Genetic transformation of black pepper (piper nigrum L) for phytophthora foot rot resistance/tolerance

Abstract

Investigations on "Genetic transformation of black pepper (Piper nigrum L.) for Phytophthora foot rot resistance/tolerance" were carried out at the Department of Plantation Crops and Spices, and Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2001-2006. Three selected black pepper varieties Panniyur 1, 4 and 6 were utilized for the study. Axenic cultures of the selected varieties were raised from nodal segments as well as ripe seeds for embryogenesis and transformation studies. Among the varieties average number of multiple shoot/explant was high for variety P­6 in ½ MS medium with BA and IAA 1.0 mg l-1. Somatic embryogenesis was induced on ripe seeds of the variety P6 at the micropylar region in ½ MS medium with inositol 1.0 g l-1. Further development of the somatic embryo observed in liquid shaking cultures of SH basal. Growth regulators like BA,2,4-D ,dicamba and thidiazuron did not give rise to embryogenesis in the different explants of black pepper. Multiple shoot induction from cotyledonary nodal explants and zygotic embryo explants were observed in all the varieties in ½ MS with 1.0 mg l-1 BA and IAA. Direct regeneration from leaf segments was also observed in the same media combination. Agrobacterium tumefaciens strains EHA105, AGL.1.1303, GV2260 and LBA4404 were used for the transformation work. Strain EHA105 contains the plasmid p35SGUSINT with gus A gene and npt II gene. The AGL.1.1303 contains the plasmid harbouring antibiotic resistant selectable marker genes (npt II and hpt IV) and GUS and GFP reporter genes. The GV2260 contain the plasmid pGV2260 with osmotin gene and npt II gene. The LBA4404 contains the plasmid pBZ100 containing alfalfa glucanase gene, rice chitinase gene and npt II gene. Sensitivity studies of black pepper tissues to various antibiotics resulted in selecting the optimum threshold level of antibiotic to be used in the screening medium. Kanamycin 25 mg l-1, 50 mg l-1, and 100 mg l- were selected as the cut off level for the selection of transformants from zygotic embryo, leaf segments and nodal segments respectively. Cefotaxime at 250 mg l-1 was selected for the effective elimination of Agrobacterium after infection. Genetic transformation was standardized with Agrobacterium strain EHA105 using leaf disc and zygotic embryo explants. Tentative protocol for transformation in black pepper include Agrobacterium inoculum density 0.9, infection time 10 min and co-cultivation period of 48 h. Acetosyringone at 50-100 µM favoured transformation. Transient gus assay revealed faint blue staining on the infected leaf explants. Explants, leaf segment, cotyledonary node and zygotic embryo were used for transformation with Agrobacterium strains AGL.1.1303, GV2260 and LBA4404. There was explant specificity for the different Agrobacterium strains used. With LBA4404 zygotic embryo explants gave maximum survival in the screening medium containing 50 mg l-1 kanamycin and 250 mg-1 cefotaxime. Direct gene transfer using gene gun attempted with pBZ100 and cotyledonary node explants. Bombarded explants were found surviving in the screening medium with kanamycin for four months. However molecular analysis of selected transformants through PCR revealed that npt II gene integration has not happened in the tissues subjected to PCR analysis.