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2015 - 20202
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Subject: Biotechnology × Author: ABHISHEK, KUMAR ×
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    ThesisItemOpen Access
    STUDIES ON RNAi-INDUCED GENE SILENCING BASED RESISTANCE TO MARSSONINA CORONARIA IN APPLE
    (UHF,NAUNI, 2020-08) ABHISHEK, KUMAR; MODGIL, MANJU
    ABSTRACT Marssonina blotch is one of the most devastating diseases caused by Marssonina coronaria (Ellis & J.J. Davis). All the available commercial apple cultivars are found susceptible to this disease. In the present study, we developed an RNAi construct to target HSP90 gene of this fungus for functional analysis. For this, the full stretch of HSP90 cloned in pTZ57R/T was obtained from a recent study in our laboratory. In silco analysed 124 bp off-targeted sequence was cloned into the pGEMT and then cloned in sense antisense orientation into the pMVR-hp vector to develop pMVR-HSP90-RNAi construct. For using the same RNAi construct for fungal transformation, HSP-90 RNAi casstte from pMVR-HSP 90-RNAi vector was transferred to the pCAMBIA1300 vector, which harbours hygromycin B phosphotransferase (hpt) gene as a selection marker. In fungal transformation experiment, no fresh growth was observed in case of mycelial plugs transformed with RNAi construct in comparison to positive control. Agrobacterial overgrowth was found to be a problem after cocuiltivation. Another construct pRI 101-AN-HSP-90RNAi developed in our laboratory was used for transformation of apple cv. Red Chief. MS medium supplemented with 5 mg/l BA and 0.2 mg/l NAA resulted in best shoot regeneration frequency (96.35 percent). Regenerated shoots were multiplied, rooted and hardened successfully. Kanamycin was found highly toxic to leaf explants and affected the regeneration rate of shoots even at lower concentrations. Infection time of 7 min was found the best for transformation of apple cultivar, which resulted 9.35 percent shoot regeneration frequency with 1.65 shoots per explant. 5 & 6 mg/l kanamycin along with 500 mg/l cefotaxime was used for the selection of the transformed shoots and resulted in 2-6 percent putative shoot regeneration. In PCR analysis of putative transformed shoots, 11 lines were found to be positive with the gene specific primers and nptII gene specific primers. Eleven transgenic lines were maintained in culture room which needs further molecular analysis.
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    ThesisItemOpen Access
    STUDIES ON IN VITROCELL SELECTION IN TOMATO (Solanum lycopersicumL.) AGAINST BUCKEYE ROT [Phytophthora nicotianae var.parasitica(DASTUR) WATERHOUSE
    (2015) ABHISHEK, KUMAR; KAUR, RAJINDER
    ABSTRACT The present investigation aimed at in vitrocell selection in tomato (Solanum lycopersicumL.) against buckeye rot (Phytophthora nicotianae var. parasitica (Dastur) Waterhouse). Leaf, internode and root explants from in vitro geminated seedlings of cv. Solan Vajr were used for callus induction. The seeds were surface sterilized by treating them with 0.1% (w/v) of bavistin and 0.5% (v/v) sodium hypochlorite for different time intervals. Treatment of leaf explant with 0.1% (w/v) of bavistin for ten minutes and 0.5% of (v/v) sodium hypochlorite for three minutes was the most effective giving 68.05% uncontaminated cultures and 60.94% survival of seedlings. the MS medium supplemented with 1.2 mg/l GA3gave the best seedling germination of 32.33%. MS medium supplemented with 3.0 mg/l NAA+ 1.0 mg/l BAP gave best growth of callus. Leaf and root explants gave 95.21 % and 81.11 % callus induction, respectively, but internode gave maximum callus induction of 100.00%. The calli were subjected to different concentrations of culture filtrate of Phytophthora nicotianae var. parasitica and calli survival at 20% as selective dose of culture filtrate was 30.64%. The selected calli did not respond very well for the regeneration. The callus turned to slightly raised green nodulation on different combination of growth regulators but no shoots were formed. Molecular studies revealed that theselected tolerant calli were different at genomic level from the normal callus and the differences do not only exist at morphological level but they are also present at DNA level. Thus it was concluded that in cv. Solan Vajr callus can be used for cell selection studies and Culture filtrate of pathogen was injurious to the survival of callus beyond 20%.