Pollen Pistil Interaction in Inter-Specific Crosses of

Pollen Pistil Interaction in Inter-Specific Crosses of Vigna sp.

Krishnasamy Thiyagu, Palaniappan Jayamani* and Nagasamy Nadarajan

Department of Pulses, Centre for plant Breeding and Genetics, Tamil Nadu Agricultural University Coimbatore-641 003, India

Received June 11, 2008; accepted August 11, 2008

Summary Inter-specific hybridization is mainly used to introgress pest and disease resistant genes.Vigna radiata and Vigna mungo are widely cultivated and consumed in India. These crops are highlyaffected by Mungbean Yellow Mosaic Virus (MYMV) disease and bruchid (Callasobruchus spp.)pest, leading to heavy yield loss. Vigna umbellata, a divergent Vigna species is found to be resistantto both MYMV and bruchids. Utilization of this species to introgress the resistance gene is marginalbecause of their inherent low crossability with the cultivated Vigna species. In the present study, V.umbellata was used as pollen donors in hybridization with V. radiata and V. mungo. Low percentageof pod set observed in the cross V. radiata�V. umbellata (12.89%) and V. mungo�V. umbellata(5.56%) indicated the presence of reproductive barriers that render introgression difficult. Aim ofthis study was to observe pre-fertilization barriers operating in V. radiata�V. umbellata and V.mungo�V. umbellata crosses. In vivo pollen germination and growth of pollen tubes were studied ininter-specific crosses using fluorescence microscope. The pollen grain germination on stigmatic sur-face was normal. A very common pre-fertilization barrier of slow rate of pollen growth, in additionto structural abnormalities in stigmatic and stylar regions was observed in both the crosses. However,the level of incompatibility was high in V. mungo�V. umbellata cross than V. radiata�V. umbellata.Measures to overcome incompatibility barriers before fertilization in these crosses to produce inter-specific hybrids are discussed.

Key words Inter-specific crosses, Pre-fertilization barriers, Fluorescence microscopy, Pollen tubegrowth, Vigna species

Pulse crops provide easily digestible, high quality protein for human consumption, green nutri-tious fodder for animal and enriches the soil through biological nitrogen fixation, hence are calledas ‘poor man’s meat’ and ‘Unique Jewels’ of Indian crop husbandry (Swaminathan 1981). Legumesplay a central role in low input production systems, particularly on small-scale farms (Graham andVance 2003). Grain legumes rank third among the grain crops behind cereals and oilseeds in worldproduction. The Vigna radiata (Greengram/Mungbean) and Vigna mungo (Blackgram/Urdbean)constitute an important dietary constituents for human which was grown in 3.21 and 3 m·ha areawith a production of 0.98 and 1.37 mt, while its productivity is very low around 482 and453 kg ha�1, respectively (Ali and Kumar 2001). This may be due to dearth of high yielding vari-eties, lack of resistance to pest and disease and cultivation under rain-starved, unhealthy soil. InIndia, Mungbean Yellow Mosaic Virus (MYMV) and bruchids (Callosobruchus spp.) cause heavydamage to both the cultivated Vigna species and estimated the yield loss up to 85% (AVRDC 1998).

Wide hybridization has an important role in producing new association of plant characteristicsthat are outside the range of variability of the parental species (Asthana et al. 1994). The introgres-sion of wild and related species of Vigna has also been reported from the 1970s (Ahuja and Singh1977, Ahn and Hartmann 1978). Some of the germplasm accessions have the cultivated form of

* Corresponding author, e-mail: [email protected]

© 2008 The Japan Mendel Society Cytologia 73(3): 251–257, 2008

Vigna species which are good plant type andresistant to pest and disease (Umamaheshwari2002, Somta et al. 2007). The use of divergentspecies namely Vigna umbellata (Ricebean) ismore desirable for introgression breeding dueto no linkage drag of undesirable traits such aspod dehiscence and it is widely consumed byhuman than the other wild species (Watanasitand Pichitporn 1996). This species were foundin Western Ghats, Eastern Ghats, and NorthWestern Himalayas, which act as a potentialsource of resistance to disease such as MYMVand to insect pest such as bruchids (Monika etal. 2005, Somta et al. 2007).

