Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet]

Lablab bean [Lablab purpureus (L) Sweet var. typicus], also known as Dolichos bean, popularly known as Val in Maharashtra state, is a member of family fabaceae having chromosome number 2n=22. In India, it is grown mainly in Maharashtra, Uttar Pradesh, Madhya Pradesh, Tamil Nadu andhra Pradesh and Karnataka. In Konkan region, it is grown in Thane, Palghar, Raigad, Ratnagiri and Sindhudurg districts on residual moisture. Lablab bean has the capacity to fix atmospheric nitrogen in the soil and it can do relatively well even under poor soil fertility conditions since it is leguminous crop. Lablab bean yield is limited because of biotic and abiotic factors especially pathogenic, entomological, agronomical, genetic etc. The lower productivity of crop is attributed to many factors, among which pest is a major factor. The crop is attacked by as many as 55 species of insects and species of mites. Among them pod borer (Helicoverpa armigera) is considered to be the most important group causing loss to the tune of 80 to 100 per cent.
 
It feeds on buds, flowers and specially pods of plants. Helicoverpa have ability to complete a seven generation in a year and is present throughout the year. As there is lot of importance of the dolichos bean in agricultural economy of the Konkan region, it is necessary to apply tissue culture technique for genetic manipulation in plant to receive attention in agricultural research programs. Because of the limited genetic variability in the crop, conventional breeding is not useful. The Agrobacterium-mediated genetic transformation is more efficient and results in integration of potentially low copy number, high co-expression of the introduced genes and preferential integration into actively transcribable regions (Gheysen et al., 1998).  The variety Konkan Bhushan being highly drought tolerant, late maturing and having good yield in Konkan conditions, but susceptible to pod borer, need to be converted as pod borer resistant. Hence the research aiming at standardization of Agrobacterium mediated genetic transformation technique in the Dolichos bean Cv. Konkan Bhushan was carried out.

The present was carried out at Plant Biotechnology Center, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Dist- Ratnagiri, during the year 2014-2017. The seeds of popular genotype of Dolichus Bean “Konkan Bhushan” were collected from Vegetable Improvement Scheme, Central Experimental Station, Wakawali, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli. Fresh and healthy seeds were washed with double distilled water and then dipped in 70 % ethanol for 5 min followed by dipping in 0.1% mercuric chloride (HgCl₂) solution for 10 min and then washed with double distilled water for several times. These seeds were soaked in distilled water for 15-16 hours and then removed seed coat and isolated the mature embryo axes with single cotyledon (MEASC) as an explant for transformation. Three different strains of Agrobacterium tumefaciens having different genes viz. cry1Aabc, cry1Fa1and cry2Aa collected from NRCPB, New Delhi were used for genetic transformation experiments (Fig 1, 2 and 3). The disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used as vector system. It contains the respective gene linked to cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene under control of nopaline synthase (nos) promoter and terminator. The Agrobacterium strains were maintained in solid YEMA medium. Sub culturing was done every fortnight in fresh YEMA medium.






 
Prior to transformation events, kanamycin sensitivity test using leaf blades of normal plants was carried out with kanamycin concentration of 0, 100, 200, 300, 400, 500, 600, 700 and 800 mg/L in MS medium. Observations were recorded on seedlings died, stunted in growth or completely became albino. In Agrobacterium transformation event, initially single Agrobacterium colony was taken from the plate with the help of sterilized loop and inoculated into 25 ml YEM liquid medium containing 250 µl kanamycin (10 mg/ml) and was incubated on shaker for 48 hrs at 26°C temperature and fresh culture was used for transformation. The different five methods of colonization and co-cultivation were tried (Table 1). Agrobacterium infected explants were co-cultivated on multiple shoot induction medium MS (Murashige and Skoog, 1962) supplemented with 2 mg/l BAP in dark for 2 days. After co-cultivation, explants were washed with 800 mg/L cefotaxime for 20 min to eliminate Agrobacterium overgrowth completely and again put on medium MS + 2 mg/l BAP. Observation on plant survival rate was recorded. The leaf discs of regenerated shoots were transferred to selection medium containing kanamycin concentration found effective in kanamycin sensitivity test. Recovery of kanamycin resistant putative transgenic plants was recorded. Recovered plants were subjected to PCR analysis using gene specific primers to confirm the transformation frequency. Survival of plants and transformation frequencies for all the methods using three cry genes were compared.T₀ generation plants were grown to maturity to produce seeds from which T₁ generation was raised. Progeny analysis for gene integration was also carried out through PCR in T₁ generation. Chi-square test for Mendelian segregation ratio of 3:1 is used for finding out the goodness of fit with the formula-

