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Title
CERTIFICATE
DECLARATION
Aknowledgements
CONTENTS
1. Introduction
1.Amature rubber plantations
2. Review of Literature
2.1. Crop improvement in H. brasiliensis: Conventional methods, achievements and constraints
2.2. Biotechnological approaches for crop improvement in Hevea brasiliensis
2.2.1. Marker assisted selection
2. 2.2. Development of in vitro plant regeneration system
2. 2.3. Somatic embryogenesis as a means of micropropagation
2.2.4. Somatic embryogenesis Hevea brasiliensis
2. 2. 5 (1) Factors influencing plant regeneration via somatic embryogenesis
2.3. Plant genetic transformation
2.3.1. Agrobacterium tumefaciens: a natural vector for genetic transformation
2.3.2. Plant genetic transformation: an overview
2. 3. 3. Genetic transfomiation in Hevea brasiliensis
2. 3. 4. Control of oxidative stress tolerance in plants
2. 3.4 (I) Role of SOD in abating oxidative stress
2. 3. 5. Tapping panel dryness (TPD)
2.3.5 (1) Hole of SOD in the prevention of TPD
2. 3. 6. Development of transgenic plant for enviornmental stress tolerance
3. Materials and methods
3.1. Development of transgenic plants
3. 1.1 Induction of callus
2.Flower buds of Hevea
3. 1. 2. Agrobacterium tumefaciens strain and binary vector.
3.Binary vector pDU96.2412 used for genetic transformation.
4.SOD, GUS and nptll genes between RB and LB of T-DNA region
3. 1.3. Preparation of antibiotics
3. 1. 4. Antibiotic kill curve tests
3. 1. 5. Preparation of Agrobacterium culture for infection
3. 1. 6. Agrobacterium infection and co-culture
3. 1.7. Preparation of co-cultivation medium
3. 1.8. Selection of transformed cell lines by GUS histochemical staining
3. 1. 9. Callus proliferation and ernbryogenic callus induction
3. 1.10. Somatic embryo induction
3. 1.11 . Embryo maturation
3. 1.12. Embryo germination and plant regeneration
3. 1.13. Culture conditions
3. I. 14. Acclimatization of the plants
3.2. Molecular analysis of transgenic plants
3. 2.1. Isolation of DNA from and control plants
3. 2. 2. DNA quantification
3. 2. 3. Isolation of plasmid DNA
3. 2.4. Polymerase chain reaction
3. 2. 5. Southern hybridization analysis
3. 2. 5 (a) Restriction digestion of genomic DNA
3. 2. 5 (b) Blotting of the DNA fragments
3. 2. 5 (c) Preparation of labeled probe
3. 2. 5 (d) Hybridization
3. 2.5 (e) Blot washing and autoradiography
3.3. SOD expression by Northern hybridization.
3. 3. 1. lntroduction of stress
3. 3.2. RNA isolation from leaf and callus samples
3. 3. 2 (a) Electrophoresis of RNA
3.3.2 (b) RNA blotting
3. 3. 2 (c) Preparation of labeled probe and hybridization
3.3. 2 (d) Washing and autoradiography
3.4. Estimation of superoxide dismutase, peroxidase and catalase enzyme activities in transformed callus
3.4.1. Induction of stress
3.4. 2. Enzyme preparation
3. 4. 3. Assay of Superoxide dismutase
3. 4. 4. Estimation of Peroxidase enzyme activity
3. 4. 5. Catalase enzyme assay
3.4 6. Protein estimation
4. Results
4. 1. Development of Transgenic plants
4.1.1. Induction of callus for Agrobacterium infection
4.1.2. Identification of ideal explant stage for Agrobacterium infection
4.1.3. Identification of the ideal antibiotic for the selection of transformed callus
5.Callus formed two months after inoculation of immature anther.
6.Proliferation of transformed callus in the selection medium.
7.Histochemical staining for GUS expression in the callus.
4. 1.4. Selection of transformed callus by GUS histochemical staining
4. 1. 5. Callus proliferation and embryogenic callus induction
4.1.6. Somatic embryo induction
8.Transformed embryogenic callus
9.Cluster of Globular embryos
4.1.7. Effect of water and osmotic stress on embryo induction
10.Effect of osmotic stress on callus fresh weight.
4. 1.8. Embryo maturation
11.Effect of water stress on callus fresh weight
12.Somatic embryos under different developmental stages.
13.Mature embryos with well-developed cotyledon
4. 1. 9. Effect of amino acid on embryu maturation
4. 1. 10. Effect of water and osrnotic stress on embryo maturation
14.Effect of amino acids on embryo maturation
4. 1. 11. Embryo germination and regeneration of transgenic plants
15.Germinating embryos with shoot and root primodia
16.Fully developed transgenic plantlet in culture tube
17 Transgenic embryo showing GUS expression
18. Transgenic plantlet showing GUS expression
4. 1. 12. Acclimation of the plants and transplantation to soil
19.Hardened transgenic plants.
20.Transgenic plant growing in polythene bag
4.2. Molecular analysis of transgenic plants
4. 2. I. Polymerase chain reaction
4.2.2. Southern hybridization analysis
21.Detection of trangenes by PCR.
4. 3. Northern hybridization analysis
22.Southern hybridization of genomic DNA from transgenic plants
23.Northern blot analysis after probing with Mn SOD cDNA.
4.4. Estimation of superoxide dismutase, peroxidase and catalase enzyme activities in transformed callus
4.4.1. Effect of water and PEG stress on SOD enzyme activity
5. Discussion
5.1. Development of transgenic plants
5.2. Molecular confirmation of gene integration
5.2.1. Polymerase chain reaction analysis
5.2.2. Southern hybridization analysis
5.3. SOD gene expression by Northern blot analysis
5.4. Evaluation of superoxidc dismutase, peroxidase and catalase enzyme activities in transformed callus
6. Summary and Conclusions
References
List of Figures
ABBREVIATIONS