The inter-specific cross hybridization andintrogression are meant to tap genes of pestand disease for cultivars improvement. In pre-vious attempts made between divergent rela-tive species of V. umbellata germplasm acces-sion and cultivated species of V. radiata and V.mungo have been less successful due to lowcrossability (Umamaheshwari 2002). An un-derstanding of the reproductive isolation barri-er systems that hamper wide hybridization andseed development would certainly assist in theproduction of inter-specific hybrids of Vigna.This could be due to both at pre-fertilizationand post-fertilization levels. The mechanism of pre-fertilization barriers was observed inwide crosses namely inhibition of pollen ger-mination, delayed pollen tube growth andstructural aberrations of pollen tubes in Cotton(Gunasekaran 1997) and in Sesame (Rajesh-wari and Ramasamy 2004, Ganesh Ram et al.2006). Considering the above, the present in-vestigations have been made with the objectiveto study the pollen pistil interaction of V. umbellata pollen tubes at various stages of pis-tils of V. radiata and V. mungo to determinewhether reproductive isolation barriers are in-hibiting the production of inter-specific hybridderivatives.

Materials and methods

Details of the plant materials used and itscharacteristic features are furnished in Table 1.The cultivated Vigna used were V. radiata cvCO 5 (2n�22) and V. mungo cv VBN1

252 Cytologia 73(3)Krishnasamy Thiyagu et al.

Tab

le1.

Det

ails

of

Vig

nasp

ecie

s us

ed in

the

stud

y

Sci

enti

fic

Com

mon

Var

iety

/C

hrom

o-

Poll

en

Cro

ss c

ompa

tibi

lity

wit

h so

me

num

ber

fert

ilit

y Fe

atur

esna

me

nam

ena

me

(2n)

(%)

Vig

nasp

ecie

s

V. r

adia

taG

reen

gram

/C

O 5

2290

.25

Ear

ly m

atur

ity,

sub

-ere

ct, g

labr

ous,

hig

h pr

otei

n an

d C

ross

es w

ith

the

wil

d V.

rad

iata

var.

subl

obat

aas

M

ungb

ean

susc

epti

ble

to M

YM

V a

nd b

ruch

ids

poll

en p

aren

t and

wit

h V.

ang

ular

is, V

. um

bell

ata,

V.

mun

goan

d V.

tril

obat

aas

see

d pa

rent

V. m

ungo

Bla

ckgr

am/

VB

N1

2289

.15

Wel

l ada

pted

, hig

h yi

eld,

ear

ly m

atur

ity,

hig

h pr

otei

n C

ross

es w

ith

the

wil

d V.

tril

obat

a, V

. mun

gova

r. U

rdbe

anan

d su

scep

tibl

e to

MY

MV

and

bru

chid

ssy

lves

tris

as s

eed

pare

nt a

nd w

ith

V. r

adia

taas

pol

len

pare

ntV.

um

bell

ata

Ric

ebea

nR

BL

122

88.5

5G

ood

plan

t typ

e, d

eter

min

ate

grow

th, g

labr

ous,

twin

y,

Cro

ss c

ompa

tibl

e w

ith

V. a

ngul

aris

as s

eed

pare

nt o

nly,

sl

ight

ly c

urve

d po

ds, h

ighe

r in

fol

iage

, ter

min

al

and

wit

h bo

th V

. rad

iata

and

V. m

ungo

as p

olle

n pa

rent

. le

aflet

leng

th, p

etio

le le

ngth

, num

ber

of fl

ower

s pe

r N

on-v

iabl

e, h

ighe

r le

thal

ity

of s

eeds

in V

. rad

iata

race

me,

pod

s pe

r pe

dunc

le a

nd p

od le

ngth

, see

d si

ze,

cros

s, w

here

as h

ybri

d st

eril

ity

in V

. mun

gocr

oss.

seed

wei

ght,

hilu

m le

ngth

and

yie

ld p

er p

lant

. Im

mun

e to

MY

MV

and

res

ista

nt to

bru

chid

s

(2n�22), a well adapted high yielding cultivars and the cultivated divergent relative species usedwas V. umbellata cv RBL 1 (2n�22), a determinate, twiny and resistant to MYMV and bruchids.The parents used in this study were maintained as homozygous stocks by continuous selfing, in theDepartment of Pulses, Tamil Nadu Agricultural University, Coimbatore, India.

