improvement. The use of interspecific
hybridization significantly enlarges the genetic base
of individual food legume species.

Interspecific hybridization of Vigna food legumes

was recently studied at the Asian Vegetable Research

 

and Development Center (AVRDC). The V. umbellata (rice

bean) is highly resistant to bean fly (Melanagromyze

 

phaseoli), Cercospora leaf spot and other diseases, but

 

of little agronomic value as a cultivated species (2, 3).
The nature of reproductive barriers between the major
Asian food legumes in the genus Vigna has been studied

(1, 14, 15, 16). Interspecific crosses were made with
difficulty. The use of large numbers of pollinations have

yielded extremely low numbers of interspecific hybrid

55

56

plants. These occasional interspecific hybrid plants
are nearly always sterile (l, 3, 13).

Bates and Deyoe (6) theorized that crossability
barriers evolved during speciation are mediated through
a specific inhibition reaction (SIR) similar to immune
responses in animals. The SIR may effect fertilization
and development of hybrid embryos in wide crosses.
Animal-effective immunosuppressants were applied to various
cereal species to test the hypothetical model of inter-
specific and intergeneric crossability barriers (6, 7).
An immunosuppressant, E-amino-n-caproic acid (EACA), did
overcome crossability barriers in interspecific and inter-
generic crosses of cereals (8, 9, 10, 21). Application
of EACA by either injecting the solution into the leaf
sheath or by foliar sprays increased embryo formation
and seed set in several intergeneric crosses of cereals.
In food legumes, injection of EACA was less effective,
while foliar sprays with aqueous solutions of EACA to the
maternal parent significantly increased viable hybrid seed

set of the interspecific cross of V. radiata x V. umbellata

 

(4, 13).

The present study was undertaken to investigate the
effects of EACA on the crossability of Vigna species to
create new genetic variation for plant breeding programs

for food legume improvement.

MATERIALS AND METHODS

Plant material. A series of experiments was conducted

 

using Vigna radiata (mungbean) as the maternal parent and

 

V. umbellata (rice bean) as the pollen parent. Two

 

cultivars of mungbean, Tainan #1 and PHLV #18, and one
cultivar of rice bean, HK, were used. These were obtained
from the Asian Vegetable Research and Development Center
(AVRDC) as AVRDC accession nos. 2013, 2184 and 4006,
respectively. Two other interspecific crosses, V. mungg

(black gram) x V. radiata and V. umbellata x V. angularis

 

 

(adzuki bean) were attempted to test their response to
EACA treatments. The cultivars used were black gram,
"T-9", and adzuki bean, "Chien Shien", which were AVRDC

accession nos. 3115 and 5124, respectively.

Cultural conditions. All plants were grown in the

 

greenhouse. During winter, temperatures were adjusted to
26°C (day) and 18°C (night) with a photoperiod of 14 h light
supplied with high intensity metal-halide lamps (10-12 klx).
Plants used as maternal parents were grown in 20 cm pots
containing either a mixture of soil, sand and peat (3:1:1)
or a “Peat Lite Mix". Plants were fertilized with 3 g/pot
of "Peter's" fertilizer (20N-20P-20K) biweekly. Two plants

were maintained in each pot.

Chemical treatments. The chemicals, E-amino-n-caproic

 

acid (EACA), gentisic acid and L-lysine HCl were tested and

S7

58

compared for their success on the interspecific crosses

of Vigna. The chemical treatments were made as aqueous
solutions applied to the maternal plant as foliar sprays

to run-off. Spray treatments were initiated prior to the
meiotic stage of floral development. In V. radiata,
meiosis occurs when the floral envelope of the bud is 2-3 mm
in size. Daily treatments of EACA were initiated at this
pre-meiotic stage and continued over a l4-day period before
cross-pollination. The surfactant, "Tween 20", was used in
all treatments. For post-pollination treatments, EACA was
applied daily for 14 consecutive days after cross-pollina-
tion. A "cotton wrap" method was also tested and compared
to foliar spray as a post-pollination treatment. In this
method, the pedicels of cross-pollinated (hybrid) pods were
wrapped with cotton into which an EACA solution was slowly

dripped once a day continuously for 14 days.

Crossing techniques. Flowers for hybridization were

 

emasculated the day before anthesis and immediately
pollinated. The hybridization technique described by
Buishand (12) and Boling et al. (11) was used. Plants
were pollinated for five to seven consecutive days after
termination of chemical treatments. Approximately equal
numbers of pollination attempts were made for each treat-
ment. After cross-pollinating for 5-7 days, all subsequent

flowers were removed to eliminate competition between hybrid

59

and naturally self-pollinated pods.

Experimental design and data analysis. Randomized

 

complete block and split-plot designs were used. The

pods were harvested individually and the number of shrivel-
led and "normal” seeds noted. Seeds were then germinated
to confirm their hybridity.

Two experiments were conducted to evaluate the chemical
effects on the development of the hybrid embryos. Fertilized
ovules were collected 12 or 14 days after pollination and
the excised embryos were examined using a dissecting
microsc0pe. The development of embryos was classified as
either normal or abnormal based upon visual examination of
their gross morphological appearance and apparent degree of
differentiation.

Analyses of variance were performed on all data after
using either square root (/x77—§) or arcsin transformations.
Duncan's multiple range test was used to compare treatment

means .

RESULTS AND DISCUSSION

Effect of EACA on V. radiata-V. umbellata hybrid cross.
Foliar sprays with a log10 concentration series of EACA
from 0 to 10000 mg/l were tested. A large number of viable
hybrid seeds was obtained (Table 1). In Experiment 1, the
treatment of 100 mg/l EACA was superior to all other treat-
ments including the control except for 1000 mg/l. The
efficiency of viable hybrid seeds produced was 50.1 seeds
per 100 attempts compared to 17.7 seeds for the control.
Treatment with the highest concentration of EACA (10000
mg/l) was toxic to the plants.

In Experiment 2, three concentrations of EACA (100,
500 and 1000 mg/l) were tested. The treatment of 1000 mg/l
EACA significantly increased the number of viable seed as
compared to the control (Table l). The numbers of viable
seed per 100 attempts for the treatment and control were
13.6 and 2.3, respectively. Differences among EACA treat-
ments, however, were not significant. The number of viable
seed obtained coincided with the percent pods that produced
viable seed; i.e., the higher the percentage of pods with
viable seeds the greater the number of viable seeds observed
in both experiments. Differences between treatments for
percent pods with viable seeds were not significant in the
second experiment.

In subsequent experiments, attempts to identify the

optimum concentration were made with no one consistent

60

61

Table 1. Effect of EACA treatments on the success of the
interspecific cross of V. radiata x V, umbellata.

 

 

 

 

 

EACA No. flowers Total viable No. of viable Pods with
treatment crossed seeds seeds per 100 viable seeds
(mg/l) obtained attempts (%)
Experiment 12
o 137 25 17. 7by ll.5ab
10 159 19 12.0b 8.9bc
100 142 69 50.1a 24.6a
1,000 124 24 22.3ab 15.7ab
10,000 76 0 O b 0 c
Experiment 2X
0 88 2 2.3b 2.3“5
100 86 6 6.9ab 6.9
500 93 8 8.5ab 7.4
1,000 96 13 13.6a 10.3

 

2Data represent the total number and the mean of 6 replications.

yMean separation in columns within experiment by Duncan's multiple

range test, 5% level.

xData represent the total number and the mean of 4 replications.

ns= Net significant.

