RESUMO
Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.
Assuntos
Morus/crescimento & desenvolvimento , Morus/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Glucuronidase/genética , Canamicina/farmacologia , Canamicina Quinase/genética , Morus/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da PolimeraseRESUMO
Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.
Assuntos
Agrobacterium tumefaciens/fisiologia , Robinia/genética , Robinia/microbiologia , Transformação Genética , Glucuronidase/genética , Glucuronidase/metabolismo , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Plantas Geneticamente Modificadas/genética , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismo , Robinia/enzimologia , Plântula/enzimologia , TransgenesRESUMO
Callus was derived from cultured cotyledons on MS medium supplemented with 2,4-D (0.25 mg/l) and NAA (0.25 mg/l). Plantlets were regenerated from the callus and nodal explants on MS medium containing BAP (2.0 mg/l) and Kn (2.0 mg/l), and further multiplied on the same medium. Addition of adenine sulphate (25.0 mg/l), ascorbic acid (20.0 mg/l) and glutamine (150.0 mg/l) in the medium resulted in enhanced axillary branching. Multiple shoots formed after 6 weeks were separated and subcultured in the fresh medium of same composition. For rhizogenesis, microshoots of 2.0-2.5 cm length were dipped in sterilized IAA solution (10 mg/l) for 24 hr followed by transfer to half strength MS medium containing activated charcoal (0.02%) resulting in rooting (75%) within 8 weeks. The rooted plants were transferred to pots containing sterilized soil and sand mixture for hardening and 71% survival was recorded. Fifty true to type plantlets of A. catechu could be obtained within seven months of culture establishment.
