GENETIC TRANSFORMATION AND EXPRESSION ANALYSIS OF COLD TOLERANT GENE IN TOMATO (Solanum lycopersicum Mill.

Abstract

Chilling stress severely reduces the productivity of tomato as it is a cold sensitive plant. CBF3/DREB1A plays a key role in generating cold tolerance in tomato by regulating the response of multiple genes under chilling stress. In this study, cold tolerant gene (DREB1A) driven by Lip9 promoter, was transformed in three tomato genotypes (Rio Grande, Moneymaker and Roma) through Agrobacterium tumefaciens, employing tissue culture dependent and tissue culture independent transformation strategies. For tissue culture dependent transformation strategy, the effects of various PGRs (IAA, NAA, ZEA, Kin, BAP and GA3) and two ethylene inhibitors (AgNO3 and CoCl2) were investigated on callus induction and in vitro shoot regeneration. The maximum callus induction frequency (67.48%) was recorded on MS basal media enriched with 2.0 mg/l IAA, 2.5 mg/l BAP in cv. Rio Grande followed by Roma (62%) and Moneymaker (58.23%). Supplementation of AgNO3 (10-15 mg/l) in MS basal media along with PGRs (2.0 mg/l IAA, 2.5 mg/l BAP) significantly yielded the highest callus induction frequency (91.83%) in cv. Rio Grande, followed by Moneymaker (82.66%) and Roma (88.33%). Similarly, in vitro shoot regeneration frequency on MS media fortified with 0.1 mg/l IAA, 1.0 mg/l ZEA and 2.0 mg/l BAP significantly enhanced with the addition of 8-10 mg/l AgNO3 in all the cultivars i.e. in cv. Rio Grande (96.65%) followed by Roma (92.66%) and Moneymaker (90%). Likewise, the highest callogenesis (75.65%) was recorded in cv. Rio Grande on callus induction medium (CIM) supplemented with CoCl2 (5.5 mg/l), IAA (2.0 mg/l) and BAP (2.5 mg/l) followed by cv. Roma whose best callus induction (73.66%) was obtained on CIM supplemented with CoCl2 (4.5 mg/l), IAA (1.0 mg/l) and BAP (2.5 mg/l). In case of cv. Moneymaker the best callogenesis (68%) was secured on CIM having CoCl2 (3.5 mg/l), NAA (2.0 mg/l) and BAP (2.0 mg/l). The xiv highest in vitro shoot regeneration (85%, 81% and 78%) was recorded in Rio Grande, Moneymaker and Roma, respectively on shoot induction media supplemented with CoCl2 (4.25 – 5.0 mg/l). During this study, various concentrations of sucrose and sorbitol were scrutinized on in vitro shoot culture. The highest in vitro shoot regeneration frequency (100, 97.69 and 99%) was recorded in Rio Grande, Moneymaker and Roma with accumulative effect of sucrose and sorbitol (30: 30 g/l). Subsequently, transformation experiments were conducted by optimizing various factors both for tissue culture based and in planta techniques. For tissue culture based method of transformation; fifteen days old in vitro seedlings, forty-eight hours pre-culture period, bacterial density (OD600 nm = 0.2), three minutes infection period, 60 μM acetosyringone, forty-eight hours co-cultivation period, pH 5.6 of co-cultivation media, six days pre-selection duration, cefotaxime (500 mg/l) and hygromycin (35 mg/l) as lethal dose were found optimum. For in planta technique of transformation, various factors such as growing medium; soil: vermiculite (1: 1), optical density (OD600 nm = 1.0) and incubation period (20 min) were found optimum for efficient transformation efficiency. Polymerase chain reaction, Multiplex polymerase chain reaction, Southern blotting and Reverse transcriptase PCR confirmed the presence, integration and expression of DREB1A in T0 - T2 transgenic lines. Physiological and biochemical analyses of T2 transgenic plants depicted that after various chilling stresses; stomatal conductance, transpiration rate, CO2 concentration rate, photosynthetic rate, relative water contents, proline contents, total soluble sugar contents, chlorophyll contents, carotenoid contents and ascorbic acid contents of transgenic lines were significantly higher than those of NT plants. These findings clearly indicate that transgenic tomato plants over-expressing Arabidopsis CBF3 gene enhanced protection and provided cold tolerance under controlled conditions in transgenic containment.