PJB-2018-1730
Transformation of tomato variety Rio Grande with drought resistant transcription factor gene ATAF1 and its molecular analysis
Muhammad Awais, Rafiq Ahmad, Nadeem Khan, Prashanth Garapati, Muhammad Shahzad, Amber Afroz, Umer Rashid and Sabaz Ali Khan
Abstract
Tomato (Solanum lycopersicum L.) being an important vegetable is cultivated and used throughout the world. It not only contributes in fulfilling the basic nutritional requirements of the human body but also has many health benefits due to its rich biochemical composition. However, its production at large scale is hampered by many limiting factors such as biotic and abiotic stresses. Among the different abiotic stresses, drought poses drastic impact on tomato yield. Drought stress is genetically regulated by many transcription factors that not only regulate the stress responsive mechanism but also facilitate the growth and development of tomato plants. NAC is an important stress related transcription factor genes family, and the ATAF1 gene, a member of this family, is involved in ABA signaling and stress response. In this study, tomato variety Rio Drande was transformed with drought resistant ATAF1 gene via Agrobacterium mediated gene transformation method. The ATAF1 gene was first cloned in the pK7WFG2 vector having kanamycin selectable marker and then it was introduced in the Agrobacterium tumefaciens strain GV3101 through heat shock method. The tomato cotyledon and hypocotyl ex-plants of variety “Rio Ggrande” were cultured on callus induction medium (MS + 2.5 mg/L IAA + 2 mg/L BAP). The calli were then infected with Agrobacterium tumefaciens strain GV3101 containing ATAF1 gene and selection was carried out on the kanamycin selectable medium (MS + 100 mg/L Kan), and were regenerated on MS medium with 1 mg/L IAA + 1 mg/L BAP. Out of 216 putative transformed calli, 13 calli were able to regenerate on the selection medium. Of the 13 calli, three transgenic tomato plantlets were recovered, and these were confirmed through PCR analysis for the presence of 432 bp fragment of ATAF1 gene. The transformation protocol reported here can be used to generate drought resistant tomato plants in future
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