Crosses were attempted between V. radiata�V. umbellata as well as V. mungo�V. umbellata.Artificial pollination was carried out following the method suggested by Bolling et al. (1961). Forhybridization, the flowers in V. radiata and V. mungo (female parents) were emasculated in theevening, between 3–5 PM. The pistils were closed using petals after emasculation to avoid contami-nation. Next day morning between 7–9 AM, the pollen from V. umbellata (male parent) was trans-ferred to the stigma of emasculated flowers. Similarly self-pollination was done at the same timeand in the same fashion for all the parents. Crosses were made between female and male parentsunder field condition to calculate percentage of pod set. The pollen fertility was assessed in threeparents using 1% I2-KI solution. The stained pollen grain was considered as fertile whereas un-stained as sterile.

2008 253Pollen Pistil Interaction in Inter-Specific Crosses of Vigna sp.

Table 2. Pollen tube growth in V. radiata, V. mungo, V. umbellata and their crosses

Genotypes/Pollen tube growth (Hours After Pollination: HAP)

Crosses2 4 6 8 10 12 18 24

V. radiata selfed � ��

V. mungo selfed ��

V. umbellata selfed � ��

V. radiata�V. umbellata � � � ��

V. mungo�V. umbellata � � � � ��

Where, �, pollen grains not germinated on stigmatic region; �, pollen grains germinated on stigmatic region; �, inhi-bition of pollen tube growth either due to stigmatic exudation or bend back of pollen tube growth on stigmatic region;�� pollen tube growth in the upper stylar region; ��, inhibition of pollen tube growth due to degeneration or lateraltube growth in the upper stylar region; ��, pollen tube growth in the middle stylar region; ��, minimum numberof pollen tube reached in the middle stylar region; ��, pollen tube growth in the bottom stylar region; ��,minimum number of pollen tube reached the ovary; ��, pollen tube reached the ovary and fertilized.

Fig. 1. Time taken for the growth of pollen tube in parents and crosses of Vigna spp.

The pollinated flowers were collected after 2, 4, 6, 8, 10, 12, 18 and 24 h after pollination(HAP) and were fixed in acetic acid, alcohol (1 : 3 v/v) fixative. The pistils were separated from theflowers and stored at 4°C for 12 h. For every collection time, 10 crossed and self-pollinated flowerseach from five tagged plants of V. radiata and V. mungo were used for microscopic observations.The pistils were transferred to 70% ethanol and stored at 4°C, until further analysis. In order to findout the pre-fertilization barriers in wide crosses of Vigna, aniline blue fluorescence technique wasfollowed. The methodology described by Parani (1998) for Sesame was modified slightly for Vignawith respect to softening period with sodium hydroxide (NaOH) and tri basic potassium phosphate(K3PO4).


Plate 1. a–l) Pollen tube growth of parents and crosses. V. radiata: Self pollinated. a) Pollen tube growth instigmatic region (2 HAP); b) pollen tube growth in middle stylar region (6 HAP); c) pollen tubereach ovule (10 HAP). V. mungo: Self pollinated. d) Pollen tube growth reached upper stylar region(2 HAP); e) pollen tube growth in bottom stylar region (6 HAP); f) pollen tube reach ovule (8HAP). V. radiata�V. umbellata cross. g) Inhibition of pollen tube due to stigmatic exudation instigmatic region (6 HAP); h) degeneration of pollen tube growth in upper stylar region (8 HAP); i)pollen tube reach ovule (12 HAP). V. mungo�V. umbellata cross. j) Bend back of pollen tubegrowth to opposite direction in stigmatic region (8 HAP); k) lateral expansion of pollen tubegrowth in upper stylar region (10 HAP); l) fertilized ovule (24 HAP).

