title: Agrobacterium-mediated in planta genetic transformation of sugarcane setts.
abstract: An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant. Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA(®). Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 µM acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time.
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.|Center for Bioenergy, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO, 65101, USA.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Mayavan
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.|Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, 9000, Ghent, Belgium.
authors.forename: Kondeti
authors.initials: K
authors.lastname: Subramanyam
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.
authors.forename: Balusamy
authors.initials: B
authors.lastname: Jaganath
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.
authors.forename: Dorairaj
authors.initials: D
authors.lastname: Sathish
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.
authors.forename: Markandan
authors.initials: M
authors.lastname: Manickavasagam
authors.affiliation: Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India. aganapathi2003@bdu.ac.in.
authors.forename: Andy
authors.initials: A
authors.lastname: Ganapathi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D031786:Saccharum / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
keywords: Agrobacterium tumefaciens; BASTA®; In planta transformation; Sonication; Southern blot hybridization; Sugarcane setts; Vacuum infiltration