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Keywords: Natural Sciences
Physiology & related subjects
Issue Date: 2010
Abstract: Tomato (Lycopersicon esculentum Mill.) is a key food and cash crop for many low income farmers in the tropics. It is the second most consumed vegetable after potato and unquestionably is the most popular garden crop. Biotic and abiotic stresses cause considerable losses both of quality and quantity. Modern breeding techniques can be substituted to enhance its productivity. A simple, time and cost saving, reproducible and efficient regeneration and transformation method is still a basic demand for large-scale transgenic tomato production. Bacterial wilt caused by Pseudomonas solanacearum is one of the severe widespread and lethal diseases of tomato and is more prevalent in Northern areas of Pakistan. Transformation with Xa21 and molecular basis of resistance and susceptibility were also undertaken in present study. Five L. esculentum cultivars including Rio Grande, Roma, Pusa Ruby, Pant Bahr and Avinash that are commonly grown in Pakistan were selected with an objective of developing a highly reproducible and efficient in vitro plant regeneration protocol. Seeds were sterilized with various concentrations of clorox and maximum contamination free seedling development was observed at 8.0% treatment in all the genotypes. Hypocotyls and leaf disc segments of 3 weeks old seedlings were used as explants source. Different growth regulators were tested for the callus induction. Media composition containing GA 3 and BAP gave optimal response for callus induction from both hypocotyls and leaf discs in all the genotypes. Higher order of callus was induced with leaf disc such as 85.4% in Rio Grande and 83.8% in Avinash, in comparison to 73.9% and 76.7% in hypocotyls. It was followed by media having IAA and kinetin, with 84.4% and 80.9% (leaf discs) and 73.84% and 75.9% (hypocotyls) in Rio Grande and Avinash, respectively. Callus obtained was sub-cultured for regeneration on different regeneration media. In regeneration medium (RM 3 ) having IAA and GA 3 , more regeneration was obtained in hypocotyls derived calli; 75.73% and 73.4% in comparison to 71.7% and 69.2% in Rio Grande and Avinash, respectively in leaf disc derived calli. Different concentrations of IAA and BAP were also tested for direct regeneration without and with GA 3 in the media. Statistically higher percentage of regeneration was recorded with inclusion of GA 3 in all the media as compared to the respective media without GA 3 . IAA and kinetin were the other growth regulators showed promising regeneration and were further tested with and without coconut water in the media. Addition of coconut water in the media produced significantly higher regeneration with the XIrespective media without coconut water. Statistically higher regeneration percentage was obtained in hypocotyls versus the leaf discs. Days required for development of shoot primordial (days to maturity) were significantly reduced by inclusion of coconut water in all the cultivars. The number of shoot primordial was remarkably increased by the addition of coconut water in hypocotyls as well as leaf discs. Soma clones obtained were established in glass house and compared to the control plants. Plant height, number of leaves, number of fruits, fruit height and fruit width were decreased in soma clones while, days to flowering and days to fruiting were increased in comparison to control. Nine isolates of P. solanacearum were isolated from the stem and eight of vascular portion from wilted tomato samples obtained from Katha Sagral Research Station Sawat. Hypersensitivity on tobacco, pathogenecity on tomato and biochemical tests was performed for the characterization of the P. solanacearum. Hypersensitivity of tobacco and biochemical tests confirmed the presence of P. solanacearum in these isolates. Pathogenicity test of Roma and Rio Grande evaluated them as resistant genotypes while Pusa Ruby and Pant Bahr as susceptible cultivars. The Xa21 gene is a member of the Xa21 gene family of rice that provides broad spectrum Pseudomonas resistance in rice. The development of bacterial wilt has become an important research objective. Genetic transformation has become a widespread tool in both basic research and commercial plant breeding programs for disease resistance. EHA101 carrying the binary vector pTCL5 containing genes Xa21 for bacterial blight resistance, Hygromycin Phosphotransferase (HPT) resistance gene as a selectable marker and GUS as a reporter gene were co-transformed into tomato cultivars using Agrobacterium mediated transformation. Different parameters affecting the transformation efficiency were optimized during the present study. Co-cultivation period, age of seedlings, pre-selection period, selection of antibiotics and acetosyringone concentrations were found to be key factors in transformation experiment. Various co-cultivation periods of 1, 2, 3 and 4 days were tested. GUS expression (80% and 77.0%) was observed in Rio Grande in hypocotyls and leaf discs when co-cultivation time period was kept for 2 days and normal bacterial growth along with explants proliferation was observed. 