Agrobacterium-mediated in planta genetic transformation of sugarcane setts.

KEY MESSAGE: 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.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

An efficient in planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting.

An efficient and reproducible Agrobacterium-mediated in planta transformation was developed in Jatropha curcas. The various factors affecting J. curcas in planta transformation were optimized, including decapitation, Agrobacterium strain, pin-pricking, vacuum infiltration duration and vacuum pressure. Simple vegetative in vivo cleft grafting method was adopted in the multiplication of transformants without the aid of tissue culture. Among the various parameters evaluated, decapitated plants on pin-pricking and vacuum infiltrated at 250 mmHg for 3 min with the Agrobacterium strain EHA 105 harbouring the binary vector pGA 492 was proved to be efficient in all terms with a transformation efficiency of 62.66%. Transgene integration was evinced by the GUS histochemical analysis, and the GUS positive plants were subjected to grafting. Putatively transformed J. curcas served as "Scion" and the wild type J. curcas plant severed as "Stock". There was no occurrence of graft rejection and the plants were then confirmed by GUS histochemical analysis, polymerase chain reaction (PCR) and Southern hybridization. Genetic stability of the grafted plants was evaluated by using randomly amplified polymorphic DNA (RAPD), marker which showed 100% genetic stability between mother and grafted plants. Thus, an efficient in planta transformation and grafting based multiplication of J. curcas was established.

Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 µM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).

Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane.

KEY MESSAGE: 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.