LAUSR

The current investigation entitled “Genetic transformation for pod borer resistance in dolichos bean (Lablab purpureus(L). Sweet)”was aimed at the development standardization technique for in vitro regeneration of Lablab bean and Dolichos bean and to study the standardization technique for genetic transformation of Dolichos bean Cv. (KonkanBhushan). In surface sterilization, treatment consisting of 70 % alcohol for 5 minute followed by HgCl2 (0.1%) for 10 minutes was found to be the most effective in establishing aseptic cultures (97.22%).Mature embryo axis with single cotyledon (MEASC) explant on MS media supplemented with 6 mg/l BAP and 0.5 mg/l NAA responded well for shoot induction frequency, multiple shoot induction in genotype konkanbhushanfollowed by other genotypes.MS media supplemented with 0.5mg/l BAP and 0.2 mg/l GA3 found better for elongation of in vitro regenerated shoots.The genotypekonkanbhushanrootedearliest in 9.30 days on medium MS + 1.0 mg/l IBA + 2 % sucrosewith the highest root induction frequency (79.51%) and maximum roots per shoot (5.04). During hardening, the potting mixture of soil, vermiculite and sand in equal proportion (1:1:1)showed the highest survival rate. Agrobacteriummediated genetic transformation experiments were carried out in the dolichos bean (KonkanBhushan) which had shown better regenerability. Three different Cry genes viz., Cry1Aabc,Cry1Fa1and Cry2Aa were used for genetic transformation experiments. In genetic transformation five methodswere employed using mature embryo axes with single cotyledon as explant for selection of putative transgenics.Kanamycin concentration of 500 mg/lwas found as optimum.Use of cefotaxime@ 800 mg/l was found effective in controlling the excess growth of A. tumefaciens.Among different methods the explant injured mildly and dipped in Agrobacterium for 20 minutes under aseptic condition and co-cultivated for 48 hours and after cefotaxime washing explants put for germination in soil with the A. tumefaciens strain having Cry2Aa gene was found with the minimum survival percentage (6.00%) but maximum transformation frequency by both kanamycin screening (4.70%) as well as PCR analysis (2.13%).Among the three Cry genes used for transformation, the Cry2Aa gene was found to be the most effective.The progeny analysis confirmed the 3:1 mendelian segregation ratio.