24 days old seedling was found to be optimum with maximum GUS expression (76.7% and 77.4 % in hypocotyls and leaf discs, respectively) was recorded in Rio Grande. 7 days pre-selection period had shown maximum transformation efficiency and was 89.3% and XII87% in hypocotyls and leaf disc of the Rio Grande, respectively. More than 7 days of pre- selection had resulted in escapes, although more explants were proliferated but GUS expression was reduced. Direct selection had resulted in blackening of explants and GUS expression was not observed. Hygromycin at 25 mg L -1 was selected as lethal dose for selection of transformed calli. At this concentration maximum explants turned brown and no proliferation was observed, so this dose was selected for selection of transformed and untransformed explants. To enhance the host bacterium interaction, different acetosyringone concentrations i.e. 0, 50, 100, 200, 300 and 400 μM were used. Callus differentiation was not observed at 0-50 μM acetosyringone application. Hygromycin resistant calli were obtained at higher concentrations of acetosyringone (200-400 μM). In particular, 400 μM acetosyringone promoted the production of hygromycin resistant calli and GUS expression (92.3% and 80%) in Rio Grande with hypocotyls and leaf discs. 400 μM acetosyringone, 2 days co-cultivation, 7 days of pre-selection and 500 mg L -1 cefotaxime were found indispensable for successful transformation. Regeneration frequency of transgenic plants varied from 4.18- 42.1% in five cultivars. Among the five cultivars tested Rio Grande showed a transformation frequency of 42.08% and 40.42% followed by Avinash i.e., 40.62% and 37.5% in hypocotyls and leaf discs, respectively. Transgenic plants in green house resulted in more than 300 plants; from them forty nine grow to maturity. PCR analysis revealed the presence of the Xa21 and the HPT genes in the transgenic green house plants. Some of the plants have only HPT. The genetic segregation to HPT and GUS gene was observed in T 1 progeny seedlings. Mendelian segregation of 3:1 was observed in T 1 progenies. Untransformed plants produced significantly taller plants and a higher percentage of fruits vis-a-vis transgenic plants. The categorization of the tomato cultivars was very useful in determination the molecular mechanism of susceptibility and resistance of the tomato cultivars. To investigate the molecular mechanisms of bacterial resistance in susceptible (Pusa Ruby and Pant Bahr) and resistant (Roma and Rio Grande) cultivars of tomato, proteins were extracted from leaves of 3-week-old seedlings and separated by two-dimensional polyacrylamide gel electrophoresis. Fifteen proteins were differentially expressed in the susceptible and resistant cultivars, and analyzed by protein sequencer. They were related to the energy, photosynthesis, protein destination and storage, protein synthesis and defense proteins. Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play a key role in defense against insects and pathogens, were used in order to XIIIfind out the pathogen related proteins produced in all the tomato cultivars. Proteomics approach was used to determine the proteins induced by JA and SA in tomato cultivars that were resistant and sensitive to bacterial wilt. Antioxidant and defense proteins were induced and up regulated by time course application of JA. RuBisCO small chain was significantly reduced by application of JA. On the other hand, translationally controlled tumor was induced in response to SA. Proteins differentially expressed due to cultivar difference were plastocyanin and hypothetical protein. Proteins related to defense, energy and protein destination/storage were suspected to be responsible for susceptibility and resistance of the cultivars. Furthermore, ABC transporter plays a crucial role in signal transduction of both JA and SA in tomato resistant and sensitive cultivars to bacterial wilt.
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