62

concentration as the "best" EACA treatment. Concentrations
of EACA less than 100 mg/l seemed ineffective, whereas
concentrations over 2500 mg/l appeared phytotoxic.
Concentrations in the range of 100 to 1000 mg/l seemed most
effective (Table 1). In general, high concentrations of
EACA (over 2500 mg/l) caused chlorosis and abnormal floral
morphogenesis on the treated plants. Morphogenetic
anomalies induced by EACA treatments have been reported

for Dolichos lab-lab (18).

 

The total number of viable seeds obtained and viable
seeds per 100 attempts are higher in Experiment 1 than
Experiment 2 for all corresponding treatments including
controls (Table l). The relative difference in degree of
response between the two experiments and subsequent
experiments was probably due to differences in environmental
conditions. The application of EACA to the maternal parent
significantly improved the deve10pment of embryos from
wheat-rye crosses, but was not independent of the temperature
regime (22) suggesting that in the present Study, depending
upon environmental conditions, the magnitude of response
could vary from one experiment to another.

To determine the effect of plant growth stage on
chemical treatment, an experiment was designed using various
durations of pre- and post-pollination EACA treatments.
Plants of the maternal parent, E. radiata, were subjected

to daily treatments of 100, 500 and 1000 mg/l EACA at various

63

growth stages. No significant differences were observed
for the number of viable seed produced by EACA treatments

made at different stages of plant development.

Post-pollination EACA effects on V. radiata-V. umbellata

 

hybrid cross. The possibility of post-pollination treat-
ments with EACA to reduce hybrid embryo abortion or break-
down was explored. Plants of the maternal parent, E. radiata,
were sprayed daily with 100 and 1000 mg/l EACA for 14 days
after hybridization. Treatments with EACA increased 2- to
4 times the number of viable hybrid seeds as compared to
the control (Table 2). A concentration of 1000 mg/l EACA
was Optimal for post-pollination treatment. At this
concentration, numbers of viable seeds were significantly
higher than that from untreated plants. As mentioned
previously, foliar application of EACA in concentration
greater than 2500 mg/l were phytotoxic. An experiment

to test cotton wraps for post-pollination EACA treatments
was conducted to possibly reduce phytotoxic effects. Two
concentrations of EACA (2500 and 5000 mg/l) were used to
compare with the control and the ”optimal" 1000 mg/l EACA
treatment. An increase in numbers of viable hybrids seed
were obtained using the "cotton wrap" method (Table 2). No
phytotoxic effect was observed from these treatments with
relatively high concentration of EACA. Although EACA

treatments significantly increased the number of viable

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64

Table 2. Effect of EACA applied as post-pollination treatments
on the success of the interspecific cross of y. radiata

x K. umbellata.

 

 

 

 

 

EACA Total viable No. of viable Pods with
treatment seeds seeds per 100 viable seeds
(mg/l) obtained attempts (%)
Foliar spray experimentZ
0 21 32.8by 23.5b
100 48 75.0b 31.3b
1,000 99 154.7a 42.2a
Cotton wrap experimentX
0 24 30.8c 22.7ns
1,000 70 87.8a 32.2
2,500 43 53.5abc 35.9
5,000 67 83.3ab 38.7

 

2Data represent the mean of 4 replications. A total of 64 pollination
attempts was made in each treatment.

yMean separation in columns within experiment by Duncan's multiple

range test, 5% level.

XData represent the mean of 4 replications. A total of 80 pollination
attempts was made in each treatment.

ns=Not significant;

65

seeds obtained, differences among treatments were not
significant suggesting that the treatment with 1000 mg/l
was equal or superior to other treatments.

To define more clearly the optimum duration for EACA
application, an experiment was conducted using 1000 mg/l
EACA applied daily as a foliar spray for 7 and 14 consecutive
days as a pre-pollination or post-pollination treatment. The
EACA applied daily either for 14 days before cross-pollina-
tion cn‘ for 7 days after cross-pollination significantly
increased the number of viable seeds relative to the control
(Table 3). There were no significant differences in effects
among EACA treatments. The relatively low number of viable
seeds obtained in all treatments, including the control,
was probably due to the high temperatures encountered

during this experiment.

Cultivar response to EACA treatment. Two cultivars

 

of Z. radiata were used to determine their crossability

with X. umbellata and their responses to EACA treatments.

 

Plants of the two cultivars, Tainan #1 and PHLV #18, were
treated with a series of EACA concentrations (0 to 4000
mg/l) for 14 days before anthesis, and then crossed with

the same cultivar (HK) of X. umbellata. Crossability was

 

higher for Tainan #1 which yielded more viable hybrid seeds
than PHLV #18 regardless of the chemical treatment (Table 4).
These data suggest that cultivar may play an important role

in the success of interspecific hybridization.

66

Table 3. Effect of duration of treatment with 1,000 mg/l EACA on
viable seed set of the interspecific cross of y. radiata

x K. umbellata.z

 

 

 

 

 

EACA Total viable No. of viable Pods with
treatment seeds seeds per 100 viable seeds
obtained attempts (%)
Pre-pollination
7 days 6.9aby 5.6
14 days 9 12.5a 9.7
Postspollination
7 days 7 9.7a 9.7
14 days 5 6.9ab 6.9
Control 1 1.4b 1.4

 

ZData represent the mean of 3 replications. A total of 72 pollination
attempts was made in each treatment.

yMean separation in column by Duncan's multiple range test, 5% level.

(n

H

[1 1

Ov-

“H

-18

67

Table 4. Response of two varieties of V. radiata to EACA treatments
by interspecific hybridization With V. umbellata.Z

 

 

 

 

 

 

variety
EACA Tainan #1 PHLV #18 Mean viable
treatment Total Viable seeds Total Viable seeds seeds/100
viable per 100 viable per 100
(mg/1) seeds attempts seeds attempts attempts
o 34 40.5cy 22 26.2c 33.4d
100 133 158.33 43 51.2b 104.7a
500 46 54.8b 63 75.0a 64.9b
1,000 52 61.9b 46 51.2b 56.5c
4,000 43 51.2bc 19 22.6c 36.9d
Mean 62 73.3 39 45.2

 

2Data represent the mean of 6 replications. A total of 84 pollination
attempts was made in each treatment.

‘nMean separation in columns by Duncan's multiple range test, 5% level.

68

Treatments with EACA were effective on both mungbean
cultivars as the number of viable seeds was increased
(Table 4). However, the cultivars responded differently
to the EACA concentrations. The interaction between cultivar
and EACA treatment was highly significant @£=.01). The
highest number of viable seed was exhibited by the treatment
of 100 mg/l EACA in "Tainan #1", while the highest number
of viable seed in "PHLV #18" was produced from the treatment
with 500 mg/l EACA. The effective concentrations of EACA

for the interspecific cross of V. radiata x V. umbellata

 

ranged from 100 mg/l to 1000 mg/l.
In an intergeneric cross of cereals, involving 10
cultivars, EACA generally enhanced total seed set of

Triticum turgidum var. durum x Secale cereale by 45%,

 

 

although the crossability of the 10 cultivars differed
while certain cultivars were unaffected by EACA treatment
(10). Such interactions between maternal cultivars and
EACA treatments confound the development of a specific
methodology for using EACA in interspecific and inter-

generic hybridization.

Effects of EACA on other interspecific crosses. Inter-
specific hybridization of V. mungo x V. radiata has not been

reported in the literature. The cross of V. umbellata x

 

V. angularis was accomplished by culturing immature embryos

 

in vitro (1). The possibility of successfully making these

two interspecific crosses using EACA treatments was explored.