 Where,
O = Observed value, E = Expected value, X2 = Chi-square value, d = Deviation.

Optimization of colonization and co-cultivation period
 
For production of transgenics, the first step is successful infection by bacterium and also successful incorporation in host genome. The site of incorporation of gene is also important for its expression. Bacterial colonization, induction of bacterial virulence system, generation of T-DNA complex, T-DNA transfer and integration of T-DNA into the plant genome are essential steps for transgenic production (Gustavo et al., 1998). Difference in susceptibility of the crop to Agrobacterium strain was also recorded in legumes such as chickpea (Islam et al., 1999), pea (Hobbes et al., 1989) and groundnut (Lacorte et al., 1991).
 
In the present transformation study, three stains of cry genes were used with five different methods as given in Table 1. Among these, cry1Fa1 showed maximum (62.50%) survival percentage when MT₁ method was followed (Table 2). However, the frequency of putative transgenics was more in MT5 (2.13%) when cry2Aa strain was used. This could be due to vir gene factors which influence the infection by different cry genes (Surekha et al., 2007).


 
It could be seen from Table 2 that the infection method employing mild injury to explant and colonization in Agrobacterium culture having cry2Aa gene for 20 minutes under aseptic conditions followed by co-cultivation for 48 hrs and after cefotaxime washing put for germination in soil (MT₅) showed highest survivability of explants (74.80%) while longer period of colonization (30 min) and 3 days co-cultivation (MT₃) reduced regeneration and survivability (17.69%). However, the lowest survival (6.00%) was observed in method MT₄ with cry1Aabc and cry2Aa gene in which bacterial suspension culture was injected into explants pre cultured in the medium supplemented with MS + 2 BAP for 2 days and co-cultivated for 48 hrs on same media. These reports are in agreement with Kumar et al., (2004), Surekha et al., (2007), Sharma et al., (2006) and Rao et al., (2005). Similar successful transformation in pigeonpea with cry1Ac gene using pricked embryo axes as explant was also reported by Kaur et al., (2016). It clearly indicates that if axillary buds properly colonized with Agrobacterium without disturbing meristematic zone with mild injury, then there will be least harmful effect of co-cultivation on regeneration. Therefore survival percentage was found more in MT₅ method.
 
Selection and identification of transformed shoots
 
In kanamycin sensitivity test, the kanamycin concentration of 500 mg/L added to MS medium turned leaf blades of normal plants completely albino (Table 3). Therefore, this concentration of kanamycin was further used for screening of putative transgenic plants. Leaf blade explants excised from well-established putative transgenic plants were subjected to kanamycin screening. Effective selection was observed at 500 mg/l kanamycin turning non transformed plants completely albino. Result of this experiment indicate that stringent selection pressure is required to identify lethal dose of kanamycin for selection of putative transgenic, in case of explants derived from well-established plants. In earlier reports viz.  Lawrence and Koundal (2001); Kumar et al., (2004); Basavanna et al., (2008) and Surekha et al., (2007) the lower concentration of kanamycin for selection of putative transgenic was mentioned. However, in the present study, at lower concentration only yellowing was observed and complete albino plants recorded at highest concentration. This indicates that dose of kanamycin for effective selection depends on age and type of tissues used for screening. At lower concentrations, tissues show only yellowing at the side of leaves and sometimes it will lead to false selection of transgenic plants. However, at the higher concentration non transgenic tissues completely became albino and confirm the integration of transgenic in green and healthy tissues only. The highest number (12) of kanamycin resistant plants was obtained in MT₂ method of transformation followed by MT₁ (Table 4). The benefit of this type of selection was that the original plants were grown without selection pressure so healthy, rooting was observed in rooting medium. It is in agreement with report of S. V. Sawardekar (2007) which emphasizes the importance of relating or even increasing concentration of selection chemical to minimize the number of putative transformed plants.
 




