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首页> 外文期刊>In Vitro Cellular and Development Biology. Plant: Journal of the Tissue Culture Association >Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato
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Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato

机译：外植体年龄，发芽培养基，培养前参数，接种培养基，pH，洗涤培养基和选择方式对土壤杆菌介导的番茄转化的影响

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摘要

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l?1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l?1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.
机译：开发了一种使用子叶外植体的农杆菌介导的番茄（Solanum lycopersicum）品种转化的有效方案。根据几种不同因素评估转化频率，包括种子发芽培养基，幼苗年龄，预培养持续时间，预培养和共培养培养基，接种培养基，培养基pH，洗涤培养基和初始选择培养基中卡那霉素浓度。从半衰期的Murashige和含有8.9μM苄基腺嘌呤（BA）的Skoog（MS）基础培养基上萌发的6日龄幼苗切下的子叶，是最合适的外植材料。在含有pH值为5.0的8.9μMBA，9.3μM激动素和0.4 mg l？1硫胺素的MS培养基中进行六天的外植体预培养和接种农杆菌培养5分钟，可显着提高转化频率。然而，添加烟草饲养细胞层并没有导致转化率的任何显着改善。在最初的10 d中，选择培养基中20 mg l？1的卡那霉素导致最高的转化频率。结合每个参数的最佳条件，可实现44.3％的整体转换效率。通过PCR和Southern印迹分析确认基因转移。在自体受精的T1后代的独立转基因品系中，发现孟德尔遗传比例为71.5％。优化的转化程序显示，对所有三个测试的番茄品种，即喀什维希什（H-86），希萨尔·安莫尔（H-24）和喀什阿姆利特（DVRT-1），都具有很高的转化频率，并且有望获得可重复的结果与其他番茄品种。

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