69

Plants of the maternal parent, V. mungg, were treated
daily with 100, 1000 and 2000 mg/l EACA for 14 days; then
cross-pollinated with V. radiata for 5 days. No viable
hybrid seeds were produced by any of the treatments
including the untreated plants. Treatments with 1000 and

2000 mg/l EACA induced phytotoxicity and premature pod

abortion. For the interspecific cross of V. umbellata

 

x V. angularis, maternal plants were subjected to daily

 

treatments with the same concentrations of EACA described
in the cross of V. mungo x V. radiata with the same results,

viz., no viable hybrid seeds were obtained.

Effects of other chemicals on V. radiata-V. umbellata

 

hybrid cross. Several chemicals including EACA, acriflavin,

 

gentisic acid, salicylic acid and chloramphenicol have been
used to enhance the crossability of interspecific and inter-
generic hybridization in cereals with varying degrees of
success (7, 8). Gentisic acid was tested to compare its
efficiency to EACA. An experiment was designed using three
concentrations (10, 100 and 1000 mg/l) of each chemical.
Maternal plants, V. radiata, were sprayed daily with
appropriate concentration of either EACA or gentisic acid

for 14 days; then cross-pollinated with V. umbellata.

 

Both chemicals were effective in increasing the numbers
of viable seeds obtained for this interspecific hybrid

cross (Table 5). The EACA was a more effective promoter of

70

Table 5. Relative effect of EACA and gentisic acid on the success
of the interspecific cross of V. radiata x‘V} umbellata.

 

 

 

Treatment Total viable No. of viable Pods with
Chemical (mg/1) seeds seeds per 100 viable seed
obtained attempts (%)
EACA 10 13 20.3ch 9.4c
100 14 21.9bc 14.1abc
1,000 36 56.3a 25.0a
khan 21 32.8 16.2
Gentisic
acid 10 ll ' 17.2bc 7.8c
100 8 12.6bc 11.0bc
1,000 20 31.3b 23.5ab
Mean 13 20.4 14.1
Control 4 6.3c 6.3c

 

2Data represent the mean of 4 replications. A total of 64 pollination
attempts was made in each treatment.

yMean separation in columns by Duncan's multiple range test, 5% level.

71

hybrid seed production than gentisic acid. Treatments
with 1000 mg/l of either EACA or gentisic acid produced
totals of 36 and 20 viable seeds or 56 and 31 viable seeds
per 100 attempts, respectively. The untreated plants,
however, yielded only 4 viable seeds or 6 viable seeds per
100 attempts. Obviously, the use of these chemicals to
accomplish the interspecific cross of V. radiata x

V. umbellata is advantageous as indicated by 5- to 9-fold

 

increases in seed numbers compared to the control (Table 5).
An analogue of EACA, L-lysine, was also compared to
EACA for overcoming crossability barriers in the V. radiata

x V. umbellata cross. Two concentrations (100 and 1000 mg/l)

 

of each chemical and two combinations in equal concentra-
tion of these two chemicals were compared with the control.
Chemical treatments were applied daily as pre-pollination
treatments described previously. Treatments with 100 and
1000 mg/l of either EACA or L-lysine resulted in increased
numbers of viable hybrid seeds (Table 6). However, the
differences between L-lysine treatments and the control were
not significant. Treatment of the maternal plants by a
combination of equal concentration of the two chemicals

(50 mg/l + 50 mg/l) was superior to other treatments in
terms of number of viable seeds produced, but it was not
significantly different from that obtained with either

chemical applied separately.

Chemical effects on V. radiata-V. umbellata hybrid

embryo development. In the previous studies, the frequency

 

72

Table 6. Relative effect of EACA and L-lysine on the success of
the interspecific cross of V. radiata x V} umbellata.Z

 

 

 

Treatment Total viable No. of viable Pods with
(mg/1) seeds seeds per 100 viable seeds
obtained ‘ attempts (%)
Control 2 2. 3c>' 2. 3c
EACA
100 8 9.labc 9.lab
1,000 11 12.5ab 11.4ab
L-lysine
100 S 5.7bc 5.7bc
1,000 9 10.3abc 9.lab
EACA + L-lysine
50 + 50 14 15.9a 14.8a
500 + 500 4 4.7bc 4.6bc

 

ZData represent the mean of 4 replications. A total of 88 pollination

attempts was made in each treatment.

yMean separation in columns by Duncan's multiple range test, 5% level.

73

of fertilization in V. radiata, using V.-umbellata as the

 

pollen parent, was relatively high as indicated by a high
percentage of pod-set. However, most embryos aborted
within 10-14 days resulting in empty pods at maturity.
Extremely low numbers of embryos developed to mature seeds
(14). An experiment was conducted using EACA and L-lysine
to determine their effects on interspecific hybrid embryo
development in yiyg. Plants of the maternal parent were
subjected to treatments with either various concentrations
of single chemicals or combinations of two chemicals for

14 days prior to hybridization and post-pollination
treatments for 7 consecutive days following cross-pollina-
tion. Hybrid ovules were sampled at 14 days after pollina-
tion. Excised embryos were examined and classified as
either normal or abnormal based on their size and morphologi-
cal appearances.

The development of young hybrid pods and seeds measured
at 14 days after pollination were not affected by the
chemical treatments (Table 7). Treatments with 100 and 1000
mg/l EACA, 100 mg/l lysine, and a combination of 500 mg/l
EACA plus 500 mg/l lysine significantly increased the
frequencies of normal embryos Gx=.05) relative to non-
treatments. Generally, embryo abortion occurred during
all stages of embryogenesis; however, the critical stage
of embryo abortion or breakdown was observed to occur

10 to 14 days after pollination. The low frequencies

74

Table 7. Effects of EACA and L-lysine on development of l4-day-old
pods, seeds and embryos produced from the cross of
V. radiata x V. umbellata.Z

 

 

 

 

Chemdcal Pod size Length Fertilized NOrmal

treatment of ovules embryos

(mg/l) Length Width seed examined (%)

(m) (nun) (mu) (no.)
Control 65.0 5.5ch 4.0 147 0.7d
EACA _

100 65.0 5.7a 4.0 131 9.3ab
1,000 58.1 5.6ab 4.1 115 7.0bc
L-lysine

. 100 64.5 5.4bc 4.3 134 8.4ab
1,000 63.9 5.3c 4.2 144 2.9d
EACA + L-lysine

50 + 50 68.7 5.4bc 4.2 155 3.3cd
500 + 500 66.2 5.4bc 4.2 144 13.1a

 

ZData represent the mean of 15 samples.

yMean separation in columns by Duncan’s multiple range test, 5% level.

75

of normal-appeared embryos observed in all treatments

were probably attributed to a delay in collecting the
hybrid ovules. Therefore, the experiment was repeated

and expanded by adding the treatments with gentisic acid.
Hybrid ovules were sampled at 12.days instead of 14 days
after pollination. The frequencies of normal—appeared
embryos in all treatments including control were correspond-
ingly higher in this latter experiment relative to those
shown in Table 7 (Table 8). The EACA concentrations of
both 100 mg/l and 1000 mg/l resulted in significantly
increased frequencies of normal-appeared embryos @K=.05).
Differences between treatments of lysine and control,
however, were not significant. The application of EACA
(500 mg/l) in combination with an equal concentration of
lysine (500 mg/l) was as effective as EACA applied alone.
The frequency of "normal" embryos produced as a result

of EACA + lysine treatments was significantly increased

as compared with that obtained from untreated, but did not
differ significantly from that of EACA applied alone. On
the other hand, treatment with 1000 mg/l gentisic acid was
found to be effective and similar to EACA treatments. It
increased the frequency of "normal" embryos by 13.8%
(Table 8). Differences between treatments with gentisic
acid and EACA, however, were not significant. The combina-
tion of gentisic acid with lysine had no effect on normal

embryo development relative to the control.

