Development of drought and salinity tolerant peanut (Arachis hypogaea L.) lines through Agrobacteriummediated genetic transformation

Abstract

A series of experiments were carried out to establish a suitable protocol for Agrobacterium-mediated genetic transformation in two locally grown varieties of peanut (Arachis hypogaea L.), namely, Dhaka-1 and BARI Badam-8. As a prerequisite of the transformation protocol, an efficient in vitro regeneration system was established for these two peanut varieties. Three different types of explants, namely, immature cotyledonary leaflet, single cotyledon attached decapitated embryo (SCADE) and deembryonated

half cotyledon (DEHC) explants were used for in vitro regeneration. The best performance of multiple shoot development was achieved in case of Dhaka-1 (72.2%) and BARI Badam-8 (68%) varieties when the explants of SCADE and DEHC when initially cultured on MS medium supplemented with 88.8 µM BAP, followed by two subsequent cultures on lower concentrations of BAP (66.6 µM BAP and 13 µM BAP) containing medium. In most of the cases the regeneration of multiple shoots was obtained in 40 days. Best response towards the formation of roots from the excised in vitro regenerated shoots was observed on half strength of MS medium supplemented with 2.5 µM IBA or 1.0 µM IAA for the two varieties of peanut. Fully developed in vitro regenerated plantlets were successfully established in soil for further growth and development. For optimization of genetic transformation protocol, Agrobacterium strain LBA4404 containing binary vector plasmid pBI121 harbouring GUS (β-Glucoronidase) and nptII (neomycin phosphotransferase) genes (construct I) was used. Transient GUS histochemical assay revealed that among the two explants used maximum transient GUS expression (88%) was observed from de-embryonated half cotyledon explants of variety BARI Badam-8. In this case the required optical density of Agrobacterium suspension was 0.8 at 600 nm with 10 minutes for the incubation of explants in that bacterial suspension. Transformed shoots were cultured on 150 - 200 mg/l kanamycin supplemented medium to select the putatively transformed shoots. During this experiment it was possible to develop a series of multiple shoots from the selected transformed explants using Agrobacterium strain having marker genes (GUS and nptII). Histochemical GUS assay of the putatively transformed shoots on optimum concentrations of kanamycin showed the integration of marker genes. However, gene specific bands were not amplified during PCR experiment. During this study Agrobacterium strain containing EHA105/pCAMBIA 1301-PDH45 harbouring PDH45 and hptII genes were used for the transfer of drought and salinity tolerant gene in the peanut plants. Single cotyledon attached decapitated embryo (SCADE) and de-embryonated half cotyledon (DEHC) explants of variety Dhaka-1and BARI Badam-8 were used for transformation experiments. Among the explants studied maximum transformation efficiency was observed in de-embryonated half cotyledon (DEHC) explants with bacterial suspension having an optical density of 0.5 at 600 nm with 25 min incubation period and 48 hrs of co-cultivation period. Transformed shoots were selected using 20 mg/l hygromycin. Stable integration of PDH45 and hptII genes were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots. In this case, transformation efficiency was found to be 1.08% in case of BARI Badam-8. A total 13 putatively transgenic plants (T ) of BARI Badam-8 were confirmed through PCR analysis. These T 0 0 peanut plants survived through acclimatization and developed till maturation. Transgenic lines were maintained in the greenhouse. T seeds were collected and raised following the Biosafety guidelines in the greenhouse for further investigation.