I= STUDIES ON PRODUCTION OF TRANSGENIC RICE PLANTS RESISTANT OF BACTERIAL BLIGHT USING AGROBACTERIUM MEDIATED TRANSFORMATION
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Title of Thesis
STUDIES ON PRODUCTION OF TRANSGENIC RICE PLANTS RESISTANT OF BACTERIAL BLIGHT USING AGROBACTERIUM MEDIATED TRANSFORMATION

Author(s)
AMANA NOOR
Institute/University/Department Details
Department of Biochemistry/ Quaid-i-Azam University, Islamabad
Session
2008
Subject
Biochemistry
Number of Pages
207
Keywords (Extracted from title, table of contents and abstract of thesis)
transgenic rice plants, bacterial blight, agrobacterium, xanthomonas oryza, basmati, basmati 370,basmati 385, basmati 2000, super basmati, pakhal, jp-5, swat-i, dr -83, ks-282, xa 21 gene

Abstract
Rice is one of the most important food crops of Pakistan and second chief commodity after wheat. It suffers from destructive Bacterial Leaf Blight disease (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo). Approximately 50% incidence of BLB disease has been reported in Kallar, a high quality rice producing belt of Pakistan. The use of resistant varieties is feasible to control the disease. For this purpose, the gene, Xa 21 for bacterial blight resistance is now being transferred into commercial varieties of rice. Since, Pakistan is one of the major rice producers, concerted efforts are underway to develop transgenic rice using local varieties as the starting material. For this study, nine indica varieties were selected including four Basmati, Basmati 370,Basmati 385, Basmati 2000 and Super Basmati and five coarse varieties i.e. Pakhal, JP-5, Swat-I, DR -83 and KS-282 on the basis of popularity due to aroma in basmati rice and availability in the local market.

High frequency callus induction and regeneration is the pre-requisite for successful transformation. Mature seed was used as explant source. The surface sterilized seeds were inoculated on different kinds of callus induction media i.e. N6 and MS for callus induction. N6 media with 2 mg/ l 2, 4-D proved to be better (84.16%) than MS (63.6%). After three weeks, scutellum derived calli were transferred to shoot regeneration medium (MS) supplemented with different concentrations and combinations of NAA (1mg /1 - 2 mg/ l) and BAP (0.5, 2.5 and 5mg/ l) or Kinetin (0.5, 2.5, 5.0 and 10 mg/l). A large variation of shoot regeneration (0-91%) was observed among the rice varieties depending upon the medium. DR-83 showed best regeneration (91.11%) on shoot regeneration medium 8 (MS + NAA 1.0 mg/l + Kin 0.5mg/1 + BAP 2.5mg/l) followed by Basmati 385 on shoot regeneration medium 3 (MS + NAA 1.0mg/1 and BAP 5.0 mg/l) i.e. 83.3 %. Water stressed condition also showed positive effect on regeneration frequency. The regenerated plants were shifted to glass house and grown to maturity. The somaclones were significantly lower than those of control plants.

Three week old scutellum derived calli of more than 5 mm size were infected with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pTCL5 containing genes Xa 21 for bacterial blight resistance, hygromycin rersistance gene as a selectable marker and GUS as a reporter gene. Different parameters affecting the transformation efficiency were optimized during the study. Hygromycin at 50mg/1 was lethal dose for the selection of transformed calli. Co-cultivation period of 2 days was found to be best for maximum GUS expression. Age of mature embryo derived calli was also a key factor in transformation process. Twenty one days old scutellum derived calli showed highest number of GUS expression. Application of 400┬ÁM acetosyringone promoted the production of resistant calli and for the maximum GUS expression. Effect of antibiotics in selection and regeneration of transformed rice calli was investigated. Result showed that both cefotaxime (500mg/l) and carbenicillin (250mg/l) were better than cefotaxime (500mg/l) or carbenicillin (500mg/l) alone for the control of bacteria. Direct selection protocol produced significantly higher number of hygromycin resistant calli as compared to indirect selection. Transgenic plants were established in glass house and were grown to maturity. A large number of morphologically normal, fertile transgenic plants were obtained. DR-83 showed a transformation frequency of 43.75% followed by Basmati 385 i.e. 42.50 %.

The susceptibility of nine rice varieties at different growth stages (30, 60 and 90 days) to bacterial leaf blight was evaluated with the twenty different local and exotic strains/isolates of Xoo by leaf clipping inoculation method. It was evident from the results that all the nine rice varieties were susceptible to Xoo at the seedling stage. The disease incidence of all the nine rice varieties was less at maximum tillering stage and at leaf flag stage.

PCR analysis of transgenic plants revealed the integration of the Xa 21 gene and hpt gene into the host genome. Transgenic TO containing the Xa 21 gene for bacterial blight resistance was evaluated in the glass house for reaction to eight races of Xoo, including three exotic and five local strains/isolates. Inoculation was done at seedling stage and at maximum tillering stage following the clipping method and the percent disease incidence was calculated 14 days after inoculation. The inoculation tests displayed that transgenic To plants were resistant. The present findings suggest that rice varieties carrying Xa 21 gene can effectively control BLB disease, however, further studies are needed to evaluate their performance in field

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
230.95 KB
2 1 Introduction And Review Of Literature
761.67 KB
  1.1 Crop 1
  1.2 Origin And Distribution 3
  1.3 Ecology 4
  1.4 World Production Of Rice 4
  1.5 Rice Varieties 7
  1.6 Export Of Rice 8
  1.7 Agro-Climatic Zones 10
  1.8 Importance And Uses 11
  1.9 Constraints Of Rice Production 13
  1.10 History Of Bacterial Leaf Blight 15
  1.11 Symptoms Of Bacterial Leaf Blight 17
  1.12 Rice Improvement 20
  1.13 Genetic Transformation 21
  1.14 Methods Of Genetic Transformation 35
  1.15 Induction Of Resistance By Genetic Manipulation 46
  1.16 Xa 21 Bacterial Leaf Blight Resistance Gene 46
  1.17 Aims And Objectives 54
3 2 Materials And Methods
4922.9 KB
  2.1 Techniques Routinely Followed In Plant Tissue Culture Work 56
  2.2 Transformation 62
  2.3 Bacterial Blight 72
  2.4 Molecular Analysis 77
  2.5 Resistance Analysis 81
  2.6 Inheritance And Mendelian Segregation 81
  2.7 Statistical Analysis 82
4 3 Results
1260.04 KB
  3.1 Part I: Tissue Culture 84
  3.2 Part 2: Regeneration 95
  3.3 Part 3: Transformation 104
  3.4 Part 4: Bacterial Leaf Blight 123
  3.5 Part 5: Molecular Analysis Of Transgenic Plants 130
5 4 Discussion
295.42 KB
  4.1 Factors Affecting Callus Induction 139
  4.2 Factors Affecting Regeneration 143
  4.3 Effect Of Water Stress 146
  4.4 Glass House Assessment Of Soma Clone Of Rice 146
  4.5 Transformation 147
  4.6 Regeneration Frequency Of Transgenic Plants And Their Gus Expression 154
  4.7 Bacterial Blight 154
  4.8 Analysis Of Transgenic Plants 159
  4.9 PCR Analysis For Xa 21 (Bacterial Blight Resistant) Gene 160
  4.10 Resistance Identification Of Primary Transgenic (To) Plants Against Bacterial Blight 161
  4.11 Inheritance Of Xa 21 Gene In Transgenic T1 Seedling 162
  4.12 Conclusion And Future Directions 162
6 5 Rferences
428.65 KB
  5.1 References 165
  5.2 Electronic-Database Information 207