76

Table 8. Effects of EACA, gentisic acid and L-lysine on development
of lZ-day-old V} radiata x‘V. umbellata embryos.z

 

 

 

Chemical Concn No . fertilized Normal embryos
treatment (mg/1) ovules examined (%)
Control 0 217 13.2c1y
EACA 100 216 35.2a
1,000 198 32.8ab
Gentisic acid 100 221 19.6cd
1,000 208 27.0abc
L-lysine 100 223 20.1cd
1,000 217 21.5de
EACA + L-lysine 50 + 50 214 20.9cd
500 + 500 204 28.63bc
Gentisic acid + L-lysine 50 + 50 193 22.2bcd
500 + 500 223 13.9d

 

2Mean of 4 replications.

yMean separation in column by Duncan's multiple range test, 5% level.

77

In general, EACA either applied alone or combined
with lysine enhanced normal embryo development by 15 to
22% (Table 8). Both chemicals consist of six-carbon-chain
skeletons carrying an amino group at the epsilon position.
Therefore, it was presumed that both chemicals might behave
similarly in promoting embryo development. Both chemicals
were reported to improve significantly the formation of
embryos in a wheat-rye cross (21) which agrees with the
present study. Such chemical treatments are promising
for plant breeding programs particularly for the inter-

specific cross of V. radiata x V. umbellata.

 

Suggested methodology. The chemical EACA, was the

 

most effective for increasing both the total numbers of
viable seeds and "normal" embryos formed among the
chemicals tested. The use of EACA provides a valuable
technique to circumvent certain crossability barriers
to facilitate the movement of germplasm between distantly
related species.

The use of EACA for interspecific hybridization of

V. radiata and V. umbellata is suggested as follows:

 

(a) Initiate daily treatments of maternal parent at pre-
meiotic (2 mm bud size) or desired stage of bud develop-
ment, approximately 14 days before anthesis; use 100 to
1000 mg/l EACA as an aqueous foliar spray to run-off. (b)
Emasculate flowers the day before anthesis and cross-

pollinate either immediately or the subsequent day; daily

78

pollination of additional flowers should be continued

for 3 or more days. (c) Continue daily spray for 7 days
after cross-pollination. (d) Remove all self-pollinated
flowers to prevent competition. (e) Harvest pods either
at immature stage (10 to 12 days after pollination) for
embryo culture or at maturity (20 to 22 days). (f) Verify
hybridity of hybrid seedlings by genetic markers and/or

cytogenetic analysis.

10.

ll.

12.

LITERATURE CITED

Ahn, C.S. and R.W. Hartmann.

1977. Interspecific

hybridization among four species of the genus
In: Proc. First International Mungbean

Vi na. __
Symposium. Asian Vegetable

ment Center, Shanhua, Taiwan,

Asian Vegetable Research and
Annual Report for 72-73.

Asian Vegetable Research and
Annual Report for 1974.

Baker, L.R., N.C. Chen and H.

of an immunosuppressant on
of the genus Vigna.

Barber, H.N. 1970.
plants. Taxon. l9:

Bates, L.S. and C.W. Deyoe.
and cereal improvement.

Bates. L.S., A. Campos V., R.

Anderson.
grains.

1974. Progress
Cereal Sci. Today

1976. Chemical
Barley Genetics

Bates, L.S.
barriers.

Bates, L.S., R. Rodriquez, R.

1976. Wide hybridization:
and rye. Wheat Newsletter

Bates, L.S.,
1977.

E. Moreno, F.J.

HortScience 10:

Econ. Bot.

Research and Develop-

ROC. p. 240-246.
Development Center. 1974.
Shanhua, Taiwan, ROC.
Development Center. 1975.
Shanhua, Taiwan, ROC.
G. Park. 1975. Effect

an interspecific cross
313 (Abstr.).

Hybridization and evolution of
154-160.

1973. Wide hybridization

27: 401-412.

Rodriquez and R.G.
toward novel cereal
19: 283-285.

manipulation of crossability
III: 271-273.

F. Waters and K.A. Mujeeb.
gene transfer from barley
22: 84-85.

Zillinsky and K.A. Mujeeb.

Effect of E-amino-caproic acid foliar spray

on seed set and embryo formation in Triticum

var.

Durum x Secale cereaIe. CHem.-Biol.

 

tur idum L.
Interactions 17:

363—365.

Boling, M., D.A. Sander and R.S. Matlock.
bean hybridization techniques.

Buishand, T.J. 1976.
spp.) Euphytica 5: 41-50.

79

1961.
53:

Mung-

Agron. J. 54-55.

The crossing of beans (Phaseolus

 

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

80

Chen, N.C., J.F. Parrot, T. Jacobs, L.R. Baker and
P.S. Carlson. 1977. Interspecific hybridization
of food legumes by unconventional method of plant
breeding. In: Proc. First International Mungbean
Symposium._Asian Vegetable Research and Develop-
ment Center, Shanhua, Taiwan, ROC. p. 247-252.

Chen, N.C. and L.R. Baker. In Press. Influence of
parental cultivar and parental heterozygosity on
crossability among four species of Vigna. (In press).

Coyne, D.P. 1964. Species hybridization in Phaseolus.
J. Hered. 55: 5-6.

Evans, A.M. 1975. Species hybridization in the
genus Vi na. IITA Tropical Grain Legume Bu11.,
Ibadan, Nigeria. Spp.

Loomis, R.S., W.A. Williams and A.E. Hall. 1971.
Agricultural productivity. Ann. Rev. Plant Physiol.
22: 431-468.

Rajendra, B.R., K.A. Mujeeb and L.S. Bates. 1979.
Effect of E-amino-caproic acid on floral morpho-
genesis in Dolichos lab-lab. Chem.-Biol. Inter-

actions 24: 117-120.

Roberts, L.M. 1970. The food legumes: Recommendations
for expansion and acceleration of research to increase
production of certain of these high-protein crops.

The Rockefeller Foundation, New York.

Swaminathan, M.S. and H.K. Jain. 1973. Food legumes
in Indian agriculture. In: Nutritional Improvement
of Food Legumes by Breeding. (M. Milner, Ed.).
PAG/UN. New York. p. 69-82.

Taira, Tomoaki and E.N. Larter. 1977. Effects of
E-amino-n-caproic acid and L-lysine on the develop-
ment of hybrid embryos of triticale (x Triticosecale).
Can. J. Bot. 55: 2330-2334.

Taira, Tomoaki and E.N. Larter. 1977. The effects of
variation in ambient temperature alone and in
combination with E-amino-n-caproic acid on develop-
ment of embryos from wheat-rye cross (T. turOidum
var. durum cv. Jori x S. cereale). Can. J. Bot.

55: 2335-2337.

