Development of yellow mosaic virus Resistant Genotypes in Mungbean through Interspecific Crosses of Wild Vigna Species. HSOA Journal of Plant Science Current Research Development of Mungbean yellow mosaic virus Resistant Genotypes in Mungbean through Interspecific Crosses of Wild Vigna Species

Mungbean Yellow mosaic disease is a main destructing viral disease in mungbean caused by Mungbean yellow mosaic virus (MYMV) which leads to severe sometime 100 percent yield reduction and it necessitates for developing MYMV resistant lines. The present investigation was carried out with an objective of evolution of MYMV resistant progenies through incorporating wide genes of same genera. An attempt was made between V. radiata x V. umbellata crosses. This agroinoculation technique was employed to examine the F 2 individuals, which were derived from a cross between VMGG012-005 (Moderately susceptible) x VGGru1 (resistant homozygous advanced line from Vigna radiata x Vigna umbellata derivative used as MYMV donor) to screen for the MYMV resistant progenies. In the field condition, MYMV infection can be evaluated by MYMV disease rating scale (1-9). Out of the 225, F 2 individuals, 48 individuals were identified as resistant to MYMV and subjected to agroinoculation. The two tandem viral constructs of MYMV, VA 221 (KA30 DNA A + KA22 DNA B) and VA 239 (RA30 DNA A + KA27 DNA B) mobilized in Agrobacterium tumefaciens strains Ach 5 and C 58 were used for Agroinoculation. The results shows that among the 48 individuals, only ten individuals namely Resplant3, Resplant6, Resplant7, Resplant9, Resplant13, Resplant19, Resplant25, Resplant37, Resplant40 and Resplant43 showed resistance to VA 221 and the same entries found to be susceptible to VA239 strain. The all 48 entries again raised as F 3 generation in next season in the field it showed resistant to MYMV. Other 177 individuals are susceptible to both strains, VA 221 and VA 239. All these field resistant individuals are forwarded to further generations and confirmed for resistance in field condition. In F 3 generation only ten genotypes which was showed resistance for VA221.


Introduction
Mungbean (Vigna radiata L.) is an important pulse crop in developing countries of Asia especially India, China, Japan , Myanmar and other countries and Latin America where it is consumed as dry seeds, fresh green pods [1]. Mungbean serves as vital source of vegetable protein (19.1-28.3%), mineral (0.18-0.21%) and vitamins. It is a native of India -Burma and is cultivated extensively in Asia [2]. India is the leading mungbean cultivating country, for mungbean improvement research has elaborately taken by mungbean breeders in India with covers up to 55% of the total area [3]. The World acreage and 45% of total production [4]. Among the biotic sources plant viruses are responsible for a significant proportion of crop disease [5]. It causes serious economic losses in many major crops by reducing economic part like seed yield and quality [6]. Mungbean yellow mosaic virus Disease (MYMV) is reported to be the most destructive viral disease among the various viral diseases, caused by Mungbean yellow mosaic virus. It causes severe yield reduction in all mungbean growing countries in Asia including India [7]. MYMV belongs to the family Geminiviridae [8]. The family Geminiviridae is divided into four genera (Mastrevirus, Curtovirus, Topocuvirus and Begomovirus) based on genome structure, type of insect vector and host range [9]. Begomovirus is the largest genus of the family Geminiviridae [10], which is characterized by a bipartite genome (DNA-A and DNA-B) or monopartite genomes that were transmitted in a circulative persistent manner by white fly Bemisia [11]. Conventional breeding methods are unsuccessful in developing Yellow Mosaic Virus (YMV) resistant mungbean lines due to rapid explosion of new isolates and also the complexity of mechanism in controlling the resistance to MYMV [12]. The major problem encountered by scientists to develop the MYMV resistant variety is the identification of MYMV resistant lines in segregating population. Identifying the resistant lines is very complicated task due to the lack of reliable screening protocol for assessing the resistance/susceptibility against MYMV. Hence the scientists are in need of any biological/molecular tool that can lead to screening of the resistant or susceptible lines for MYMV. Developing host resistance to the disease or the vector has therefore been considered as the only solution to control this disease [6]. Plant genetic transformation is of particularly benefit to molecular genetic studies and crop improvement programmes [13]. The entry VMGG012-005 is a extra early duration genotype, tolerant to high temperature with drought. If improve this culture for high level of MYMV resistant which can be used directly as variety and also used as donor for short duration, drought tolerant and high temperature resistant along with MYMV donar.
The emerging field or genetic engineering endows with a new technique agroinoculation was successful for screening virus resistant plants [14,15]. Jacob SS et al., [16] demonstrated the feasibility of using an in vitro molecular protocol to screen for resistance/susceptibility against. MYMV and proved that agroinoculation can be successfully adopted for screening MYMV resistant mungbean genotypes. Exploitation of this reproducible and less expensive technique may lead to the development of a MYMV resistance genotype. With this background knowledge this present investigation was carried out with the aim of identifying the MYMV resistant progenies through agroinoculation.

