title: An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.
abstract: Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.
authors.affiliation: CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, India.
authors.forename: Manju
authors.initials: M
authors.lastname: Shri
authors.forename: Arti
authors.initials: A
authors.lastname: Rai
authors.forename: Pankaj Kumar
authors.initials: PK
authors.lastname: Verma
authors.forename: Prashant
authors.initials: P
authors.lastname: Misra
authors.forename: Sonali
authors.initials: S
authors.lastname: Dubey
authors.forename: Smita
authors.initials: S
authors.lastname: Kumar
authors.forename: Sikha
authors.initials: S
authors.lastname: Verma
authors.forename: Neelam
authors.initials: N
authors.lastname: Gautam
authors.forename: Rudra Deo
authors.initials: RD
authors.lastname: Tripathi
authors.forename: Prabodh Kumar
authors.initials: PK
authors.lastname: Trivedi
authors.forename: Debasis
authors.initials: D
authors.lastname: Chakrabarty
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D003545:Cysteine / Q000494:pharmacology; D012275:Oryza / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D003545:Cysteine