SECTION III
TECHNIQUES IN CIRCUMVENTING CROSSABILITY
BARRIERS IN THE INTERSPECIFIC CROSS,
VIGNA RADIATA x V. UMBELLATA

 

 

SUMMARY
Application of techniques such as partial detachment
of pods, defoliation, pollen mixture, interspecies grafting,
species bridge and amphiploidy were studied to facilitate

the crossing of Vigna radiata x V. umbellata. Number of

 

 

viable Fl seeds was increased in pods with partially
broken pedicels and in pods excised from the plant 10-14
days after hybridization and cultured in ying. Partial
or complete removal of leaves from the maternal plants 4
days after pollination also increased the number of viable
F1 seeds. The "side-grafting" of two species, and the use
of a third species bridge were effective in reducing embryo
abortion and increasing the number of viable F1 seeds.

Hybrid sterility was circumvented by doubling the
chromosome number. Five triploid plants were derived from
cultured embryos of the backcross of the amphidiploid to
V. radiata. The triploid plants grew vigorously and

flowered profusely with partial fertility.

81

INTRODUCTION

Vigna radiata (L.) Wilzcek (mungbean) is an ancient
and important pulse crop in Asia and parts of Africa. It
is an excellent source of protein, but its yield potential
is low and it is susceptible to many insect and disease

pests (AVRDC, 1974). The V. umbellata (Thunb.) Ohwi 8

 

Ohashi (rice bean), a closely related species of V.
radiata, was reported to be highly resistant to bean fly

(Melanagromyze phaseoli) but of little agronomic value

 

 

as a cultivated species. V. umbellata is also highly

resistant to Cercospora leaf spot, powdery mildew and root

 

diseases (AVRDC, 1975). The present study was to incorporate

this resistance from V. umbellata to V. radiata.

 

Interspecific hybridization between V. radiata

and V. umbellata has been reported with limited success

 

(Dana, 1967; AVRDC, 1974, 1975; Ahn & Hartmann, 1977;
Chen et a1., 1977). The interspecific cross was only
successfully made when V. radiata was used as the female
parent, with difficulty due to low numbers of hybrid
plants obtained. Most of the embryos were observed to
abort prematurely (AVRDC, 1976; Ahn G Hartmann, 1977;
Chen 8 Baker, in press). The hybrid plants obtained grew
vigorously, but were highly sterile. Apparently, there
were barriers to crossability between these two species.
Chen and Baker (in press) found that foliar applica-

tions of E-amino-n-caproic acid significantly increased

 

the number of viable V. radiata x V. umbellata Fl seeds.

82

83

In the reciprocal cross between Phaseolus vulgaris and

P. coccineus, Ibrahim and Coyne (1975) used the techniques

 

of partially breaking the pedicels of pods or pod culture
in Ziploc plastic bags to obtain several viable F1 seeds.
Mixed species pollen were used to overcome inter-
specific incompatibility for a number of plant taxa
including Populus (Stettler 8 Guries, 1976), Sesamum
(Sastri & Shiranna, 1976) and Ipomea (Guries, 1978).
The incompatible or "foreign" pollen is generally used
with a killed compatible, or "mentor" pollen which can
ultimately effect interspecific fertilization. The use
of third species "bridges" to overcome interspecific
incompatibility have been successfully used (Dionne, 1963;
Hermsen G Bamanna, 1973; Ronald G Ascher, 1976; Sams et
a1., 1977).

For the interspecific cross, V. radiata x V. umbellata,

 

hybrid sterility was overcome by doubling the chromosomes
to induce amphidiploid (Dana, 1967; Ahn 5 Hartmann, 1977).
The fertility of the amphidiploids was increased up to
80% as indicated by pollen stainability. However, the

use of V. radiata x V. umbellata or of the amphidiploids

 

in a practical breeding program has not been reported.
This paper reports on the use of several techniques to
overcome crossability barriers in the interspecific

cross of V. radiata x V. umbellata and their possible

 

implications in plant breeding.

MATERIALS AND METHODS

 

Plant materials. The cultivars used in the inter-

 

specific hybridization were V. radiata cv. Tainan #1 and

V. umbellata cv. HK. Three other species, V. mungo,

 

V. angularis and V. radiata var. sublobata, were used as

 

 

"species bridges”. The cultivars used were V. mungo,

”T-9", V. angularis, ”Chien Shien" and V. radiata var.

 

sublobata, "81" and "82". All materials were obtained

 

from the Asian Vegetable Research and DeveIOpment Center
(AVRDC). A spontaneous amphidiploid "MRSl" (AVRDC, 1976)

of V. radiata x V. umbellata was used to backcross to

 

V. radiata to derive triploid progenies.

Cultural conditions. All plants were grown in a glass-

 

house. During the winter, temperatures were adjusted to
26°C (day) and 18°C (night) with a photoperiod of 14 hr
light supplied with high intensity mercury lamps (10-12 klx).
Plants were grown in 20 cm pots using a "Peat Lite Mix”.
The fertility program was to apply 3 g/pot of 20N-20P-20K

biweekly.

Crossing Method. Flowers for hybridization were

 

emasculated the day before anthesis and immediately
pollinated. The hybridization technique used in crosses
was described by Boling et a1. (1961). After hybridiza-
tion, all subsequent flowers were removed to eliminate

competition.

84

85

Partial detached and detachedpods. The techniques

 

of partial pod attachment and excised pods were described
by Ibrahim and Coyne (1975). In this study, pedicels

of cross-pollinated pods were partially cut with a
sterilized razor 10-12 days after hybridization. For
excised pods, pods were removed from the plant at 10-12
days after pollination and surface sterilized with 10%
chlorox followed by four rinses in sterilized water and
were placed in sterile petri dishes and sealed with
"parafilm". The dishes were cultured in the laboratory
at 220 1 20C supplied with 16 hr artificial light. To
determine the best time for detaching the pods, hybridiza-
tion was made to obtain pods that were 10 to 18 days old.

The pods were either partially detached or detached.

Defoliation treatments. All leaves were excised
from the plant 8-12 days after cross-pollination. To
determine the most effective time for defoliation,
hybridizations were made at two-day intervals to provide
6 ages, from 2 to 12 days post-pollination. Four defoliation
treatments were compared including a control. They were:
(a) excise one leaflet from every leaf (approx. 33%);
(b) excise two leaflets from every leaf (approx. 67%);
(c) excise all leaves from the plant (100%); and

(d) control.

Pollen mixture. The use of pollen mixture

 

to increase the percent of the hybridization

between V. radiata and V. umbellata was

 

86

through the use of the third species, V. angularis.

 

Pollen from the third species was mixed with pollen of

V. umbellata for cross-pollination with V. radiata and

 

compared to the use of V. umbellata pollen alone (control).

 

The mixing was accomplished by successively touching

stigmata containing pollen of V..umbellata and V. angularis

 

 

to the stigma of V. radiata.

Interspecies grafting. Grafting between two species

 

was made to increase the number of viable Fl seeds in the

cross of V. radiata x V. umbellata. Two types of grafting

 

were used; viz., side-grafting and approach-grafting.
In side-grafting, the scions from V. radiata were side-

grafted onto the plant of V. umbellata. In approach-

 

grafting, plants of V. radiata and V. umbellata were grown

 

side-by-side and the approach-grafting was made at 3 weeks
after planting. Grafted plants were placed in a mist
chamber for 2 weeks to facilitate survival of the grafted
plants without excising the shoots or roots of either

plant.

