Nigerian J Genet. 18(2003):36-43

Nigerian J Genet. 18(2003):36-43

CYTOGENETIC STUDIES OF F, HYBRIDS BETWEEN COWPEA (VIGNA

UNGUICULATA (L.) WALP) AND v. RHOMBOIDEA BURTT. DAVY

B. Aliyu

Department of Crop Production and Horticulture,

Federal University of Technology

P.M.B. 2076 Vola, Adamawa State ABSTRACT

Vigna rhomboidea is a hairy wild relative of cowpea that is a source of useful genes for

pubescence for incorporation into cultivated cowpea for the improvement of cowpea crop production

through insect resistance. Four hundred and eighteen interspecific and control pollinations were

carried out between a cultivated cowpea (IT84S-2246) and a V rhomboidea accession (Tl/nu 1455)

with the aim of obtaining viable interspecific hybrids. Pod set was observed to be low (2.9% from 268

crosses) on cow pea as seed parent and V rhomboidea pollen parent. Cytogenetic investigations on

chromosome homology on the FI hybrid plants suggest that the reduced fertility of the hybrids could

be due .to genetic imbalances in the gametes resulting from multivalent chromosome associations.

centricfusion of chromosomes, chromosome loops, chromosome breakages as well as chromosome

laggards.

INTRODUCTION Cowpea, an important pulse crop in many

developing countries of the world is susceptible to

several insect pests that significantly reduce its yield

potential. Sources of resistance to some of the insect

pests have been identified in some wild Vigna species

(Birch, et al., J 985; Singh, et al., 1990; Anon, 1988;

1996). Unfortunately, the

exploitation of interspecific hybrids for crop

improvement is complicated by incompatibility barriers

(Baudion and Marechal, 1991). The nature of the

crossability barriers between species and their mode of

origin has been

described by Smartt (1970) as being complex. During

hybridization studies with Phaseolus species, he

concluded that the genome and plasmon must both have

a common origin for normal fertility to occur. Monti

(1992) reviewed

the circumstances surrounding cross ability barriers

between species. He noted pre- fertilization and/or post

fertilization barriers as factors that prevents success in

crosses. Barone and Ng (1990) examined the cytological

abnormalities in pollen tube growth following crosses

between cowpea (V unguiculata) and wild V vexillata.

They concluded that lack of fertilization and/or collapse

of the fertilized ovules was responsible for the failure of

the

crosses. The failure of interspecific hybridization due to

embryo degeneration has also been reported (AI- Yasiri

and Coyne, 1960). Aliyu (2001; 2002) reported a

crossability percentage between cowpea (TVu t 627)

and a

Wild V rhomboidea accession (TVnu 1473) of

1.1% out of 116 crosses and observed that developing

crossed. pods always aborted. Although pollen-pistil

incompatibility .is an important barrier to interspecific

hybridization,

Stebbins (1958) cited in Barone et al. (1992) observed

that the failure of pollen tube growth and fertilization is

rarely the primary cause of reproductive isolation

between closely related species. It was shownthat

resources competition and not cross-genetic

incompatibility is responsible for the abortion of

embryos. The solution to overcoming crossability

barriers in interspecific hybridization involves

histological and cytological analysis in studying the

reproductive biology of the species, the FI embryo

development and the F I plants as well as

the use of molecular markers. This paper reports the

cytological abnormalities observed in the F I hybrid

plants in

crosses between cow pea variety IT84S-2246 and V

rhomboidea accession TVnu 1455.

MATERIALSANDMETHODS Materials

The seeds of the cowpea variety and V.

rhomboidea- accession were obtained from the Genetic

Resources Unit, International Institute of Tropical

Agriculture, liT A, Ibadan Nigeria. wherethe work itself

was done. Seeds of V. rhomboidea were first scarified

with a surgical blade to facilitate early germination. The

seeds were then pre-germinated in a controlled

Aliyu: Cytogenetics of hybrids of V. unguiculata x V. rhomboidea

environment (conviron) within the laboratory.

