Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble).
The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000199:Actins / Q000235:genetics; D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic; D012038:Regeneration; D012231:Rhizobium / Q000235:genetics* / Q000254:growth & development; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014908:Triticum / Q000235:genetics*
D000199:Actins; D004269:DNA, Bacterial; D015084:2,4-Dichlorophenoxyacetic Acid; D005966:Glucuronidase