Species bridges. Two F1 hybrids, V. radiata x V. mungo

 

and V. umbellata x V. angularis, were used to test the

 

 

”bridging species”, V. mungo and V. angularis. The hybrid

 

of V. radiata x V. mungo was hybridized with V. umbellata

 

while the hybrid of V. umbellata x V. angularis was crossed

 

 

with V. radiata. Two accessions of V. radiata var. sublobata

 

87

were crossed with V. radiata. The resultant hybrid plants

were hybridized with V. umbellata which used V. radiata

 

var. sublobata as a bridging species for the interspecific

 

cross of V. radiata x V. umbellata.

 

Amphiploidy. To induce amphiploids, cuttings from

 

sterile hybrid plants were treated with 0.25% (2.5 mg/ml)
colchicine for 8 hr; and then, rooted in a mist chamber.
The induced amphiploids were backcrossed to V. radiata
and the backcross embryos cultured in ylgrg on a
Linsmaier and Skoog medium (1965). The successful plants
derived from cultured embryos were grown to flowering and

backcrossed again to V. radiata.

Experimental design and data analysis. Randomized

 

complete block and split-plot designs were used. Data
were taken on the number of flowers crossed, total number
of seeds obtained, number of viable seeds and percentage
of pods that produced viable seeds. The authenticity of
hybrid plants from viable seeds was confirmed by several
genetic markers (Chen 8 Baker, in press). The analysis
of variance was performed and L.S.D. or Duncan's multiple

range test used to compare the treatment means.

RESULTS AND DISCUSSION

Growth of pod, seed and embryo. Pods from the cross

 

of V. radiata x V. umbellata and self-pollinated pods from

 

V. radiata were excised at 4-day intervals and the length
of pods, seeds and embryos measured. The growth rate of
hybrid pods, seeds and embryos was relatively slow as
compared to the selfed (Fig. l). The growth of hybrid
embryos increased from 4 days after pollination to a

maximum at 12 days; whereupon, growth ceased.

Partial and detached pods. Partially detached and

 

detached pods contained significantly more viable seeds
than the control "attached" pods (Table l). Partially
detached pods was more effective than detached. However,
more defined techniques for pod sterilization and cultural
conditions were needed to further improve the success of
13 vitro pod culture. For example, the relatively poor
results obtained from the detached pods in Experiment 1
were mainly due to incomplete sterilization that permitted
the pods to rot before attaining maturity. Furthermore,
the high humidity conditions in the petri dishes also
caused the seeds to germinate (viviparity) before pod
maturity. Thus, it is necessary to carefully control
both sterilization procedures and relative humidity
conditions to assure pod maturation.

The detaching technique used to overcome embryo

abortion was based on the assumption that inhibiting

88

89

 

 

 

08588§

 

 

LEMSTHMI)

 

 

 

 

8 _. Ham
6 . SELF-“'1‘. /.
q .. / HYBRID
. J: _____ ,
2 i / ,”’
’,.—o
0 LI 8 12 16

DAYS AFTER POUJNATIG‘I

Figure 1. Comparative growth of pod, seed and embryo of natural
self of V. radiata and the interspecific hybrid,
V. radiata x V. umbellata.

90

.:0wumomaaou hog moon mm mm:0wumowfiaoc v we coo:

 

 

 

N
.coHumUMHQo» Log mp0; om mm:0wumuflaaoh a mo :82H
¢.m~ - ~.m m.- - m.~ wm
.a.m.4
m.n~ mm m.o o.o~ HM m.N Hocucou
a? 8 mg: 92 2 o.m 88
@9336:
moon
v.mm me~ w.mm m.wm ow m.n~ vozomuow
Hmflupmm
mumEouum muaeouum

mwoom mHan> ooH pod A.ocv mwoom omnmw> ooH pom A.o:v

cu“: moon w mwoom manmw> mnoom oaamw> and: moon w muoom oanmw> mmoom oHan> ucosumopk
NN ucoefipmmxm Hg acmeflpomxm .

 

.mumfifionss aM.x mumwwmh .Mw.mm0po
owwfloommpoucm ecu mo mmooosm oz» :0 moon nonomuoc can vocomuow Hmwupmm may we uuowwm .H magma

91
substances are prevented from being translocated to the
pods (Ibrahim 6 Coyne, 1975). Partially detaching the
pedicels of pods permitted sufficient water absorption
to continue physiological processes, but may have reduced
the translocation of inhibitors. 13 XEEZE pod culture
the inhibiting substances are eliminated and the pods
continue necessary physiological and developmental
functions under controlled temperature and light
conditions. The best time to apply these techniques
to reduce F1 embryo abortion was 10 to 12 days after
pollination (Table 2). The highest number of viable
Fl seeds (12) was obtained from the 10 day-old pods
with partial detachment, while the maximum number of

viable Fl seeds (7.3) was produced from the 12 day-old

pods with 12 vitro pod culture.

Defoliation treatments. As mentioned above,

 

inhibiting substances may be translocated from the

leaves to the pods and cause embryo abortion. Thus,
partial or complete defoliation might reduce or eliminate
hybrid embryo abortion. Complete removal of leaves from
the maternal plants significantly increased the numbers

of viable F seeds from 5 to 12 times as compared to the

1
controls (Table 3). The defoliation treatments also
increased the percentages of pods that produced viable
seeds (from 17 to 29%). Generally, the greater defolia-

tion resulted in the highest number of viable seeds (Table

4). Regardless of the time of defoliation, the average

92

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N
.coflumoflflaoh pom mcom m mmcoflumUMHaou e we :82H
coma ON ooo.a EON ON oo.~ - - Hotooou

wen oH ow.o omN mm om.a ow.q a¢.wo mm

oooom cm oom.H oAa om om.¢ am.a ma.oo 0H

23mm ow oao.v omm QNH oo.o no.4 oo¢.4o 4H

amA ova om.A aAo mMN om.HH oo.m ooq.mm NH

ooamq om oom.N mom OVN ao.- ow.~ Nom.mm CH

memom mugsouum mwoom mumsouam
oHan> OOH too A.o:v oaooa> OOH too A.o:V REEL Asst cozoacaaaoo
goo: mooo a mooom ofioaa> mooom oanaa> sou: mooo » mooom oaooa> mooom oaooa> soot: aowcoa zoowo mama
moon cocomuo: moon wonoauow Hmwupmm owwm won

 

H.mumHHonez am.x mumwnmp .Mw.mm0ho owwfiuommuoucw ogu mo
mmoouzm oz“ co moon nocomuoo vcm wozomuop Hmwupma pom med“ sewumcwafiom-umoa mo uuomwm .N ofinmb

Table 3. Effect of defoliation on the success of the interspecific
cross, V. radiata x V. umbellata.

 

 

 

 

 

Treatment Viable seeds Viable seeds % pods with
(no.) , per 100 viable seeds
attempts
Experiment 11
Defoliated 28.7 73.3 26.7
Undefoliated 2 .3 7 . 5 3 . 3
L.S.D. 5% 4.6 4.7
Experiment 22
Defoliated 12.7 66.3 38.4
Undefoliated 1.5 8.8 9.4
L.S.D. 5% 1.1 12.0
Experiment 32
Defoliated 7.6 48.4 26.6
Undefoliated l . 5 9 . 6 9 . 4
L.S.D. 5% 0.9 8.4

 

1Mean of 3 replications.

2'Mean of 4 replications.