Seedlings at two-leaf stage were transplanted into

plastic pots in a screen house. Each pot was 20 cm in diameter and filled with garden soil. Two

seedlings were transplanted into each pot and this

was replicated four times. The pots were 1 m

apart within rows and 1.5 m between rows. Cowpea seeds were seeded directly in the plastic

pots at three weeks after transplanting V.

rhomboidea seedlings. The pots .were

completely randomized in the screen house.

Water was supplied by sub irrigation with a

minimum of top watering.

Crossing

268 interspecific crosses in addition to self

pollinations on the parent plants to serve as

controls were carried out. . Emasculation and

pollination were done according to the methods

of Rac hie et al. (1975)

Cytogenetic Protocols

Flower buds were collected at 7.00am,

9.00am, 10.00am and. 1 ).00am . inthe morning

hours. The buds were immediately fixed in a

freshly prepared mixture of acetic-ethanol in a

ratio of 1 part acetic acid to 3 parts ethanol and

stored in 70% ethyl alcohol.

Each flower bud was first placed on paper

towel to drain the alcohol. The bud was then placed

on a clean slide which was placed on a • Wild TYP

181300 binocular dissecting microscope with an

intralux 5000 illuminator. With the aid of 2 transfer

needles held in both hands, the bud was carefully

dissected and the anthers liberated on the slide. A

drop of 1 % aceto-carrnine was added. A cover slip

was then placed on the aceto-carmine-immersed

anthers. With the right hand thumb, the slide was

gently pressed between folded blotting paper. The

slide was then gently warned with a spirit lamp with

periodic tapping and addition of few drops more of

aceto-carmine. The preparation was finally

observed under a Leitz compound microscope.

Micrographs were taken with a WILD MPS 46/52

Photoautomat attached to the Leitz microscope.

The stainability of pollen of parental plants

as well as of the F 1 hybrids was examined. This

would provide additional information about

genetic affinities between V rhomboidea and V.

unguiculata. For each plant, five opened flowers

were picked at random. For

eachflower, pollen grains were shed on a clean slide

by using transfer needles. A drop of 1% aceto-

carrnine was added to the pollen on the slide.

Artifacts and debris were removed from the stained

slide with the tip ends of two transfer needles held

in both hands. A cover slip was finally placed on

the stain. Five slides were

made for each plant. Each slide was analyzed by

surveying five different fields at random on, the

microscope. Thus 25 fields were surveyed for each

plant. This was carried out for each of the parental

and F 1 plants and used to estimate the average

fertility of the pollen.

RESULTS Pod set within one week of crossing was

observed to be 13.8% out of which only 2.9% reached

maturity. The matured pods were malformed and the

seeds were shrunken. Pod set and maturity in the

control pollinations were, however, normal (Table 1).

The stainability of pollen in the hybrid was 41.6% as

compared to 98.0% for the cowpea and 95.1 % for the

wild parent. For a bud of 3 mm size, meiosis is

complete and the only structures to be seed would be

those of tetrad stage of pollen and matured pollen

.grains as well as disorganizedpollen grains that made

up 3 5.0% (Table 2). In this study young flower buds

of approximately 2 ,mm size yielded more meiotic

stages. At the same time buds collected and fixed

between 7.00am-8.00am yielded good meiotic stages.

However, slightly smaller or larger buds collected at

other times also yielded meiotic stages. Pachytene

bivalents (Figs. I and 2) and multivalent associations

with chromosome

breakage (Figs. 3 and 4) were observed. Metaphase

chromosomes of the first meiotic division (Metaphase

l )were also observed (Fig. 5). In this plate, bivalents

were clear and they can be seen to orient themselves

on the

equatorial plate. A notable feature in this pollen

mother cell is the presence of two ring bivalents at the

polar ends 0 the metaphase plate. Bivalents were also

seen undergoing separation after synapsis in

pachytene (Fig. 6). Centromeric regions were

conspicuous in some bivalents.

