Crossability Studies of Interspecific Hybridization among Vigna Species

A total of 80 interspecific crosses i.e., 36 each of V. radiata × V. umbellata (V. radiata as female) and V. mungo × V. umbellata (V. mungo as female) and 08 crosses of V. radiata × V. mungo (V. radiata as female) were attempted to study the crossability relationship among these three Vigna species. Among the crosses of V. radiata × V. umbellata the crossability was observed highest in HUM 12 × RBL 9 (16.27%) followed by HUM 12 × RBL 9 (15.78%). In case of V. mungo × V. umbellata, the maximum crossability of 11.36% was noticed in cross, Mash 338 × RBL 9. For V. radiata × V. mungo, the highest crossability was visualized in hybrid, ML 1464 × Mash 338 (37.5%). The study indicated that different kinds of pre and post fertilization barriers are responsible for complete sterility to low fertility. RBL 1 and RBL 9 gnotypes of ricebean showing substantially high percent of crossability and better seed set with different cultivars of mungbean and blackgram may be utilized for genetic improvement of the mungbean and blackgram.


Introduction
Legumes are next to cereals in terms of their economic and nutritional importance as human food. They also play an important role in maintaining soil fertility and sustainability of production systems. Among several pulses grown mungbean and blackgram are one of the important grain legumes grown throughout the year. Being a short duration crop they can be a better option for enhancing the pulse production of pulses. However, the total production and productivity of mungbean is affected by a number of biotic and abiotic factors. Among biotic factors, Mungbean Yellow Mosaic Virus (MYMV) transmitted through whitefly, i.e., Bemesia tabaci is a major constraint to the cultivation of grain legumes in India, particularly mungbean and blackgram. The weather parameters play a vital role in survival and multiplication of white fly and influence MYMV outbreak during monsoon season. Management of this disease is only possible by the way of reducing the whitefly population using insecticides which are ineffective under severe infestations making complete destruction due to virus. Therefore, development and use of virus resistant cultivars turns out to be the most effective and economical strategy against MYMV [1]. Basic reason for limited success had been due to the limited variability prevailed among the mungbean and blackgram genotypes used for hybridization in most of the studies.
Interspecific hybridization plays a significant role in alien gene introgression and is the probable option for transferring the desirable genes of qualitative and quantitative characters in mungbean and blackgram. Ricebean [V. umbellata (Thunb.) Ohwi and Ohashi], a long duration (90-120 days) minor legume which is genetically close to mungbean and blackgram (all three species 2n = 2x = 22) possess resistance for MYMV, CLS, bruchids, and powdery mildew. Successful hybridization primarily depends on the intercrossing potential/ crossability of the parents involved as well as development of the hybrid embryos including fertility of the F 1 hybrids and their derivatives. In interspecific crosses of food legumes failure of interspecific hybridization due to embryo degeneration is common [2,3]. Interspecific hybridization among mungbean, blackgram and ricebean with varying degree of success has been reported. Keeping this in view, the present piece of investigation was initiated to study the crossability relationship among three Vigna species viz. V. radiata (mungbean), V. mungo (blackgram) and V. umbellata (ricebean).

Materials and Methods
For the present experiment, a total of six diverse genotypes/ varieties, each of mungbean viz. Pusa 0672, ML 1464, SML 1455, HUM 12, KM 2241, TM 96-2, ricebean namely RBL 1, RBL 6, RBL 9, RBL 33, RBL 140, RBL 141 and blackgram viz. Mash 338, Mash 114, Co 5, Palampur 93, Shekhar 2 and T 9 were selected. The experimental material was planted in crossing block in cemented pots at two different dates of sowing of 10 days intervals (August 10 and August 20, 2014) at Agricultural Research Farm, Banaras Hindu University Varanasi during Kharif season, 2014 [4][5][6]. Buds of optimum size of the female parent were emasculated the day before anthesis (1600 to 1800 HRS) and pollinated in the next morning (0600 to 0800 HRS). 8 to 12 flowers per plant per day were emasculated besides picking the self-pollinated flowers/ pods to avoid any severe load. Hybridization technique using hand emasculation and pollination was followed [7]. A total of 80 interspecific crosses i.e. 36 each of V. radiata × V. umbellata (V. radiata as female) and V. mungo × V. umbellata (V. mungo as female) and 08 crosses of V. radiata × V. mungo (V. radiata as female) were accomplished. Observations were recorded on the number of buds emasculated, pollinated, pod initiated and matured pods harvested.

