Crossability Barriers in Interspecific Hybridization of Ricebean [Vigna umbellata (Thunb.) Ohwi and Ohashi] with Other Vigna Species

Ricebean [Vigna umbellata (Thunb.) Ohwi and Ohashi] is a multipurpose grain legume of Mid-Himalayan region mainly cultivated for food, fodder, green manure and has emerged as a good alternative to other pulse crops such as blackgram and greengram which do not flourish in this region due to their susceptibility to cold temperature stress. It is a warm season annual legume mainly grown in East Asia (Chen et al., 2016). It is an annual pulse of high nutritional status which has remained in general, neglected for its improvement by breeding either by hybridization or other methods. Legumes are significant source of carbohydrates and largest producer and consumer of legumes in the world are in India. It well reported that the nutritional value of rice bean is higher as compared to many other legumes of the Vigna family and has some superior qualities greater than greengram, blackgram and cowpea. It is also resistance to drought, pests and diseases during growth period, synchronising habit of pod maturity, resistance to attack of storage pests and a high percentage of seed viability. 
 
Despite having all the favourable traits; it is not much popular among the farmers due to the late maturity and indeterminate growth habit. Since variability for these traits is very low in this crop, inter-specific hybridization of ricebean with other Vigna species for the incorporation of earliness, synchronous maturity and non-shattering can lead to its improvement. Thus, in the present study inter-specific hybridization was performed between ricebean, adzukibean, mash and mung to introgress desired traits into otherwise high yielding ricebean genotypes.

Sowing plan
 
Eight ricebean (Vigna umbellata), two urdbean (Vigna mungo) and one adzukibean (Vigna angularis) genotype(s) were sown during the year 2015 in the glasshouse, Department of Organic Agriculture, CSK HPKV, Palampur, Himachal Pradesh (Table 1). Staggered sowing was done at seven days interval starting from first week of November to first week of December, 2015. The accessions of ricebean were sown twenty days prior to each sowing date of urdbean and adzukibean so as to synchronize flowering.
 

 
Crossing plan
 
Inter-specific crosses among ricebean (V. umbellata), urdbean (V. mungo) and adzukibean (V. angularis) were attempted under glasshouse conditions by performing emasculation from 3-5 PM and pollination at 8-9 AM upon flowering.
 
Data recording pertaining to development of F₁ hybrids
 
Data were recorded with respect to:
Number of flower buds emasculated and pollinated.
Number of mature pods obtained.
Number of mature seeds obtained.
 
Sowing of F₁ hybrids
 
F₁ hybrids were evaluated during kharif, 2016 in the Experimental Farm, Department of Organic Agriculture, Palampur. The inter-specific hybrids along with their parents were raised in pots containing mixture of soil, sand and vermi-compost in 2:1:1, in a Completely Randomized Design (CRD) with unequal replications as number of F₁ seeds varied for different crosses (Image 1).


 
Recording of observation
 
Data was recorded for various morphological traits on five randomly taken plants for each F₁ progeny and parent.
 
Molecular characterization of F₁ hybrids
 
The parents as well as their F₁’s were subjected to confirm hybridity through SSR markers using standard protocol. Forty four randomly chosen primers were screened, out of which six were found polymorphic (details of SSR primers is given in Table 2). The experiment was carried out in the molecular Lab of the Department of Genetics and Plant Breeding, CSK HPKV, Palampur, Himachal Pradesh.
 

Generation of inter and intra-specific hybrids and crossability studies
 
Since the main objective of the present study was to incorporate earliness and determinate habit into Vigna umbellatta; the donor parents used were genotypes from Vigna mungo and Vigna angularis. Thus, crosses of six genotypes of Vigna umbellate (RBHP-36, RBHP-38, RBHP-43, RBHP-61, RBHP-107 and RBHP-108) with Vigna mungo (Him Mash-1 and Palampur-93) and Vigna angularis (HPU-51) were attempted with the objective to transfer genes for earliness and determinate trait, under glasshouse conditions in the year 2015.
 
The crossability percentage (Table 3a and 3b) ranged from 0-4%. In the present study the crossability was possible only among the genotypes of Vigna umbellata and Vigna mungo. Differences in percentage of pods harvested were highly significant among the crosses indicating presence of reproductive barriers that renders introgression difficult (Thiyagu et al., 2008). The cross combinations RBHP-36 × Him Mash-1, RBHP-107 × Him Mash-1 and RBHP-108 × Him Mash-1 resulted in highest pod set percentage. It was observed that pod set percentage was maximum when ricebean was used as female parent as compared to the crosses where urdbean was used as female parent. In most of the cross combination the pod set percentage was 0% when urdbean was taken as female. In remaining crosses the pods developed normally but the seeds obtained were wrinkled.

