Traditional rice varieties consists of many important traits such as medicinal and nutritional value, drought and pest resistance. Efficient tissue culture and transformation techniques are important in uncovering such genes from traditional rice varieties. Aim of this study was to optimize an efficient protocol of callus induction and transformation of Sri Lankan traditional rice varieties.
Dahanala, Kaluheenaty, Kalubalawee, madael, Kahatawalu, Kaharamana, Gonabaru, Alwee, Wannidahanala, Kottiyaran, BG380 and H4 were selected for the study. Twelve different concentration combinations of 2, 4-D and kinetin were used to determine the effect of hormone concentrations on callus induction. Calli were transformed using Agrobacterium mediated transformation. For calli transformation Agrobacterium tumefaciens GV3101 containing pBI121 vector was used. Transformed callus were detected using GUS analysis. Efficiency of transient transformation was calculated for each variety. There was a significant difference of 12 treatments of callus induction of tested varieties except Gonabaru. Kaluheenaty, Madael, Gonabaru, Kottiyaran and H4 have shown the best callus induction for the same hormone concentration combination of 2.5mg/L 2,4-D and 0.5 mg/L kinetin. Other six varieties showed best callus induction for different concentration combinations with majority of them being in the region of 2.5mg/L 2,4-D and 0.5 mg/L kinetin.
Results showed that callus induction was genotype dependant. Higher transient transformation efficiencies were reported for most varieties. Depending on transient transformation efficiency, tested varieties could be divided into significantly different seven groups. Results of this study indicate the possibility of using optimized hormone concentrations and Agrobacterium mediated transformation for genetic transformation of selected Sri Lankan traditional rice varieties.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENT
TABLE OF CONTENTS
LIST OF TABLES
LIST OF PLATES
LIST OF FIGURES
CHAPTER
INTRODUCTION
1.1 Background
1.2 Justification
1.3 Objectives
CHAPTER
LITERATURE REVIEW
2.1 Rice
2.1.1 Sri Lankan traditional rice varieties
2.1.2 Importance of traditional rice varieties
2.2 Genetic transformation of plants
2.2.1 Gene transferring methods
2.2.1.1 Agrobacterium mediated method
2.2.1.2 Chemical methods
2.2.1.3 Electroporation
2.2.1.4 Particle gun method
2.2.1.5 Micro injection
2.2.2 Detection of transformants
2.2.2.1 Scorable reporters
2.2.2.2 Selectable reporters
2.3 Development of rice transformation systems
2.3.1 Agrobacterium mediated transformation of rice
2.3.2 Agrobacterium mediated transformation of Sri Lankan rice varieties
2.4 Suitable rice explants for genetic transformation
2.4.1 Callus induction and regeneration media for rice
2.5 Applications of Agrobacterium mediated transformation of rice
2.5.1 Transformation of rice for crop development
2.5.2 Transformation of rice for functional genomics
CHAPTER
MATERIALS AND METHODS
3.1 Plant material
3.2 Culture media
3.2.1 Callus induction medium
3.2.2 Culture medium for Agrobacterium strain
3.3 Vector and bacterial strain
3.4 Callus derivation
3.4.1 Surface sterilization of seeds
3.4.2 Inoculation of seeds and incubation
3.5 Agrobacterium mediated calli transformation
3.6 Detection of transformants using GUS analysis
3.7 Experimental design and Data Analysis
3.7.1 Callus induction
3.7.2 Transformation efficiency
CHAPTER
RESULTS
4.1 Callus induction
4.1.1 Effect of growth regulators on callus initiation
4.1.2 Effect of growth regulators on callus growth
4.2 Genetic transformation of rice
4.2.1 Efficiency of genetic transformation of callus
CHAPTER
5.1Rice explants for callus formation
5.2 Surface sterilization of seeds before inoculation
5.3 Nutrient media for callus induction
5.4 Callus initiation and growth
5.4.1 Effect of growth regulators on callus initiation
5.4.2 Effect of growth regulators on callus growth
5.4.3 Effect of genotype on callus induction
5.5 Effect of other minor nutrients on callus induction
5.6 Efficiency of genetic transformation of callus by - mediated method
5.6.1 Factors affecting Agrobacterium mediated
transformation of rice
5.6.2 Efficiency rice calli transformation using other gene transfer methods
CHAPTER
CONCLUSIONS AND SUGGESTIONS
6.1 Conclusions
6.2 Suggestions
REFERENCES
APPENDIX I – Composition of MS medium
APPENDIX II – Compostion of LB medium
-
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X. -
Upload your own papers! Earn money and win an iPhone X.