Chromosome laggards at anaphase I (Fig. 7)

and pachytene bivalents with loops (Fig. 8) were

also observed.

Nigerian J. Genet. 18(2003):36-43

Table1. Fertility (expressed as percent pod set) of eowpea, V rhombJlideq;,and their F, hybrid

F, Hybrid Number of Pollen stainability Pod set within 1 Mature

d Pollinations (%) week pods

Cowpea (IT84S-2246) 100 98.0 94.1 87.3

F, Hybrid 268 41.6 13.8 2.9

V rhomboidea (TVnu

1455)

50 95.1 81.0 73.5

Table 2. Percent frequency ofthe pollen mother cells for different stages of meiosis in the Ft hybrid of the cross IT84S-2246 x TVnu 1455

Size of flower stage of meiosis percent pollen

1 pre-PMC 8

2 Leptotene 2

Pachytene 13

Diplotene 3

Diakinesis 2

Metaphase I & II 15

Anaphase I & II 17

Resting 40

3 Tetrad stage 10

Pollen grains 55

Disorganized pollen grains 35

Interspecific hybridization is designed in breeding

programmes to transfer genes or relatively small

blocks of foreign chromosomes from a wild species to

a cultivated genotype.But incompatibility barriers

complicate the exploitation of interspecific hybrids

for crop improvement. Cytogenetic examinations

carried

out in this study showed the presence of ring bivalents

and multivalent associations in the F, hybrid plants.

Chromosome associations with a maximum of 4 and

3 bivalents per cell have been observed and were

reported to indicate a low degree of homology

between Elymus shandongesis x E. semicostatue (Lu

et al .. 1989). The presence of chromosome laggards

observed at anaphase 1 was an indication of delayed

chromosome movement during cell division. Ng

(1992) suggested that hybrid sterilityis due to

chromosomal and not genie

causes. However, in a hybrid with delayed separation

of chromosomes, infertility is under

genicmechanisms (Farms, 1963). In this study, .e low

fertility of the hybrids was due to imbalancesin the

genetic constitution of the geneticsTheobserved

chromosomal fragmentationalso agrees with the

findings of

Mehra and Rai (1970) who records chromosomal

fragmentation at prophase together with chromosomal

stickiness, arreste chromosomal movement and

distortion (normal location and orientation of

chromosom

at metaphase L

The presence -of laggards at anaphase indicates

that gametes will be nongeneticall balanced as a result

of the chromosoma differences. This, together with

the observe' multivalent associations, centric, fusion

chromosome breakage during division, as well as the

formation of chromosome loops isresponsible for the

low fertility of the F I hybrids whose chromosomes

were observed. Unfortunately, chromosome

homoiogy was not observed for the cowpea and V.

rhomboidea parents. Although cell division and

chromosom homology in these parent plants is

assumed to be normal, the opportunity to compare the

chromosome complements of the three genotypes was

not obtained. Consequently,conclusivedata for

chromosome association could not be provided.

Further studies on ' subject, incorporating the parental genotypes K recommended

.

Aliyu: Cytogenetics of hybrids of Jt: unguiculata x V. rhomboidea

Figure 1

Figure 2

Cbromosomes at pachytene (with artifads in the background) (Figure 1); and eleae. pachytene

. bivalents (Figure 2). (dOOO)

Nigerian J. Genet. 18(2003)."36-.:/3

Figure 3

Figure 4

Multivalent association (Figure 3); and chromosome breakage (Figures 3 and 4). (x1000)

Aliyu: Cytogenetics of hybrids of v: unguiculata x Jt: rhomboidea

Figure 5

F

i

g

u

r

e

6

Figure 6

y. . u: Cytoge=":« of hybrids ofT. unguiculatax V. rhomboidet:

REFERENCES Aliyu, B. 2001. Inheritance of pubescence and genetic

compatibility between cowpea (Vigna unguiculata

L) ( Walp) and V. rhomboidea Burtt. Davy.

tJnpublishtdPh.D. Thesis, University of lbadan.