Plant materials
The experimental material for the present investigation consisted of 225 F 2 individuals, 48 F 3 progenies and parents VMGG012-005 (short duration, high temperature drought tolerant genotype and moderately susceptible to MYMV), high yield under high temperature with drought condition. The entry VMGG 012-005 is derived from Vigna radiata (VRM (Gg)1) x Vigna radiata (Pusa bold), developed for the purpose of Moderately resistance / tolerance to Mungbean yellow mosaic virus. It was developed through intraspecific hybridization followed by pedigree selection and VGG ru1 ( high level MYMV resistant derivative of Vigna radiata x Vigna umbellata), which were raised at Agricultural Research Station , Virinjipuram, Tamil Nadu Agricultural University, Vellore, and VMGG012-005 developed at Virinjipuram is an agronomically superior high yielding with short duration variety but Moderately susceptible to MYMV, tolerant to drought and also high temperature tolerant with shortest duration. VGGru 1 is a field resistant genotype to MYMV.

Phenotyping of mapping population
In the field condition, the MYMV infection can be evaluated by infector row method as described [12]. The variety Co5 greengram is used as susceptible check at augmented design. The test materials were scored after 80% of plants showed MYMV incidence. The 225 individuals and progenies in the F 2 and 48 F 3 generation plants were scored for MYMV infection using 1-9 rating scale [17] is adopted.

Agroinoculation
The study on agroinoculation was conducted in the Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. The tandem viral constructs of MYMV, VA 221 (KA30 DNA A + KA22 DNA B) and VA 239 (KA30 DNA A + KA27 DNA B) mobilized in Agrobacterium tumefaciens strains Ach 5 and C 58 were collected from Madurai Kamaraj university, Madurai and used for further studies. Agroinoculation was done on surface sterilized overnight sprouted seeds of the parents (VMGG012-005 and VGGru 1) and F 2 individuals. Agrobacterium tumefaciens strains harbouring the appropriate partial tandem repeat clone were grown to 1 Optical Density at 600 nm in 2 mL AB minimal medium pH 7.0 containing the antibiotics like streptomycin (150 mg L -1 ), spectinomycin (50 mg L -1 ) and tetracycline (5 mg L -1 ) at 28°C at 220 rpm. From this, 1 mL of the culture was taken to inoculate, another 50 mL of AB minimal medium (pH -7.0) containing the above mentioned antibiotics and grown to 1 OD L 600 nm at 28°C at 220 rpm. The culture was spinned at 4000 rpm for 10 min at 25°C. Cells obtained were re-suspended in 50 mL of AB minimal medium (pH -5.6) with 100 in acetosyringone (100 µrn). Seed coat of the sprouted seeds was removed by using forceps and pricked around the hypocotyl region and were immediately immersed in the appropriate culture of A. tumefaciens. After the overnight incubation, seeds were washed with distilled water and sown in pots containing autoclaved sand and vermiculite in the ratio of 1:1. Agroinoculated plants were maintained in a growth chamber at 25°C, 60-70% relative humidity and a photoperiod of 16/18h. The Hoagland's solution was applied twice in a week for proper growth of the plants and transferred to green house after 15 days for symptom observation [6] and used the same protocol for agroinoculation test [6]. For this study also followed the same inoculation procedure.

