Khurana Jigyasa, Chugh Archana, Khurana Paramjit
Department of Plant Molecular Biology, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110 021, India.
Indian J Exp Biol. 2002 Nov;40(11):1295-303.
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).
本研究建立了四倍体二粒小麦（Triticum dicoccum）的再生方案，并优化了农杆菌介导转化的条件。成熟胚和未成熟胚的再生过程分两步完成，第一步是在2,4-D存在的情况下诱导愈伤组织，第二步是在不含2,4-D但含有细胞分裂素的培养基（RM1）上进行再生。较高浓度的2,4-D（4 mg/l）虽然有利于愈伤组织的形成（成熟胚中为89.39%，未成熟胚中为96%），但对进一步再生不利。较低浓度的2,4-D（1 mg/ml）虽然愈伤组织形成不理想（成熟胚和未成熟胚分别为56.8%和84%），但在RM1培养基上的再生响应最高（成熟胚和未成熟胚分别为64.4%和56.6%）。总体而言，未成熟胚的再生响应比成熟胚低10 - 12%。由于成熟胚易于获得，因此在两个印度品种DDK1001和DDK1009中，选择成熟胚来源的愈伤组织作为农杆菌介导转化的靶组织。组织化学GUS表达显示成熟胚来源的愈伤组织适合此类研究。在所使用的CaMV35S和Act1启动子中，单子叶植物启动子Act1与LBA4404（pBI101::Act1）共培养时，在成熟胚来源的愈伤组织中显示出较高的GUS基因活性。
