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Subject: Biotechnology × Author: KAPOOR, BHUVNESH × Start date: 2020 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    AGROBACTERIUM MEDIATED DEFENSIN GENE TRANSFER STUDIES IN TOMATO (SOLANUM LYCOPERSICUM L.)
    (NAUNI,UHF, 2020-11) KAPOOR, BHUVNESH; SHARMA, RAJNISH
    ABSTRACT Tomato (Solanum lycopersicum L.) is one of the most economically important horticultural crops consumed globally. The crop known as ‘Red Gold’ in the farming community is suffered every year due to number of pathogenic diseases which are country-wide in occurrence. Considering the limitations of conventional methods of crop improvement, present study was taken to develop the transgenic lines of tomato by expressing defensin gene (TvD1) having antimicrobial activity. As a prerequisite to the genetic transformation procedures, regeneration protocol for tomato cv. Solan Lalima was standardized by using different explants. The cotyledon explants exhibited best shoot regeneration response on MS medium augmented with various growth regulators with maximum average number of shoots per explant, highest mean shoot length, minimum average days taken for the initiation of shoot induction, respectively as compared to hypocotyl and leaf explants. The in vitro regenerated shoots were further successfully rooted and hardened. However, cotyledons and hypocotyl explants were found to be physiologically more responsive, hence, were further considered for carrying out genetic transformation experiments. The kanamycin sensitivity experiments established that kanamycin concentrations 50 mg l-1 for cotyledon and 100 mg l-1 for hypocotyl explants were found to be effective for inducing selection pressure. The bacteriostatic antibiotic timentin revealed maximum shoot regeneration response with maximum average per cent shoot regeneration in cotyledons (97.70±1.33) and hypocotyls (87.66±1.33). Agrobacterium cell density of 0.2 at O.D600 and infection time of 2 min was found optimum for sufficient infection. Timentin at concentration of 500 mg l-1 was found efficient in controlling Agrobacterium growth. The 48 hrs of pre-culturing followed by 48 hrs of co-cultivation showed highest 13.33±0.11% and 26.66±0.33% shoot regeneration in cotyledon and hypocotyl explants, respectively. Out of total 15 putative tomato transformants considered for PCR analysis, four showed amplifications of nptII and TvD1 gene. Further, three transformed tomato lines exhibited expression of TvD1 gene through semi-quantitative RT-PCR. Therefore, it was inferred that present investigations would be helpful in developing resistance in this economically important horticultural crop by targeting its various devastating fungal and bacterial pathogens.