.Hm>oH Nm .umou omcwp oaawufiss m.:mo::: x5 mcssfioo :N sewumgmdom cam:

 

 

94

 

 

 

m

.:oNum:wHHoa goumm mxmaN

.cwflmom uofid ufifimm mmcoflumowamog v we :32H

- - em moa mm wNH Hm mNH NH ed cam:

Hampo>o

nmN omN omN UNA nva Una swam omN no mo NH
an nvdfi mom. mocN ammo gamma nva onmmHN mm mm OH
nmv gum muo oanH swm gamma ammo onmwm mmH mmfi w
now nww mom unmc anew unmc poem pmmHN mma and a
two mVNN was mmNm mooH moom mmm mmmN mmN mmm o
nwm onmm mwm enema amom upon nmN onmo mNH mama N
Amxmov

mooom muanuum mcoom mumsouum mvoom mumeouum mcoom muqsouuw mwoom muaeouum :ofip

2an 9: too 3%? 2: 8o 2an 2: too 2%.? 2: too Boos 2: too 5:88
new: mwoom no“: mcmom new: mcoom zuflz mwoom an“: mcoom mo

88 to 38$ moon 1” 28$ 88 a 28:, 88 a 23> 88 a 2%; Nos:

coo: Nooa who “mm No
Hampo>o :oNumNHommo mo oopwoo

 

Hg mama ..>.
.mmouo onNUonuoucfl ecu mo mmouuzm any so coflumwfiomov mo ooamow van mafia mo uuommm .v manmh

95

numbers of viable seeds per 100 attempts were 123, 128
and 145 for 33, 67 and 100% defoliation, respectively,
compared to 14 for the control. Complete defoliation
(100%) following pollination caused a large number of
pod abscission. The time of defoliation seemed to
affect embryo abortion. Defoliation at 4 days after
pollination was the most effective for obtaining F1
seed as opposed to leaf removal 12 days after pollination
(Table 4).

Adverse effect of defoliation on grain yield has
been reported for several legume creps (Stewart et
a1., 1978; Enyi, 1979). The mechanism by which defoliation

facilitated hybrid embryo development in this inter-

specific cross of V. radiata x V. umbellata is unknown.

 

Defoliation of the maternal plant may have eliminated
or reduced the synthesis of inhibitory substances in the

leaves and be transported to the pods and seeds.

Pollen mixture. Use of pollen mixture combining a

 

compatible pollen with a foreign species pollen to over-
come interspecific incompatibility has been successful
in Populus (Stettler 8 Guries, 1976). Application of

pollen mixture to the cross of V. radiata x V. umbellata

 

was made to learn regards its possible effect on embryo
abortion.

Pollen of a third species, V. angularis, which was

 

partially compatible with V. radiata (Ahn G Hartmann, 1978;

96

Chen G Baker, in press) was mixed with V. umbellata

 

and used to pollinate V. radiata. The use of pollen
mixture increased the number of viable hybrid seeds

3 times that of the control (Table 5). The number of
viable seeds and percent pods producing seeds were

increased. Possibly, either the pollen of.V. angularis

 

and/or the hybrid embryos of V. radiata x V. angularis
stimulated the growth and development of the hybrid

embryos. The effect of mixed pollen might be similar
to that of "double pollination" described by De Vaulx

and Pitrat (1977).

Interspeciesygrafting. Grafting between V. radiata

 

and V. umbellata was successful. The grafted plants grew

 

vigorously and produced seeds in both the scion and stock
species. Moreover, a large number of viable hybrid seeds
resulted by grafting as compared to control (Table 6).

The use of side-grafting in which V. radiata was used as

the scion on V. umbellata was the most effective in over-

 

coming embryo abortion in the interspecific cross of

 

V. radiata x V. umbellata. The number of viable seeds

per 100 attempts was 187 using side-grafting as compared
to 7 for the ungrafted control (Table 6). An increase

in percentages of both viable seeds and pods that produced
viable seeds was realized for the side-grafted plants.

Approach-grafting was not as successful as side-graft.

Table 5. Effect of mixed pollen on facilitating interspecific
hybridization of V.radiata and.V§ umbellata.l

97

 

 

 

 

 

Treatment Total seeds Viable seeds Viable seeds % pods with
obtained No. per 100 attempts viable seeds

Experiment 1

Nfixed

pollen 232 9 4.0 22.5 17.7

Control 246 3 1.2 7.5 7.5

Experiment 2

Mixed

pollen 487 25 5.1 31.3 18.8

Control 455 7 1.5 8.8 8.8

 

lMixed pollen of V} umbellata and V. angglaris were cross-pollinated
onto V. radiata.‘ A total of 40 p6111nation attempts was made in

each‘freatment.

98

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ozu mo mecca pew muoomm o>wwvommog wowmwuxo uzomuaz.:30pm who: mofiuomm ozu nouwmhm ecu mo

mucmfim .MUOHm mumHHmaE: .>.m co :oNom ecu mm mumwwmp «M wcwm: xn owns mm3.m:«ummpm-ocwm

H

 

 

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o.mN N.oN o.¢ NN ooN m4 omoaw-;oooooo<
c.oN m.owa N.om NN oMN mm omatw-oon

39553

comm oHnmN> ooH hon w .02 woCMmuno commONU ucoeumopu

nu“: moon N mwoom o~nmw> mooom ofinmw> mwoom HmHOH mpoonm .oz Huwmko

 

.mam-03€: .wa mumwwmg .M mo mmouu uNmNuommuoucw

use Now mpowpamn xuflfifinmmmopu mcweoopo>o co mcfluwmpw “wowoommhoucw mo ouco:~m=~

.c oHnmh

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g 1
. ';k?“.NOTQ-h&1"“-=
+ . -

V. mungo, V.

   
 

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hlf‘ fl

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p.
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99

Species bridge. Three potential bridging species,

 

angularis and V. radiata var. sublobata,

 

were tested. Pod-set was observed for the crosses of

 

(V. radiata x V. mungo) x V. umbellata and (V. umbellata

x V. angularis) x V. radiata, however, the pods abscised

 

prematurely and no viable seed was produced. One hybrid

plant was obtained from the cross, (V. radiata x V. mungo)

x V. umbellata. The hybrid plant grew normally but was

 

sterile and succumbed soon after flowering.
A promising potential bridging species was found
for this cross. By using V. radiata var. sublobata as a
bridging species, considerably higher numbers of viable
seeds were produced from the crosses of (V. radiata x
V. radiata var. sublobata) x V. umbellata as compared to
that of the cross, V. radiata x V. umbellata (Table 7).
‘The number of viable seeds per 100 attempts were 497
and 296 for where a third species was involved as compared
to only 11 for direct hybridization of the two species.
V. radiata var. sublobata has been considered a
wild pregenitor of cultivated V. radiata and V. mgggg
(Arora et a1., 1973). It was originally classified as
Phaseolus sublobatus Roxb., but it has been separated

from Phaseolus and given varietal status under V. radiata

 

by Verdicourt (1970). The morphological diversity in
V. radiata var. sublobata and its possible value as a
source of resistance to yellow mosaic virus have been

-v'7

reported (Arora et a1., 1973; Singh 8 Ahuja, 197,).

100

 

 

a; z a: N com 3 g x a
a x
9: SN . max 3 o: “N am .5 gum x as
g x
4.3 34 a? at EN om :m .5 3m on go
comm mumsouum
manmw> OOH he; w .02 nmcwmuno nommONU mmONU

 

5w: mfiOQ mm mflmmm OHQQH> mfimom OHDQM> Wfivom HQHOH ka‘OHw .OZ

 

.mumHHonED .m.m:m mymmpmp .quo
xuwfifinmmmONU may o>ouaEH ou mowoodm owwwpn m mm mumnofinom .um> mumflcmp .> we uommwm .N magma

101

The use of V. radiata var. sublobata as a bridging species

 

provides another means to circumvent barriers to cross-

ability in the interspecific hybridization of Vigna.