Results and Discussion
Most of the ruling mungbean varieties are susceptible to MYMV accepted by the farmers. It is necessitates for developing MYMV resistant varieties because mungbean is important pulse food crop in Asia. Most of previous screening studies for MYMV resistance in Asia were conducted in field condition under high MYMV epidemic pressure. Evolution and identification of MYMV resistant lines through conventional breeding method relies on field screening. Even though it is time consuming one and requires evaluation at hot spot area [12]. Sometimes the screening based on natural occurrence in the hot spot areas also does not give consistent results due to the stain presence and environmental condition. For identifying the real resistant source and combination with plant breeding approaches will likely to be needed for the improvement of crops [18,19]. Interspecific hybridization in Vigna radiata x Vigna trilobata for MYMV donar development study revealed same result for many characters reported by [20]. Diversity analysis also made among the Wild Vigna species for many characters same results were reported [21].
Pathogen Derived Resistance (PDR) is a very effective genetic engineering approach to control plant viruses [22]. Advancements in the field of genetic engineering provide a new technique called agroinoculation. It is an effective method by which infectious viral clones can be introduced into plants using A. tumefacians [14,23]. reported agroinoculation is all efficient method to employ the virus resistant plants. In earlier report successful agro-inoculation of mungbean with MYMV was recorded by [16,24] proved for MYMV studies in mungbean. Genetic transformation in combination with conventional breeding increases the efficiency of the breeding programme especially the incorporation of the disease resistance into the varieties and is the most predominant and a powerful tool to achieve the goal. The parents VMGG012-005 special features (Table 1) and VGGru 1 initially subjected to agroinoculation, revealed that VGGru1 did not develop any mosaic or leaf curling symptoms upon inoculation with VA 221 strain but at the same time they exhibited susceptibility against VA 239 strain. VMGG012-005 is the moderately susceptible mungbean, developed typical yellow mosaic and leaf curling symptoms in the trifoliate leaves upon agroinoculation with both the Agrobacterium strains (VA 221 and VA 239). Similar to the present finding reported [24] and also observed the leaf curling and mild yellow mosaic symptoms from agroinoculation in Vigna sp. using two different MYMV isolates. The 225 F 2 individuals with MYMV infection, evaluated under field condition indicated that 48 individuals were identified as resistant and their seeds were harvested and subjected to agroinoculation which aims to identify the MYMV resistant mungbean progenies in the segregating population.
The list of the MYMV field resistant genotypes subjected to agroinoculation screening is presented in Table 2.
The results of the agroinoculation showed that, among the 48 individuals only ten individuals showed to be resistant. The ten individuals namely Resplant3, Resplant6, Resplant7, Resplant9, Resplant13, Resplant19, Resplant25, Resplant37, Resplant40 and Resplant43 did not develop any mosaic or leaf curling symptoms upon inoculation with VA 221 strain. But at the same time they exhibited susceptibility reaction against the strain VA 239. The remaining thirty eight individuals developed typical yellow mosaic symptom for both the strains (VA 221 and VA 239). The ten resistant, individuals were found to have behaved the same way as that of their resistant, parent VGGru1. The same result revealed in mungbean [25][26][27][28].
The agroinoculated mungbean plants started developing yellow mosaic symptoms from the 17 th day to 23 th day and there were no symptoms in the control plants.

Sl.No
Name of the variety VMGG 012-005    At the 25 th day, the yellow mosaic symptoms were clearly seen on the leaves. The average infectivity of MYMV strains through agroinoculation in mungbean ranged from 0 to 100%. Average per cent infection of resistant VGGru in the strain VA 221 is 0.00 and VA 239 strain is 42.33. The moderately susceptible parent VMGG012-005 recorded an average infection of 97.00 and 100.00% in VA 221 and VA 239, respectively. In blackgram , high yield with MYMV disease resistance were achieved through interspecific cross combination, this study as similar results reported in mungbean [25,28] and in blackgram [29].  [30].

V. umbellata is single resistant parent is having both MYMV and
Bruchids resistant which is used for development MYMV resistance in mungbean this result agree [31,32]. The six wild Vigna species were taken for development of MYMV resistance in mungbean the same result exhibited [33]. In intraspecific crosses were made among Vigna radiata x Vigna radiata for MYMV resistance agree with results [19,34].Various wild Vigna species cross combinations showed various level of pollen fertility percentage agree [35]. The broadening of genetic base derived from the wild crosses followed by mutation same result agree with [31]. Thirteen wild Vigna species crossed with Vigna radiata for MYMV donar development and all the characters studied now is revealed same result for many characters reported [3]. Thirteen wild Vigna species crossed with Vigna radiata for MYMV donar development and genetic diversity for variation studied completed, this study revealed same result for many characters [27]. In blackgram , high yield with MYMV disease resistance were achieved through interspecific cross combination, this study results was similar to that [29] Table 3). These findings are nearly in close conformity with the reports [14] in agro inoculating mungbean with 71 to 95% of MYMV [19,21,25,28].  To verify the non presence of viral DNA inside the host genome phenotypic showed in the field condition at consecutive sown. The same 10 genotypes which was resistant for VA 221 raised in the field condition showed resistance again. These results are in accordance with the reports [14, 19,35].The indicating the presence of viral DNA in agroinoculated symptomatic plants and their absence in asymptomatic plants with coat protein specific primers for the DNA A and B components [19,25,36,37]. The identified resistant individuals namely, Resplant3, Resplant6, Resplant7, Resplant9, Resplant13, Re-splant19, Resplant25, Resplant37, Resplant40 and Resplant43 were similar to their resistant parent, exhibiting the presence of viral coat protein gene for both the strains. This clearly depicts their pure inheritance pattern of the resistance for MYMV. For further confirmation, the individuals which showed resistance in field (F 2 generation) and agro inoculation, were alone forwarded to next generation (F 3 ) and as per this, the ten individuals were forwarded to next generation along with five susceptibles. In F 3 generation MYMV infection can be evaluated by MYMV disease rating scale. Interestingly, the ten individuals which showed resistance in field (F 2 generation) and agroinoculation, are found to be resistant in F 3 generation and F 4 generations under field conditions also.

Conclusion
The results of this study show that the ten progenies namely Resplant5, Resplant22, Resplant28, Resplant35, Resplant88, Re-splant92, Resplant123, Resplant156, Resplant157 and Resplant5168 are confirmed for resistance in both agroinoculation and field screening. From these five progenies, a MYMV resistant mungbean genotype can probably be developed by adopting future breeding programmes.