After fixation, the pistils were washed with distilled water and treated with 6 N NaOH solu-tions for at least 9 h to clear and soften the tissue. The softened pistils were put in distilled water for1–2 h, giving 4–5 washings and then stained with 0.1% water-soluble aniline blue prepared in 0.1 MK3PO4 for 24 h. The stained pistils were mounted in a drop of 50% glycerol on a glass slide andwere covered with cover slip, which was pressed gently into a thin layer. The slides were kept indark until observed under a dark field microscope (Olympus BX 60) with fluorescent attachmentusing 390–420 nm barrier filter coupled with a 450 nm excitation filter. The pollen tube growth wasobserved and images were captured with an Olympus digital camera model E500.

Results

The crosses were made between V. radiata cv CO 5 and V. mungo cv VBN1 (as female) and V.umbellata (as male) under field condition to produce hybrids. The crosses between V. radiata and V.umbellata showed 12.89% pod set whereas V. mungo and V. umbellata recorded 5.56%. V. radiata, V.mungo and V. umbellata parental species noticed 90.25%, 89.15% and 88.55% pollen fertility re-spectively, this is normal to effect fertilization.

The in vivo pollen tube growth study was observed in selfed-parents as well as crosses underfluorescence microscope. The pollen grain germination upon self-pollination in parental specieswas normal at the stigmatic region (Fig. 1, Table 2). In V. radiata, pollen tubes passed through thestigmatic surface within 2 HAP, and reached the bottom stylar region within 8 HAP, then reachedthe ovary and get fertilized within 10 HAP (Fig. 1, Plate 1a–c), and similar observations were alsoobserved in V. umbellata (figure not shown). In V. mungo, pollen tubes passed through the stigmaticsurface and reached upper stylar region within 2 HAP, the bottom stylar region reached within 6HAP, then reached ovary and get fertilized within 8 HAP (Fig. 1, Plate 1d–f).

Germination of V. umbellata pollen grains upon pollination with V. radiata as well as V. mungowas normal. In cross between V. radiata and V. umbellata, the rate of pollen tube growth was re-duced at all levels in V. radiata pistils (Fig. 1, Table 2). Pollen tube passed through the stigmatic sur-face only at 4 HAP, and even at 10 HAP growing pollen tubes were still observed in the mid stylarregion (Table 2). Inhibition of pollen tube penetration and accumulation of stigmatic exudation instigmatic region (Plate 1g, Table 2) were observed. The bursting and degeneration of pollen tubeswere also commonly observed in upper stylar region (Plate 1h, Table 2). The minimum number ofpollen tube reached the ovary at 12 HAP and fertilization taken place within 18 HAP (Plate 1i,Table 2).

The rate of pollen tube growth was drastically reduced in all stages of V. mungo�V. umbellatacross and most frequently in stigmatic region (Fig. 1, Table 2). The pollen tube passed through thestigmatic region only at 8 HAP, and the pollen tube penetrating even at the upper stylar region 10HAP (Fig. 1). The alien pollen tubes growing with reverse direction towards the stigma were ob-served (Plate 1j, Table 2). Pollen tubes with swollen and collapsed pollen tubes were frequently no-ticed (Plate 1k, Table 2). Within 18 HAP, the reduced number of pollen tubes passed to the ovularregion and effected fertilization to minimum number of ovules in 24 HAP (Plate 1l, Table 2).

Discussion

Fertilization between diverse species within the same genus will be successful only if there issome means of compatibility between both the parents. The successful completion of a series of se-quential events following pollination requires a perfect blend of co-ordination between genes andgene complexes of pollen and the ovule parents (Hogenboom 1973). The present investigation wasmainly focused on the rate of pollen tube growth and structural abnormalities of alien pollen on thecultivated pistils to understand how the reproductive isolation barriers operate on pollen tube pene-


tration in different stages of pistils. The divergent relative species namely V. umbellata confers moredurable resistance to insect pests and diseases (Watanasit and Pichitporn 1996). In the present study,low pod set was observed in V. radiata�V. umbellata (12.89%) and V. mungo�V. umbellata (5.56%)crosses. However the pollen fertility was found to be normal. Low pod set observed in the abovecrosses could be due to the operation of pre-fertilization barriers. Similar results were noticed in theinter-specific crosses of Vigna species (Umamaheshwari 2002).