The F₁’s showed very low percentage of seed germination (Table 4) ranging from 20 to 42.85 per cent. The germination percentage was recorded highest one in the cross RBHP-61 × Palampur-93 (42.85%) while lowest one in the cross Palampur-93 × RBHP-38 (20%).
 

 

 
Reciprocal crosses among Vigna umbellata and Vigna mungo showed lowest pod set percentage ranging from 0 to 3 per cent. The reciprocal difference in crossability between ricebean and urdbean suggests interaction between genic and cytoplasmic factors (Stebbins, 1958). This interaction may be the cause of hybrid embryo degeneration when Vigna mungo is used as the female parent (Ahn and Hartmann, 1977). There are no external barriers, which prevent cross-pollination between  Vigna umbellata and Vigna mungo because the timing of anthesis, dehiscence of anthers and receptivity of the stigma are identical for both the parental species. Absence of seed set and abscission of crossed flowers within four days from pollination in crosses Vigna mungo × Vigna umbellata indicates that complete sterility is the result of delay in pollen tube entry into the ovule. This may be  due to difference in length of style of Vigna umbellata and Vigna mungo which leads to inability of pollen tube to germinate and penetrate stigma and style (Chowdhury and Chowdhury, 1977) and ovary (Gopinathan et al., 1986) and slow rate of pollen growth (Thiyagu et al., 2008). These factors are reported to be significant pre-fertilization barriers.

The failure of endosperm nuclei to divide or the delayed endosperm nuclear divisions are responsible for abortion of embryo and the subsequent abscission of young fruits in the interspecific crosses. The production of shrivelled seeds from these crosses is probably associated with the failure of embryo to reach maturity. Such sterility barriers have also been recorded in the interspecific crosses between Vigna radiata × Vigna umbellata by Bharathi et al., 2006.
 
Inter-specific hybridization possesses the presence of pre-fertilization barriers confirmed by the frequency of pod set and post-fertilization barriers as F₁’s exhibited reduced germination and sterility. The frequency of inter-specific hybridization and radical and plant production percentage revealed the genotype specific response of both the species (Shayla, 2016). Thus, recovery of desirable recombinants is reduced, as hybrids exhibit varying levels of sterility (Rashid et al., 2013), inviability, lethality and genotype specific response (Dhiman et al., 2013). These different kinds of pre and post fertilization barriers are also responsible for complete sterility to low fertility in the back crosses involving the F₁ hybrid and both its parents.
 
Confirmation of inter-specific hybrids through SSR markers
 
Out of 70 SSR markers used for parental polymorphism survey, 8 were found polymorphic between different parents for inter-specific hybrids. These polymorphic markers were used for F₁ hybrid confirmation. F₁ hybrids were confirmed using two primer pairs namely CEDG043 and CEDG037 polymorphic between parents and hybrids. Both these primers showed robust and reproducible bands as shown in Plate 1.  All the inter-specific hybrids were true hybrids as confirmed by molecular marker analysis. Similar result was observed by Chaisan et al., (2013) who confirmed hybridity of inter-specific hybrids between mungbean and ricebean. They screened forty random amplified polymorphic DNA (RAPD) primers for polymorphism among the parents and finally choose two specific primers for testing of hybridity. All putative F₁ hybrids were confirmed as the inter-specific hybrids. To observe their fertility, some of the hybrid seedlings were transplanted. The hybrid produced flowers profusely but failed to set pods. To overcome the sterility, plants were induced to become tetraploid by colchicine treatment in vitro. Nandini et al., (2020) also used SSR markers to detect water use efficiency in parental lines. Similarly Prine Lekhie et al., (2018) also attempted interspecific hybridization in Vigna radiata and V. mungo genotypes. The purity of hybrids were tested through microsatellite markers. All the F1 plants gave resistant reaction to mungbean yellow mosaic virus (MYMV) indicating the introgression of resistance gene(s) from V. mungo to V. radiata.


 
Sterlity in inter-specific crosses is the result of various pre and post fertilization barriers (Nishant Bhanu et al., 2018). Though very little success has been achieved till date in this field but it is necessary to create a wide genetic variation for breeding programs through interspecific hybridization specially in the crops like ricebean where genetic variability is low but the production potential is very high. Alternative methods like embryo rescue, ovary and ovule culture and chromosome doubling holds considerable promise for the development of new cultivars incorporating genes from wide species.