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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• 作者：Govind Kumar Rai (1) (2)
  Neha Prakash Rai (1)
  Sanjeev Kumar (1)
  Akhilesh Yadav (1)
  Sushma Rathaur (2)
  Major Singh (1)
  
• 关键词：Agrobacterium transformation ; Tomato ; Solanum lycopersicum ; DREB1A/CBF3 gene ; Transformation efficiency ; Kanamycin
• 刊名：In Vitro Cellular & Developmental Biology - Plant
• 出版年：2012
• 出版时间：October 2012
• 年：2012
• 卷：48
• 期：5
• 页码：565-578
• 全文大小：868KB
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• 作者单位：Govind Kumar Rai (1) (2)
  Neha Prakash Rai (1)
  Sanjeev Kumar (1)
  Akhilesh Yadav (1)
  Sushma Rathaur (2)
  Major Singh (1)
  
  1. Indian Institute of Vegetable Research, P.B. No. 01, P.O. Jakhini-Shahanshahpur, Varanasi, 221 305, India
  2. Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, 221 005, India
  

文摘

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? 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? 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.