hygromycin-a and acetosyringone

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.

Topics: Acetophenones; Agrobacterium tumefaciens; Cinnamates; DNA, Plant; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Hygromycin B; Musa; Plants, Genetically Modified; Sonication; Tissue Culture Techniques; Transformation, Genetic; Vacuum

Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 muM), infection of explants for 30-45 min and 3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l(-1)). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency of 4% was achieved for CN explants using this protocol.

Topics: Acetophenones; Agrobacterium tumefaciens; Cinnamates; Cotyledon; DNA, Plant; Gene Transfer Techniques; Genetic Vectors; Hygromycin B; Plants, Genetically Modified; Sesbania; Transformation, Genetic; Transgenes

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.

Topics: Acetophenones; Agrobacterium tumefaciens; Cefotaxime; Cinnamates; Gene Transfer Techniques; Genetic Vectors; Hygromycin B; Plants, Genetically Modified; Poaceae; Regeneration; Seedlings; Transformation, Genetic