Results and Discussion
Introgression of desirable gene into cultivated species could lead to development of high yielding varieties coupled with resistance for biotic and abiotic stresses. Close relatives of mungbean and blackgram have been used in the breeding programme. However, recovery of successful hybrid is difficult due to crossability barriers. In spite of these technical hitches, interspecific hybridization among Vigna speices has been successfully accomplished by many workers [8,[9][10][11][12]. Further, in any interspecific hybridization, crossability is the pre-requisite for gene transfer. A better understanding of crossability relationship among the species had been helpful in opting methods for making successful crosses and also in drawing the phylogenetic relationship among species. V. umbellata possessing many desirable components coupled with resistance to MYMV, CLS and bruchids and powdery mildew can be useful in developing high yielding resistant varieties of mungbean and blackgram by transferring these genes into the cultivated species. The present investigation was carried out attempting interspecific hybridization with an objective to transfer useful traits from the V. mungo and V. umbellata into V. radiata and V. umbellata into V. mungo. The extent to crossability and ovule fertility was studied. The result of crosses pertaining to crossability and ovule fertility are furnished in (Table 1). Even though crossability barriers were predominant, it was possible to recover interspecific hybrids. The crossability of Vigna radiata × Vigna umbellata and Vigna radiata × Vigna mungo was successful only when Vigna radiata was used as female and of Vigna mungo × Vigna umbellata when Vigna mungo was used as female. The percent crossability among different sets of crosses varied from species to species. The differences in pod setting among different set of crosses might be because of wide variation in their genetic architecture leading to differences in cross compatibility. In V. radiata × V. umbellata, best combination recorded was HUM 12 × RBL 9 and HUM 12 × RBL 9 with the highest pods set percentage viz., 16.27% and 15.78% respectively. In V. mungo × V. umbellata, the maximum crossability of 11.36% was noticed in cross, Mash 338 × RBL 9. Similar crossability success were also reported in V. radiata × V. umbellata (29.63%), V. radiata × V. trilobata (8.48%), V. radiata × V. aconitifolia (7.69%) [13] and in V. radiata × V. trilobata (10.25%) [14]. similarly, highest pod set of 40.8% was observed in V. unguiculata × V. unguiculata var. spontanea [15].
Further, inter-specific hybrids involving three cultivars of urdbean (PDU-1, Palampur-93 and UG-2018) and six of ricebean (Naini, BRS-1, BRS-2, PRR-1, PRR-9301 and Local) exhibited differential response of crossability involving different genotypes [16,17]. The timings of anthesis (between 0500 to 0900 HRS), dehiscence of anthers (10 to 14 hours before anthesis) and receptivity of the stigmas (from the time of anthesis up to 6 to 8 hours after anthesis) were identical for the parental species. The length of style was different in the three species-it was 19 mm in V. umbellata, 23 mm in V. radiata and 21 mm in V. mungo. There are no external barriers, which prevent cross-pollination between V. radiata and V. umbellata, and V. mungo × V. umbellata, because the timing of anthesis, dehiscence of anthers and receptivity of the stigma are identical for both the parental species. Normal pollen germination in both selfed and cross flowers shows that the stigma does not act as barrier.
Absence of seed set and abscission of crossed flowers within 72 hours from pollination in crosses V. radiata × V. umbellata (TM 96-2 × RBL 6; Pusa 0672 × RBL 140; KM 2241 × RBL 6; ML 1464 × RBL 140; ML 1464 × RBL 141; HUM 12 × RBL 141 and SML 1455 × RBL 33) and V. mungo × V. umbellata (Mash 338 × RBL 140; Co 5 × RBL 141 and Palampur 93 × RBL 140) demonstrate that the first barrier responsible for complete sterility is the delay in pollen tube entry in to the ovules. This might be expected because of the difference in the length of style of three species. Such barriers are known in many other interspecific crosses as well [13,18,19]. In addition, relatively more number of crosses of V. radiata × V. umbellata showed high abscission of crossed flowers than V. mungo × V. umbellata which further supports that the difference in length of style is responsible for complete sterility.Pre fertilization barriers are absent in the interspecific crosses V. umbellata × V. radiata and V. umbellata × V. mungo as evident from normal pollen tube growth in both selfed and crossed flowers and low abscission rate of crosses flower within 72 hours from pollination. However, in V. radiata × V. mungo, the highest crossability was visualized in hybrid, ML 1464 × Mash 338 (37.5%). The relatively high number of pods harvested for V. radiata × V. mungo suggests that there were no barriers in crossing of these two species for the parental cultivars used. However, barriers were observed in embryogenesis as both inviable and viable seeds were produced, but completely inviable seeds in the reciprocal cross, V. mungo × V. radiata. The reciprocal difference in crossability of V. radiata and V. mungo suggests interaction between genic and cytoplasmic factors [20], which may be the cause of hybrid embryo degeneration when V. mungo is used as the female parent [2,21]. The high rate of abscission of young fruits between 3 to 30 days after pollination and low seed set in crosses of V. radiata × V. umbellata, V. mungo × V. umbellata and V. radiata × V. mungo are suggestive for the presence of post fertilization barriers. The failure of endosperm nuclei to divide or the delayed endosperm nuclear divisions is responsible for abortion of embryo and the subsequent abscission of young fruits in the interspecific crosses. The failure of embryo to reach maturity might be the probable cause of the production of shrivelled seeds from these crosses.
These Crossability barriers between the cultigen and its wild relative constitute somatoplastic sterility [22]. Such sterility barriers have been recorded in the interspecific crosses between Phaseolus lunatus × Phaseolus vulgaris [23]. No differences in pod set between the parental cultivars were found when V. radiata or V. mungo were used as the female parent. However, significant differences in numbers of seed set were obtained for the interspecific cross V. radiata × V. umbellata and V. mungo × V. umbellata. The difference between the V. umbellata cultivars as the pollen parents was highly significant. Based on the percentage pod set and ovule fertility, out of the six ricebean genotypes used, RBL 1 and RBL 9 showed substantially high percent of crossability and better seed set with different cultivars of mungbean and blackgram suggesting that these two genotypes may be utilized for genetic improvement of these crops.

Conclusion
Different kinds of pre and post fertilization barriers are responsible for complete sterility to low fertility. Despite this, novel genes and alleles from exotic germplasm and related species must be exploited and accordingly hybridization should be utilized to create a wide genetic variation for breeding programs in the Vigna species. Significant progress has been made in basic techniques of tissue culture and in development of techniques to transfer genes from more distantly related taxa. The application of embryo rescue, ovary and ovule culture, chromosome doubling and induced chromosomal exchanges through tissue culture techniques holds considerable promise for the development of new cultivars incorporating genes from wide species.