title: Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane.
abstract: An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant. Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA(®) and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Mayavan
authors.forename: Kondeti
authors.initials: K
authors.lastname: Subramanyam
authors.forename: Muthukrishnan
authors.initials: M
authors.lastname: Arun
authors.forename: Manoharan
authors.initials: M
authors.lastname: Rajesh
authors.forename: Gnanajothi
authors.initials: G
authors.lastname: Kapil Dev
authors.forename: Ganeshan
authors.initials: G
authors.lastname: Sivanandhan
authors.forename: Balusamy
authors.initials: B
authors.lastname: Jaganath
authors.forename: Markandan
authors.initials: M
authors.lastname: Manickavasagam
authors.forename: Natesan
authors.initials: N
authors.lastname: Selvaraj
authors.forename: Andy
authors.initials: A
authors.lastname: Ganapathi
mesh_terms: D000098:Acetophenones / Q000737:chemistry; D016960:Agrobacterium tumefaciens; D018744:DNA, Plant / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005838:Genotype; D030821:Plants, Genetically Modified / Q000235:genetics; D031786:Saccharum / Q000235:genetics*; D012639:Seeds / Q000235:genetics*; D013010:Sonication; D013501:Surface-Active Agents / Q000737:chemistry; D014170:Transformation, Genetic
chemical_list: D000098:Acetophenones; D018744:DNA, Plant; D013501:Surface-Active Agents; C051667:acetosyringone