145p. Aliyu, B. 2002. Interspecific hybridization

between cowpea (Vigna unguiculata (L) Walp) and

V. rhomboidea Burtt. Davy through embryo rescue.

Nig. J. Biotechn. 13(1): 18-22. AI-Yasiri, S. A. and D. P ..Coyne. 1966. Interspecific hybridization

in the genus Phaseolus. Crop Science. 6:59-61. Anon, 1988. Annual report and research highlights.

p. 73. International Institute of Tropical Agriculture,

Ibadan, Nigeria. Anon, 1996. Project 16

(Conservation and Genetic Enhancement of plant

Biodivcrsity). Annual Report. International Institute

of Tropical Agriculture, Ibadan, Nigeria. Pp. 10-11. Barone, A. and N. Q. Ng. 1990. EmbrologicaJ studies

of crosses between Vigna Unguiculata and V.

vexillata. In Cowpea Genetic Resources. Ng , .N

Q. and L. M. Monti eds. liT A, lbadan. Pp. 151-160. Barone, A., A. Del Guidice and N. Q. Ng. 1992.

Barriers to intcrspecific hybridization between

Vigna unguiculata and V. vexillata. Sexual plant

ReproductionS: 195-200. Baudoin, J. P. and R. Marechal. 1991. Taxonomy and wide crosses in

pulse crops with special reference to Phaseolus and

Vigna. InCrop Genetic Resources of Africa. Vol.

.nNg, N. Q., Perrino, P. Attere, F. and H. Zedan

eds. liT A, Ibadan. pp. 287-302. Birch, N., B. J. Southgate and 1., E. Fellows. 1985. Wild and semi-cultivated legumes as potential

sources of resistance to Bruchid beetle for crop

breeders. A study of Vigna IPhaseolus. In Plants

for Arid Lands.

Wickens, G. E., J. R. Gooden and D. V. Field eds.

George Alien and Unwin. London. Pp. 103-320.

Faris, D. G. 1963. 'Evidence for the West African origin

of Vigna sinensis (L ) Savi. Unpublished Ph.D.

Thesis, University of California, Davis. 94p.

Lu, B. R., B. Salomon, and R. Von Bothmer. 1989.

Cytogenetic studies of progeny from the

intergeneric cross Elymus muidroh xand E. x

secale. Genome 33,425,432 Mehra, R. C. and K. S Rai. 1970. Cytogenetic studies of

meiotic abnormalities in Collinsia tinctoria. I .

Chromosomal stickiness. Canadian Journal of

Genetics and Cytology, 12:560-569. Monti, L. M. 199.2. The use of wild species in crop

improvement. InBiotechnology: Enhancing

Research on Tropical Crops in Africa.

Thottappilly, G., Monti, L. M., Mohan' Raj, D. R.

and A. W. Moor eds.

CT A - lIT A. Pp. 55-62. Ng, N. Q. 1992. Wide crosses in Vigna food legumes.

In Biotechnology: Enhancing Research on

Tropical Crops in Africa. Thottappilly, G, L. M.

Mohan Raj, D. R. and A. W. Moor eds. CT A- lIT A. Pp. 77-80.

Rachie, K. 0., K. Rawal and J. D.

Franckowaik. 1975. Arapid method of handrossing cow

peas Technical Bulletin no. 2, lIT A, Ibadan, Nigeria.

18p.

Singh, S. R., L. E. N. Jackai, J. H. R. dos Santos and C.

B. Adalla 1990. Insect Pests of Cowpea. In Insect

Pests of Tropical Food Legumes S. R. Singh ed.

John Wiley. pp 43-89.

Smartt, J. 1970. Jnterspecific hybridization between

cultivated American species of the Genus

Sphaseolus. Euphytica 19:480-489.