الفهرس 
INTRODUCTION
MicropropagationOnly 14 pages are availabe for public view
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Abstract
This study was conducted at Greenhouse and Tissue Culture Laboratory, Department of Horticulture and Landscape Architecture, College of Agriculture, Purdue University, Indiana, United States of America during the period 2012 to 2014.
The objectives of this dissertation were
1- Establishment of in vitro propagation and regeneration system of four grape cultivars (Concord, Thompson Seedless, Beauty Seedless and King Ruby) in order to utilize for further studies of grapevine transformation procedure.
2- Develop a grapevine transformation protocol to produce transgenic plants with sHSP gene.
3- Study the role of sHSP to enhance thermotolerance in tomato as model plant.
The main results can be summarized in the following points:
Micropropagation
1- Chlorox (10%) for 10 minutes was the most effective for explants sterilization.
2- The combination of 1.0 mg/L BA and 0.01 mg/L NAA produced the maximum sprouting rate (100, 91.6, 100 and 83.3%) and the least number of days for bud sprouting (6, 9, 5 and 11) of Concord, Thompson Seedless, Beauty Seedless and King Ruby cultivars, respectively.
3- the greatest number of shoots/explant (5.50, 5.27, 4.95 and 4.69) and the highest shoot length (5.30, 5.22, 5.25 and 4.91 cm) of Concord, Thompson Seedless, Beauty Seedless and King Ruby, respectively were obtained from MS medium supplemented with1.0 mg/L BA and 0.01 mg/L NAA.
4- The third subculture induced the maximum response of sprouting frequency and the optimum number of shoots/explant for the four cultivars.
5- For in vitro rooting, half strength MS medium supplied with 1.0 mg/L IBA resulted in the best root formation (90, 100, 80 and 70%) for Concord, Thompson Seedless, Beauty Seedless and King Ruby cultivars, respectively.
Regeneration
1- The maximum embryogenesis rate 10% for Thompson Seedless and 5% for Concord was produced on MS medium fortified with 0.5 mg/L 2, 4-d + 1.0 mg/L BA.
2- The highest organogenesis rate 10% for Concord cultivar was obtained on LS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. The other genotypes failed to produce regenerated shoot through organogenesis.
3- Regeneration through indirect organogenesis from axillary buds and nodal segments was superior to embryogenesis and direct organogenesis from leaf explants. LS medium fortified with 2.0 mg/L BA + 0.5 mg/L NAA produced the best regeneration rate (80 and 60%) with the optimum number of shoots/explant (5.1 and 4.0) for Concord, Thompson Seedless, respectively,. While the best regeneration medium for Beauty Seedless was LS medium contained 3.0 mg/L BA + 0.5 mg/L NAA that resulted in significant regeneration frequency 30% and the number of shoots/explant (2.0). King Ruby recorded the maximum regeneration 20% combined with the optimum number of shoots (2.0) on LS medium supplied with 1.0 mg/L BA + 0.5 mg/L NAA.
4- Darkness conditions for 1, 2 and 3 weeks for the four cultivars did not increase the regeneration frequency (%) in comparison to the regeneration under light conditions.
5- The addition of STS at different levels to the regeneration medium of the four cultivars did not enhance shoot formation but enhanced root formation.
6- The inclusion of putrescine and spermine at different concentrations did not imrove the regeneration rate for the four cultivars compared to the control treatment except for the regeneration of Concord at 0.5 µM putresciene and Thompson Seedless at 1.0 µM spermine.
7- For Kanamycin sensitivity, the concentration of 200 mg/L kanamycin inhibited the growth for Concord cultivar, 150 mg/L was the inhibitory level for Thompson Seedless and King Ruby. While, 100 mg/L was the most suitable concentration for Beauty Seedless.
Transformation of grapevine with sHSP
1- Phenylalanine at 2.0 mg/L was the most effective to resolve the necrosis problem of grapes tissue in response to Agrobacterium infection.
2- Ticarcillin at 150 and 200 mg/L and timentin at 200 mg/L compeletly inhibited Agrobacterium over-growth in the selection medium after transformation process.
3- Positive transgenic events for Concord (6 shoots) and for Thompson Seedless (2 shoots) were obtained when acetosyringone was added to the Agrobacterium culture overnight and to the co-cultivation medium at 100 µM and also the Agrobacterium culture density was 0.6 OD600.While, no transgenic shoots were obtained from Beauty Seedless or King Ruby.
Characterization of sHSP in tomato as model plant.
1- For in vitro experiment, the expression of sHSP in over-expression lines were better at high temperatures 30 and 35ºC than unstressed conditions 25ºC. In regardless of genotypes, exposure to high temperatures 30 and 35ºC 24 h for 10 days increased root length, root growth fresh weight and vegetative growth fresh weight of (1-2, 2-3 and 3-4 cm) over-expression lines compared to wild type.
2- For greenhouse experiment, plantlets of over-expression lines exhibited thermotolerance compared to wild type and antisense lines at high temperatures of 30, 35 and 40ºC for four hours daily. Fresh and dry weight of vegetative and root growth of over-expressed lines increased in response to heat stress 30, 35 and 40ºC in comparison to wild type and antisense lines.
Recommendation:
To produce transgenic grapes, axillary buds and nodal segments were the best explants in LS medium supplied with different concentrations of BA and NAA at 0.5 mg/L. Phenylalanine at 2.0 mg/L was the most effective to solve the necrosis problem. Ticarcillin at 150 mg/L and timentin at 200 mg/L inhibited the overgrowth of Agrobacterium. The addition of AS at 100 µM with Agrobacterium OD600 0.6 increased the transformation efficiency.
It is higly recommended to produce genetically modified grapes with sHSP to enhance plants thermotolerance under elevated temperatures and apply transformation techniques to produce genetically modified plants tolerance to other biotic and abiotic stresses.