Agrobacterium mediated genetic transformation of ginger (zinbiger officinale rosc.)

Abstract

Investigations on genetic transformation in ginger (Zingiber officinale Rosc.) variety Rio-de-Janeiro using Agrobacterium tumefaciens strain EHA 105 harbouring antibiotic resistant selectable marker genes (npt II) and GUS reporter genes were carried out at the Department of Plantation Crops and Spices and Plant Tissue Culture Laboratory, CPBMB, College of Horticulture, Vellanikkara, Thrissur during the period from 1999 to 2005. Axenic shoot bud cultures of ginger variety Rio-de-Janeiro was raised under in vitro condition to generate explants with reduced contamination for transformation. Half strength MS medium with BA 3 mg l-1 was found to be the best for establishing shoot bud cultures. In order to standardise a regeneration protocol, MS medium supplemented with varying concentration of auxin and cytokinin were tried on different explants. Embryogenic calli were induced from bud explants of ginger supplemented with MS + 1.0 mg l-1 2,4-D + 0.5 mg l-1 BA, followed by plant regeneration on MS medium + BA 3 mg l-1 + 2,4-D 0.5 mg l-1. Bactericidal effect of antibiotics towards different strains of Agrobacterium and sensitivity of ginger tissues to different antibiotics were also studied to standardise the optimum level of antibiotics. Cefotaxime at a concentration of 300 mg l-1 was selected for eliminating the bacteria after co-cultivation. Kanamycin 100 mg l-1 was used to discriminate between transformed and untransformed cells. Agrobacterium strains were collected, recombinants were made and the presence of the construct confirmed in the native strains of Agrobacterium before starting transformation experiments. Agrobacterium strain EHA 105 p35SGUSINT was used for standardising the optimum conditions by comparing the levels of transient GUS expression in inoculated buds. A suitable transformation protocol would include 3 days preculture of explants, bacterial dilution of 1:20 (v/v), infection time of 5 min, co-cultivation of 48 h and post cultivation on callus induction medium with 100 mg l-1 kanamycin + 300 mg l-1 cefotaxime in darkness for 2 weeks and then under 16/8 h photoperiod. Use of acetosyringone in the co-cultivation medium (200 µm) and vir induced Agrobacterium strain (200 µm), increased the efficiency of transformation. Histochemical GUS assays were employed to study and compare the transient GUS expression, stable expression from putative transgenics. Further confirmation was made by PCR assays. The regeneration protocol as well as transformation protocol could be effectively used for further transformation.