Recovery of transformed plants
 
In all circumstances the desired product of transformation must be regenerated to express the introduced gene. It is, therefore important that the cells, tissues and explants selected for transformation exhibit high regeneration. Frequency of the transformed cells is influenced by the method of transformation and the presence of selecting agent (e.g. an antibiotic) and other antibiotic used for elimination of Agrobacterium in the culture medium (N. K. Mhatre, 2011). Sensitivity to the transformation system of different bacterial strains may also affect the transformation frequency. The type of regeneration is also critical in the recovery of transformed plants. Transformed plants are obtained only if regeneration occurs within the region of the explants where the cells can come in contact with Agrobacterium.
 
In the present investigation, among different methods followed, MT₅ method showed highest recovery of putatively transformed plants (2.13%). This indicate that for efficient transformation and regeneration of transgenic plant, one should follow 5 important points :i) Selection of regenerable explants having meristematic tissues ii) pre-culturing of explants and use of growth regulators during co-cultivation iii) Avoidance of longer time co-cultivation and colonization iv) Avoidance of selection pressure immediately after co-cultivation and v) Elimination of Agrobacterium from explants.
 
Molecular characterization: Integration and expression
 
One of the easiest ways to detect the integration of transgenic is through polymerase chain reaction (PCR). Among the different methods followed MT₅ method of transformation showed highest transformation frequency of 2.13 per cent (Table 4). Results obtained from PCR assay for detection of cry2Aa gene construct (1.2 kb) in T₀ generation transformed plants is presented in Fig 4. Amplification at the range of plasmid of respective gene construct indicates the stable integration of gene under study.



In other pulse crops it ranged from less than 0.1 per cent (Kumar et al., 2004; Basavanna et al., 2008 and Surekha et al., 2007) in Agrobacterium–mediated and other methods of transformation.
 
In this study, transgenic dolichos bean was produced by Agrobacterium- mediated transformation using mature embryo axes with single cotyledon (MEASC) as an explant. The ability to regenerate dolichos bean in in vitro as well as use of positive selection system may provide a means for recovery of genetic transformation. Till now, limited studies were conducted, but it has been not commercialized. However the present study showed that Agrobacterium mediated transformation is a possible approach to develop and commercialize transgenic plants in dolichos bean.
 
Progeny analysis
 
In the present investigation, emphasis was given for the development of heritable pod borer transgenic dolichos bean. The results obtained from study conducted on progeny analysis by chi-suare test indicated that most of these plants showed Mendelian segregation pattern (Table 5). Some plants also showed non mendelian segregation pattern. In all the progenies PCR positive plants were identified (Fig 5). Same kind of segregation pattern was reported by many scientists in different crops including pulses as Kaur et al., (2016) in pigeonpea being one of the recent ones; Sawardekar et al., (2007) in chickpea, Budar et al., (1986) in tobacco, Feldmann and marks (1987) in Arabidopsis etc. It indicates the heritable transformation of cry2Aa gene in dolichos bean cv. Konkan Bhushan.

The present study showed that dolichos bean can be effectively transformed by in vitro Agrobacterium mediated transformation. In dolichos bean we report 2.13 per cent transformation frequency.

The authors very sincerely acknowledge the facilities provided by Plant Biotechnology Centre and Department of Agricultural Botany, College of Agriculture, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli and also UGC, Government of India for supporting the present research work by a research fellowship viz. Rajeev Gandhi National Fellowship (RGNF).

All authors declare that they have no conflicts of interest.