Paper Details

Establishment and optimization of agrobacterium-mediated transformation and regeneration of tomato (Solanum lycopersicum L.)

Bhupendra Koul, Sugandha Srivastava, Devindra Vijay Amla, Indraneel Sanyal

DOI: https://dx.doi.org/10.12692/ijb/4.10.51-69

Int. J. Biosci. 4(10), 51-69. May, 2014. (PDF)

Abstract:

An efficient procedure for Agrobacterium-mediated transformation of tomato using in vitro derived cotyledon and leaf-disc explants for the recovery of non-chimeric stable transgenic plants has been developed. Several parameters including pre-incubation of explants, Agrobacterium cell density, co-cultivation conditions, selection regime and media fortification, have been extensively optimized. Fortification of 3 % maltose in shoot induction medium (SIM), 3 % sucrose in shoot elongation medium (SEM) and 2 % sucrose in root induction medium (RIM) showed best results using in vitro leaf-discs (2–5 mm), whereas 2 % sucrose was found optimum using cotyledon (8 mm) explants from in vitro germinated seeds. Leaf explants showed 96.2 % response with 5.8 mean numbers of elongated shoots per explant in tomato cultivar PED. Optimal hormone additives were 2.5 mg l–1 6-benzyladenine (BA) + 0.5 mg l–1 indole-3-acetic acid (IAA) in SIM, and 1.0 mg l–1 gibberellic acid (GA3) in SEM for leaf discs; versus 1.0 mg l–1 zeatin (ZET) and 0.2 mg l–1 IAA for cotyledon explants. Half-strength MS medium with 0.5 mg l–1 indole-3-butyric acid (IBA) in RIM was optimum for rooting, from both explant sources. The two-step selection cycle resulted in transformation efficiency from 3.17 to 21.38 % for cotyledon explants and 21.83 to 35.70 % for leaf explants in tomato cultivar PED using selection based on either nptII, hptII or bar genes. The optimized conditions for co-cultivation, in vitro screening and development of transgenic plants were suitable for the transfer of genes of diverse origin into Indian cultivars of tomato.