Soniya E V, Das M R
Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuram 695 014, India.
Indian J Exp Biol. 2002 Mar;40(3):329-33.
The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.
在流行的黄瓜品种（黄瓜品种Sheetal）中，研究了生长调节剂和培养条件对子叶叶片圆盘形态发生反应的影响。在添加不同浓度苄基腺嘌呤和吲哚丙酸的Murashige和Skoog培养基上，直接诱导器官发生，无需任何中间愈伤组织阶段。在4 mg/L苄基腺嘌呤和1 mg/L吲哚丙酸存在下获得了最佳结果（93%）。将伸长的芽在含有1 mg/L吲哚丁酸的基础培养基中生根，硬化并转移到田间条件下。通过用携带二元质粒pBI121的根癌农杆菌菌株LBA4404感染子叶外植体，建立了黄瓜品种Sheetal的遗传转化系统，该质粒在CaMV 35S启动子下含有可评分标记β-葡萄糖醛酸酶和选择标记nptII。当外植体用农杆菌感染15分钟并在共培养基中共培养2天时，感染最有效。在卡那霉素（50μg/ml）存在下，从子叶叶片外植体直接再生芽并进行分析。Southern印迹分析证实发生了转化。该方法将通过引入具有重要农艺性状的基因实现该作物的遗传改良。
