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Title of Thesis

Iqbal Hussain
Institute/University/Department Details
Department of Botany/ Faculty of Sciences/ Arid Agriculture University Rawalpindi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
fungal pathogens, potato, genetic transformation, agrobacterium, chitinase gene, cardinal, diamant, ultimus, hygromycin gene, fusarium, phytophthora, callus induction

Agrobacterium mediated transformation protocols for introduction of rice chitinase gene have been established to induce resistance against fungal pathogens in potato variety cardinal. Optimization of well established tissue culture system is an important step before genetic manipulation of desired gene. Initially tissue culture protocols have been established for three commercially important varieties of potato which were cardinal, diamant and ultimus. Different ex-plant nodes, intemodes, leaf disks, microtuber discs and shoot apices were tested for their in vitro responses such as direct regeneration, callus formation and regeneration through callus. The highest frequency of direct regeneration was achieved from nodal explants with 17.6 number of shoots/culture from variety cardinal, 14.3 from variety diamant and 9.0 from variety ultimus. The best media optimized for high regeneration was Murashige and Skoog (MS) media containing BAP 4.0 mg/l and IAA 0.5 mg/l. Maximum callus induction frequency (100 percent) was obtained from the MS media having 2, 4-D at the concentrations of 4.0 or 5.0 mg/l from all the explants tested except micro tuber discs. Regeneration of plantlets was higher i.e. 9.20 percent for calli derived from nodal explants, 3.60 percent leaf discs, 3.0 percent shoot apices, and 2.80 percent intemodes. Microtubers did not show any regeneration. Protocols for microtuberization of potato have been established for variety cardinal and maximum numbers of tubers 16.4 were obtained and 15.6 number of tubers/culture from MS media containing cholorocholine chloride (200 mg/l) or Sucrose (90 g/l) respectively. Statistically there was no significant difference between cholorocholine chloride and sucrose on microtuber induction. After establishing different tissue culture protocols genetic transformation of potato was carried out by using Agrobacterium tumefaciens strain ERA 101. The plasmid used in this study was a binary vector pBI333-EN4-RCG3 having chitinase gene, kanamycin and hygromycin resistant genes as selectable marker. Chitinases are considered as examples of antifungal cell-wall degrading enzymes that have been successfully used in genetic engineering in various economically important plant species in inducing resistance against fungal diseases. Chitinases catalyse the hydrolysis of chitin, B-l,4-linked polymer of N-acetyl-d-glucosamine that is the major component of the cell wall of most filamentous fungi. Nodal explant was used as explant source for transformation. Co-infection period of 2 minutes followed by 7 days of pre-selection was best suitable to achieve maximum transformation efficiency. Cefotaxime at 500 mg/l was found appropriate concentration to control the excessive bacterial growth. Transgenic potato plants were obtained on selection media containing 20 mg/l hygromycin. Presence of hygromycin and chitinase gene was confirmed by PCR analysis. Control plants did not show the presence of chitinase and hygromycin genes. Transgenic potato plants evaluated for fungal pathogens revealed the induction of high resistance against Fusarium while slight resistance was also observed against Phytophthora infestans. The non-transformed (control) plants of potatoes showed the symptoms of diseases when infection was given with Fusarium and Phytophthora infestans.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
1611.4 KB
2 1 Introduction 1
662.62 KB
3 2 Review Of Literature 7
3653.96 KB
  2.1 General Review 7
  2.2 Conventional Methods Of Potato Improvement 15
  2.3 Modern Methods For Potato Improvement 17
  2.4 Genetic Transformation 22
4 3 Materials And Methods 39
2993.19 KB
  3.1 Virus Free In Vitro Plantlet Production 39
  3.2 Microtuberization In Potato 47
  3.3 Callus Induction And Regeneration 50
  .3.5 Genetic Transformation 52
  3.6 Transformation Of Chitinase Gene In To Potato 59
  3.7 Molecular Analysis For Hygromycin And Chitinase 64
  3.8 Pathogenesity 67
5 4 Results And Discussion 71
6684.21 KB
  4.1 Virus Free In Vitro Plantlet Production 71
  4.2 Direct Regeneration 82
  4.3 Microtuberization 91
  4.4 Callus Induction And Regeneration 97
  4.5 Transformation Of Chitinase Gene In To Potato 111
  4.6 Factors Affecting Transformation Of Potato 116
  4.8 Pathogenesity 128
  4.9 Implication Of Genetic Transformation 132
  4.10 Implications For Potato Improvement 133
6 5 Summary 135
4858.85 KB
  5.1 Literature Cited 138
  5.2 Appendices 183