Amphiploidy. To circumvent hybrid sterility, the

 

ploidy of sterile hybrid plants can be doubled with
colchicine to produce amphiploids (Dana, 1967; Ahn G
Hartmann, 1977). Amphiploids were induced by treating
cuttings of the sterile hybrid with 0.25% colchicine
(Fig. 2). The amphidiploid plants were partially fertile
with pollen stainability to 81%. However, the vigorous
vegetative growth, photOperiod sensitive, and poor pod-set
suggest that it will not succeed as a new crop without
improvement. 50, one method to transferring the desirable
genes is by backcrossing the amphidiploid to V. radiata.
A spontaneous amphidiploid (AVRDC, 1976) was used
to backcross with V. radiata. Five triploid plants were
obtained with the aid of embryo culture (Fig. 2). The
triploid plants grew vigorously and flowered profusely
with infrequent pod-set. Cytogenetic studies (Machado,
1978) showed meiosis in the triploid plants (amphidiploid
x V. radiata) was irregular with 11 bivalents and 11 uni-
valents at metaphase I. The second division was also
abnormal with multipolar division and non-congregating
and lagging chromosomes. A number of progenies from the
triploids were produced by natural self-pollination and

by backcrossing to V. radiata (Fig. 2). Variation in

Figure 2.

102

Plants of interspecific hybrid V. radiata x‘V} umbellata,
amphidiploid, triploid and progenies of {he trip101d.
Z-a: Hybrid plants (2X, right) and amphidiploid (4X3.
Z-b: Triploid plants derived from embryo culture
(amphidiploid x V. radiata). 2-c: Plant of triploid

x V. radiata, shaing EBnormal monoleaflet of leaves.
2J3: Progeny of the triploid derived from natural
self-pollination.

103

 

 

 

 

 

 

U ‘ a '
i" 41’: “If
.-‘_(_ 4 (I ’ , | ‘
3“,?“ “as .04

mag} ,— 2......
,1" ‘h‘ '

 
 

 

 

104
morphological characteristics and levels of fertility
were found among these progenies. Mitotic studies
revealed that 2n=22 and 2n+l=23 somatic chromosome
numbers occurred in these triploid progenies (Machado,
1978). Improvement and selection of economic traits
could be accomplished by backcrossing to V. radiata

to recover desirable traits.

REFERENCES

Ahn, C.S. and R.W. Hartmann. 1977. Interspecific
hybridization among four species of the genus Vigna.
lg: Proc. First International Mungbean Symposium.
Asian Vegetable Research and Development Center,
Shanhua, Taiwan. p. 240-246.

Asian Vegetable Research and Development Center (AVRDC).
1974. Annual Report for 72-73. Shanhua, Taiwan.

Asian Vegetable Research and Development Center. 1975.
Annual Report for 1974. Shanhua, Taiwan.

Asian Vegetable Research and Development Center. 1976.
Mungbean Report for 1975. Shanhua, Taiwan.

Arora, R.K., K.P.S. Chandel and 3.5. Joshi. 1973.

Morphological diversity in Phaseolus sublobatus Roxb.
Curr. Sci. 42: 359-361.

Boling, M., D.A. Sander and R.S. Matlock. 1961. Mungbean
hybridization techniques. Agron. J. 53: 54-55.

Chen, N.C., J.F. Parrot, T. Jacobs, L.R. Baker and P.S.
Carlson. 1977. Interspecific hybridization of food
legumes by unconventional method of plant breeding.

In: Proc. First International Mungbean Symposium. Asian
VEgetable Research and Development Center, Shanhua,
Taiwan, p. 247-252.

Chen, N.C. and L.R. Baker. In Press. Influence of parental
cultivars and parental heterozygosity on crossability
among four species of Vigna. In Press.

Chen, N.C. and L.R. Baker. In Press. Effect of E-amino-
n-caproic acid on interspecific hybridization in the
genus Vigna. In Press.

Dana, S. 1967. Hybrid from the cross Phaseolus aureus
Roxb. x P. calcaratus Roxb. J. Cytology GIGEnet.
2: 92-977

 

 

De Vaulx, R.D. and M. Pitrat. 1977. Relation of the
interspecific cross between Capsicum annum L. and
Capsicum baccatum. In Interspecific Hybridization in
Plant Bre€ding. ProET 8th EUCARPIA Congress, Madrid,
Spain, 1977. p. 327-333.

 

 

105

106

Dionne, L.A. 1963. Studies of the use of Solanum
acaule as a bridge between S. tuberosum and species
in the series Bulbocastana,_Cardiophylla, and
Pinnatisecta. Euphytica 12: 263-269.

 

Enyi, B.A.C. 1975. Effect of defoliation on growth and
yield in groundnut (Arachis hypogaea), cowpea (Vigna
unguiculata), soybean*(Glycine max) and green gram
(Vigna aureus). Ann. Appl. Biol. 79: 55-66.

 

 

 

 

Guries, R.P. 1978. A test of the mentor pollen technique
in the genus Ipomoea. Euphytica 27: 825-830.

Hermsen, J.G. Th., and M.S. Bamanna. 1973. Double-bridge
hybrids of Solanum bulboeastanum and cultivars in
Solanum tuberosum. Euphytica 22: 557-566.

 

 

Ibrahim, A.M. and D.P. Coyne. 1975. Genetics of stigma
shape, cotyledon position, and flower color in
reciprocal crosses between Phaseolus vulgaris L. and
Phaseolus coccineus (Lam.) and implications in breeding.
J. Amer. Soc. Hort. Sci. 100: 622-626.

 

 

 

Linsmaier, E.M. and F. Skoog. 1965. Organic growth
factor requirements of tobacco tissue cultures.
Physiol. Plant. 18: 100-127.

Machado, M. 1978. Personal communication. Dept. of
Horticulture, Michigan State University, East Lansing.

Ronald, W.G. and P.D. Ascher. 1976. Lilium x "Black
Beauty" - A potential bridging hybrid 1n Lilium.
Euphytica 25: 285-291.

Sams, D.W., P.D. Ascher and F.I. Lauer. 1977. Cross-
ability of some green-peach-aphid-resistant tuber-
bearing Solanums, potential bridging species, and
Solanum tuberosum ssp. tuberosum. Amer. Potato
J. 54: 355-364.

 

 

Sastri, D.C. and K.R. Shivanna. 1976. Attempts to over-
come interspecific incompatibility in Sesamum by
using recognition pollen. Ann. Bot. 40: 891-893.

Singh, B.V. and M.R. Ahuja. 1977. Phaseolus sublobatus
Roxb.: A source of resistance to yellow mosaic
virus for cultivated mung. Ind. J. Genet. Plant
Breed. 37: 130-132.

 

Stettler, R.F. and R.P. Guries. 1976. The mentor pollen:
phenomenon in black cottonwood. Can. J. Bot. 54: 820-
830.

107

Stewart, K.A., R.J. Summerfield and B.J. Ndunguru. 1978.
Effect of source-sink manipulations on seed yield of
cowpea (Vigna unguiculata (L.) Walp.). I-Defoliation.
Trop. Agric. (Trinidad). 55: 117-125.

Verdicourt, B. 1970. Studies in the Leguminosae-
Papilionoideae for "Flora of Tropical East Africa."
IV Kew Bul. 24: 507-569.