Standarizing in Planta Agrobacterium Tumefacience Mediated Genetic Transformation Protocol to Develop New Events by Transforming G. Hirsutum Cotton based on Cry1Ac-Cry1Ec Genes

Abstract

Cotton breeding for insect resistance has been limited by a lack of sufficient genetic variation in the existing germplasms. Therefore, genetic engineering provides the possibility of creating varieties carrying new properties coming even from heterologous source. Exogenous pesticidal transgenes can be introduced into plants. Agrobacterium mediated plant transformation offers advantages like reducing copy number of the transgene and little co-suppression. Inter specific hybrids are known to be more susceptible to biotic stress. It is hence important to develop Bt version for inter specific hybrid. Presently, the Bt gene commercialized are owned by private sector. It is necessary to develop public sector's Bt event and commercialize them. UAS Dharwad is involved in developing public sector Bt cotton genotypes. One variety, RCR4 (Gossypium hirsutum, L.) was used in the present investigation. Cry1Ac-Cry1Ec genes are to control Helicoverpa armigera and Spodoptera litura. The seedlings in pots were co-cultivated with solid Agrobacterium culture after cutting the meristematic tip with sharp knife. The number of seedlings co-cultivated, number of seedlings established and the number of seedlings showing transformed status are presented in this study. PCR was performed to confirm the presence of the transgene in the plants that were selected to be advanced further. The results showed that non of plants had trangenes Cry1Ac-Cry1Ec as detected through PCR amplification. In planta genetic transformation was carried out and the plants were tested in T 0 generation by means of PCR amplification for the genes Cry1Ac-Cry1Ec. The results obtained were not amplified the Cry1Ac-Cry1Ec. Hence the transformation of the genes was not up to mark and the plants of T 1 generation are also not confirmed.