Establishment of Regeneration and Transformation System of Lycopersicon esculentum MicroTom
Zhengjing Wu, Shuwei Sun, Fenghua Wang, Dalong Guo
Biotechnology Journal International · pp. 53–60 · Published 5 Jul 2011
10.9734/BBJ/2011/356Abstract
In this article in order to build up an efficient regeneration system for cotyledons or hypocotyls of tomato MicroTom, two kinds of seed disinfectant (3% NaClO and 0.1% HgCl2 used for 5, 10, 15, 20 min), two types of basal medium (MS: Murashige and Skoog，1964, and B5: Gamborg et al.,1968), different ratio of Indole Butyric Acid (IBA: 0.05, 0.1, 0.2 mgL-1) and 6-benzylaminopurine (6-BA: 1.0, 1.5, 2.0, 3.0 mgL-1) were tested. As for gene transform system, kanamycin (Kan: 0, 25, 50, 75, 100, 150mgL-1), carbenicillin, cephalosporin, cefoperazone sodium and sulbactam sodium for injection (Carb, CS, CSSS respectively) at the rate 100, 200, 300, or 500 mgL-1, respectively were added in medium B5 in order to find the suitable concentration for bacteriostasis and shoot regeneration. Moreover, cell concentration of Agrobacterium EHA105 (OD600: 0.3, 0.5, or 0.8）and infection time (5, 10, 15 min) were screened and optimized in this article. The results showed that 3% NaClO for 20 min is optimal as for the disinfect efficiency on the seed surface. The most suitable medium to induce adventitious bud is basal medium B5+0.05 mgL-1 IBA＋1.5 mgL-1 6-BA. The cotyledons and hypocotyls were cultivated in culture medium B5+IBA @ 0.05 mgL-1 for 2 d, then infected by Agrobacterium EHA105 (OD600=0.5) for 10 min, then plantlets were transferred to a fresh regeneration medium which contained 50 mgL-1 Kan and 300 mgL-1 CSSS, which was proved to be the most suitable transformation system for MicroTom.
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