Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium
Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium
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DOI：
10.1007/s00299-006-0284-5
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发表时间：
2007-06-01
影响因子：
6.2
通讯作者：
Mii, Masahiro
中科院分区：
文献类型：
--
作者：
Chin, Dong Poh;Mishiba, Kei-ichiro;Mii, Masahiro
将含有β-葡萄糖醛酸酶（gus）、潮霉素磷酸转移酶（hpt）和新霉素磷酸转移酶II（npt II）基因的根癌农杆菌EHA 101（pIG 121 Hm）与兰类原球茎共培养，获得转基因植株。将三个基因型的原球茎保持在液体新Dogashima培养基（NDM）中，进行转化试验。用农杆菌接种的PLB在转移到含有10 g L-1蔗糖、20 mg L-1潮霉素和40 mg L-1美罗培南的2.5 g L-1结冷胶固化的NDM上4周后产生次级PLB。转化效率受基因型和共培养期间乙酰丁香酮的存在影响。以基因型L4的原球茎为外植体，在含100 μ M乙酰丁香酮的培养基上与农杆菌共培养，转化效率最高。通过GUS组织化学分析、PCR分析和Southern杂交证实了从PLB不同接种部位再生的潮霉素抗性植株的转化。
Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L-1 gellan gum-solidified NDM containing 10 g L-1 sucrose, 20 mg L-1 hygromycin and 40 mg L-1 meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 mu M acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.