Development of disease resistant lentil (Lens culinaris Medik.) lines through Agrobacterium-mediated genetic transformation

Abstract

Agrobacterium-mediated genetic transformation was carried out to integrate fungal diseases resistant gene in microsperma varieties of lentil (Lens culinaris Medik.) cultivated in Bangladesh. As an integral part of Agrobacterium-mediated genetic transformation in vitro regeneration studies were carried out using various explants from Barimasur (BM) varieties, namely, BM - 1, BM - 4, BM - 5 and BM - 6. Among these explants cotyledon attached decapitated embryo (CADE) from BM - 6 appeared to be the best responsive explant towards in vitro regeneration compatible to Agrobacterium-mediated genetic transformation. Highest percentage (96%) of multiple shoots as well as healthy roots were obtained through direct organogenesis from CADE explant on MSB5 medium supplemented with 1.0 mg/l zeatin and 0.1 mg/l NAA. More than 90% of such developing explants produced effective, elongated and healthy roots when they were transferred to MS medium without any hormonal supplement. On the other hand, in vitro regenerated shoots those failed to develop roots were exploited to induce in vitro flower as well as seed to overcome the constraints created in producing in vitro roots in lentil to obtain complete plantlets. MS and half-strength of MS medium supplemented with various concentrations and combinations of IAA, IBA and NAA were employed for this purpose. The best responses regarding the development of in vitro flowers and pods were observed on half-strength of MS medium containing 20 mg/l IBA and 0.5 mg/l NAA. Healthy seeds obtained from this pods produced seedlings which were successfully transplanted to soil for further growth and development. Transformation experiments were performed using three strains of Agrobacterium tumefaciens, namely, LBA4404 harboring binary plasmid pBI121 containing GUS and nptII gene (Strain I), EHA105 harboring plasmid pGIIMH (strain II) containing bar gene resistant to phosphinothricin and also chitinase gene (as an antifungal gene) and the third one was LBA4404 containing binary plasmid pCAMBIA2300 (strain III) conferring nptII gene resistant to kanamycin and AFP gene as an antifungal protein gene. Strain I was mainly used for the optimization of transformation protocol and to select the suitable lentil variety as well as explant for transformation. Transformed shoots were selected using 2.0 mg/l phosphinothricin (Strain II) whereas 200 mg/l kanamycin was used to select transformed shoots in case of strain I and III. Transformation efficiencies for strain I, strain II and strain III was 2.15, 0.36 and 0.47% respectively. Stable integration of desired gene within the lentil genome was confirmed through PCR, RT-PCR, Southern and Northern hybridization techniques. Seedlings developed through transformation were successfully transferred to soil for the development of transformed progenies in lentil.