Annual Report 2011-2012 - Nabi.res.in

NATIONAL AGRI-FOOD BIOTECHNOLOGY INSTITUTEinduction without an intermediary calluso n M S m e d i a c o n t a i n i n g 6 -B e n z y l a m i n o p u r i n e ( B A P ) a n dThioridazuron (TDZ) (Figure 18 A).ii. Culture was maintained as the continuoussource of shoots.iii. Shoots were separated from the multipleexpression studies in transgenic wheat.a. Callus based regeneration oftransgenic wheat: The transient andstable expression of reporter gene wasnoticed as blue colour on the embryosand callus-derived shoot bud,respectively (Figure 19 A and C). NoFigure 19: GUS histochemical assay. (A) Transientexpression in embryos (blue color). (B) Non-transgenicembryos. (C) Stable expression in callus-derived shootbud (blue color). (D) Non-transgenic shoot bud.shoot clumps and rooted on MS basalmedium containing Indole-3-butynic acid(IBA) and Naphthaleneacetic acid (NAA)(0.5mg/l each) (Figure 18 B).2. Establishment of genetic transformation:Several factors were considered for theoptimization of Agrobacterium-mediatedgenetic transformation. Mature embryoderived callus and direct multiple shootinduction based in-vitro regeneration wasused for reporter (GUS-Intron) geneexpression of GUS was noticed in thecontrol embryos and shoot bud (Figure19 B and D).l· Direct multiple shoot induction basedregeneration of transgenic:a. Stable expression was noticed as bluecolor on the shoots developed withoutan intermediate callus (Figure 20 A andB). No expression of GUS was noticedin control shoots (Figure 20 C).Figure 20: GUS histochemical assay. (A and B) Transformed germinated shoots (blue color). (C) Nontransformedshoots.17