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Studies on chitinase gene transfer in tomato (Solanum lycopersicum L.) and molecular analysis of transgenic plantlets

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Title Studies on chitinase gene transfer in tomato (Solanum lycopersicum L.) and molecular analysis of transgenic plantlets
 
Creator SHARMA, POORNIMA
 
Contributor SRIVASTAVA, D.K.
 
Subject regeneration, vegetables, planting, genes, genetic processes, transgenics, antibiotics, concentrates, iaa, biological phenomena
tomato cv. Solan Vajr, fungal resistance, Agrobacterium tumefaciens strain
 
Description ABSTRACT
Genetic transformation studies were carried out in tomato cv. Solan Vajr to standardize a protocol for fungal
resistance gene transfer using Agrobacterium tumefaciens strain containing hpt and chi11 genes in binary vector pCambia
bar- ubi- chi11. Plant regeneration studies were carried out using four different types of explants viz. leaf, petiole, cotyledon
and hypocotyl. Leaf and petiole explants were procured from twenty to twenty five days old glasshouse grown seedlings
whereas, cotyledon and hypocotyl explants were used from ten to fifteen days old glasshouse grown seedlings of tomato.
Petiole explants showed better shoot regeneration as compared to other explants. High efficiency shoot regeneration was
obtained in leaf (68.08%), petiole (80.01%), cotyledon (74.04%) and hypocotyl (79.61%) explants on MS medium
supplemented with 1.0mg/l BAP + 0.5mg/l IAA, 1.0mg/l Kn + 1.0mg/l IAA, 2.0mg/l TDZ and 1.25mg/l TDZ + 0.088mg/l
IAA, respectively. MS medium supplemented with 0.20mg/l IBA was found best for root regeneration from in vitro
developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat, then transferred to sand: soil: FYM
mixture in earthen pots, where they responded well upto flowering and fruit formation. All the regenerated plantlets were
morphologically similar. Hygromycin sensitivity experiment was carried out to study the effect of antibiotic on relative
growth of leaf and petiole tissues and to select transgenic shoots during transformation experiment. Hygromycin sensitivity
(5-30mg/l) was studied by fresh weight of the leaf and petiole explants, which was measured at the interval of 7days till
35days. From the relative growth of the explants, it was found that the concentration as low as 5mg/l was toxic to the
explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in leaf and petiole
explants and a gradual decline in per cent shoot regeneration potential of tomato with the gradual increase in the
concentration of cefotaxime was observed. Effect of different concentrations of cefotaxime and hygromycin (25mg/l) were
studied on the growth of agrobacterial cells and regeneration potential of leaf and petiole tissues after co-cultivation. In both
the explants, the growth of agrobacterial cells was controlled at concentration of 400mg/l cefotaxime. The regeneration
potential of the leaf and petiole explant was significantly not affected with the increase in the different concentrations of
cefotaxime i.e. 100mg/l to 500mg/l. Effect of preculturing and co-cultivation was studied on the transformation frequency.
Pre-culturing of leaf and petiole explants for 96 hours and co-cultivation with agrobacterial cells for 48 hours worked out to
be the best treatment as it gave the highest transformation frequency (10.34%) and (11.11%) in respective explants. Effect of
different concentrations of acetosyringone was studied in leaf and petiole explants to enhance the transformation frequency.
The maximum per cent of putative transgenic shoot regeneration was obtained from leaf (17.26%) and petiole (19.42%)
explants respectively, when cultured on shoot regeneration medium containing 100μM acetosyringone at standardized preculturing
and co-cultivation time interval i.e. 96 hours and 48 hours. The integration of hpt and chi11 into the genome of
tomato was confirmed by PCR using gene specific primers. For PCR analysis, 40 putative transgenic shoots/plantlets were
randomly selected and out of 40 putative transgenic shoots/plantlets, 27 shoots were found to be positive for the integration
of transgene i.e. hpt and chi11 into the genome of tomato. The expression of the transgene i.e. hpt and chi11 was further
confirmed at transcriptional level by RT- PCR (Reverse Transcriptase- Polymerase Chain Reaction) and Real Time- PCR
analysis where 15 out of 21 PCR positive shoots were RT- PCR positive. RT- PCR positive transgenic plants showed lower
ct value as compared to control plantlets, using Real Time- PCR analysis. To study the expression of transgene at
translational level, bioassay was carried out in six Real Time- PCR positive transgenic plants using Fusarium oxysporum f.
sp. lycopersici and one plant out of the six transgenic plants, was found to be tolerant (healthy) even after, with the increase
in the incubation period. A protocol for high frequency plant regeneration and genetic transformation with chi11 gene
transfer in tomato (Solanum lycopersicum cv. Solan Vajr) has been standardized
 
Date 2016-06-14T14:16:54Z
2016-06-14T14:16:54Z
2014
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/67362
 
Language en
 
Format application/pdf