Establishment of Regeneration and Transformation System of Lycopersicon esculentum MicroTom

Abstract

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.