title: High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.
abstract: To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.
authors.affiliation: Plant Transgenic Lab, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India. mehrotrameenakshi@gmail.com
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Mehrotra
authors.forename: Indraneel
authors.initials: I
authors.lastname: Sanyal
authors.forename: D V
authors.initials: DV
authors.lastname: Amla
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D029863:Cicer / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000472:pathogenicity; D007915:Lepidoptera / Q000187:drug effects* / Q000472:pathogenicity; D010572:Pest Control, Biological; D018515:Plant Leaves / Q000378:metabolism / Q000469:parasitology; D058439:Plant Somatic Embryogenesis Techniques / Q000379:methods; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D012038:Regeneration; D012639:Seeds / Q000254:growth & development / Q000378:metabolism; D013010:Sonication; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis