Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants
Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants
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DOI：
10.1007/s12298-011-0059-5
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发表时间：
2011-04-01
影响因子：
3.5
通讯作者：
Jaiwal, Pawan K.
中科院分区：
文献类型：
--
作者：
Chhabra, Gulshan;Chaudhary, Darshna;Jaiwal, Pawan K.
在含蔗糖（1.0%）、2,4- d （5.0 μ M）和2- ip （50.0 μ M）或2,4- d （50.0 μ M）和TDZ （5.0 μ M）的B-5培养基中，利用根癌农杆菌和BAP预处理的子叶外植体培养出了硬结节细胞群“结节愈伤组织”，首次培育出了一株印度小草属植物的转基因植株。这些愈伤组织与含有内含子和新霉素磷酸转移酶基因的二元载体的瘤胃杆菌EHA105共培养。在卡那霉素选择培养基上选择转化细胞再生成叶片，通过组织化学GUS活性测定、PCR分析和Southern杂交证实其转基因性质。获得的转化率为3.8%，从外植体开始培养到完全生长的转基因叶片需要11-13周的时间。结果表明，BAP预处理、非离子表面活性剂的使用、共培养培养基中乙酰丁香酮的存在、共培养时间3 d和培养过程中16 h的光周期对小白莲愈伤组织的诱导、叶片的再生和转化至关重要。该转化体系可用于生产具有重要药用价值的蛋白质和生物修复。
Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B-5 medium containing sucrose (1.0 %) with 2,4-D (5.0 mu M) and 2-iP (50.0 mu M) or 2,4-D (50.0 mu M) and TDZ (5.0 mu M) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for beta-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11-13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.