In V. radiata�V. umbellata cross, the pre-fertilization barriers at all levels of tissue were signif-icant, as indicated by moderate pollen tube penetration and growth. The slow rate of pollen tubegrowth was noticed in inter-specific crosses of rice (Sarker et al. 1983) and maize (Manickam1996). Although the pollen tube growth was slowed down by the structural aberrations like stigmat-ic exudation and degeneration of pollen tubes (Plate 1g and h), eventually pollen tube penetratedinto the bottom stylar tissues and reached the ovary. Similar types of structural malformations werereported in Cotton (Gunasekaran 1997). Umamaheshwari (2002) reported low percentage of pod setin the same cross using different accessions of parents. The techniques such as pollen mixture, application of growth hormones and protoplast fusion may rectify the defects before fertilizationand lead to development of new inter-specific hybrids from related species.

The pre-fertilization barriers were predominant in V. mungo�V. umbellata cross and operatedin all the stages as delayed or inhibited penetration of pollen tube growth and disorientation ofpollen tubes in stylar tissues. The pre-fertilization barriers were not restricted to any one particularstage of development, but had operated very gradually and mildly in varying degree. The commonpre-fertilization barriers found to occur in many wide crosses are pollen-pistil incompatibility.These barriers are influenced by delayed pollen grain germination and pollen tube growth of onespecies on the stigmas of another species (Monika et al. 2005, Hodnett et al. 2005).

Events starting at pollination and terminating at fertilization in wide crosses involve complexand harmonious interactions between the microgametophyte and the sporophyte of the pistil parent.It is understood that proper recognition of protein substances, pollen tube penetration on the stigmaand co-ordination between the pollen and pistil proteins are necessary to effect normal pollen tubegrowth. When pollen grains succeed in crossing the stigmatic surface, growing pollen tubes areguided to the micropyle by signals originating in the style and embryo sac (Lord and Russell 2002).During discordant situations, normal metabolism of the pollen tube was obstructed and pollen tubecollapsed, reducing further growth to the micropyle. Hodnett et al. (2005) indicated that adversepistil-pollen interactions that include the pollen tube growth inhibition in wide crosses, which mightbe viewed as consequence of inharmonious genetic interactions due to genetic divergence of thespecies involved.

In the present study, apart from the slow rate of pollen tube growth several other types of aber-rations were observed in V. mungo�V. umbellata cross. Alien pollen tubes (V. umbellata) typicallyhad shown a reverse direction of growth path towards the apex of stigma (Plate 1j). Such irregulari-ties in pollen tube development were furnished in pistils of cultivated sorghum with the pollen tubeof Sorghum intrans and S. interjectum wide crosses (Hodnett et al. 2005) and also in Gossypium spp.(Gunesekaran 1997). Another common aberration of alien pollen tube growth in cultivated Vignapistils observed in this study was the lateral expanded or swollen pollen tubes in stylar tissue (Plate1k). This phenomenon has been found in other species as well. The cross between wheat (Triticumaestivum) and rye (Secale cereale), swollen rye pollen tubes has been noticed in wheat pistils (Jalaniand Moss 1980). Wild species of Sesame (Sesamum laciniatum) swollen tube and a twisted growthpattern was found in cultivated Sesame (Sesamum indicum) style (Ganesh Ram et al. 2006). Al-though some degree of abnormalities observed in the present study namely slow rate of pollengrowth and structural aberrations, the least number of pollen tubes were reached the embryo sac andfertilized one or two ovules. It was evidenced by the presence of few seeds in the crossed pod. Lowseed set per pod was observed in crosses of different Vigna species (Umamaheshwari 2002).


It was suggested to use techniques such as bud pollination, application of growth hormones, invitro fertilization and protoplast fusion (Chen et al. 1989) to promote pollen germination, andpollen tube penetration in stylar and ovular tissues to effect fertilization in the wide crosses withpronounced pre-fertilization barriers. Hence, the use of one or a combination of these techniquescould be utilized for the production of inter-specific hybrids and introgression of MYMV andbruchids resistance genes from the V. umbellata to the cultivated V. radiata and V. mungo.

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Pollen Pistil Interaction in Inter-Specific Crosses of