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
1. INTRODUCTION
1.1. Background
1.2. Justification
2. 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 system
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 improvement
2.5.2. Transformation of rice for functional genomics
3. 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
4. RESULTS
4.1. Callus induction
4.2. Genetic transformation of rice
5. DISCUSSION
5.1. Rice 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
5.5. Effect of other minor nutrients on callogenesis
5.6. Efficiency of genetic transformation of callus by Agrobacterium mediated method
6. CONCLUSIONS AND SUGGESTIONS
6.1. Conclusions
6.2. Suggestions
Research Objectives and Key Topics
This research aims to optimize protocols for callus induction and Agrobacterium-mediated genetic transformation in selected traditional Sri Lankan rice varieties, providing a basis for functional genomics and the development of genetically improved crops.
- Optimization of callus induction using hormone combinations (2,4-D and Kinetin).
- Evaluation of amenability of diverse traditional rice genotypes to transformation.
- Application of Agrobacterium tumefaciens strain GV3101 for T-DNA delivery.
- Assessment of transformation efficiency using GUS reporter gene analysis.
- Correlation of genotype and hormonal factors with transformation success.
Excerpt from the Book
1.1. Background
As the major staple food, rice has high demand with the increasing population in Sri Lanka. There is an enormous need to increase yield. Increasing productivity of cultivated varieties is a better approach than increasing cultivated area, as land and labor are critical limiting factors. In addition to high yield, rice with improved nutritional value is needed to improve nutritional condition of the population in areas where diversity of food intake is a concern. Rice varieties with high yielding and improved nutritional value can be obtained through genetic modification. There are a dearth of genes, responsible for good qualities in genomes of traditional rice varieties. However knowledge on important target genes requires functional genetic studies in traditional rice varieties. Transgenic rice system can be applied to identify new genes, promoters, and enhancers. Their functions could be introduced in to new rice varieties. Method for production of transgenic varieties involves in-vitro plant regeneration and genetic transformation methods.
Genetic transformation of plants is a widely used tool in improvement of crop species and basic studies in Biology. Incorporation of stress tolerance, pest resistance, herbicide resistance, improved performances and value adding traits are used in crop improvement. Plant transformation is also used in studies of gene expression and biochemistry. Transformation is the incorporation of foreign DNA into plant genomes. The first transformation of plants was carried out through insertion of kanamycin resistant gene into tobacco genome in 1n 1986 (James and Krattiger, 1996). With the development of transformation techniques today many plants have been transformed successfully. The first transgenic rice plants were obtained in 1988. (Toriyama et al., 1988; Zhang et al., 1988; Zhang and Wu, 1988)
Summary of Chapters
INTRODUCTION: Outlines the necessity of increasing rice productivity in Sri Lanka and the potential of genetic transformation for crop improvement.
LITERATURE REVIEW: Examines existing knowledge on rice classification, transformation methodologies, and the specific role of Agrobacterium in functional genomics.
MATERIALS AND METHODS: Describes the plant materials, specific hormone combinations, and technical procedures used for callus derivation, Agrobacterium-mediated transformation, and data analysis.
RESULTS: Presents findings on the effect of different growth regulator combinations on callus initiation, growth, and the varying transformation efficiencies across twelve rice varieties.
DISCUSSION: Evaluates the experimental results, comparing them with established literature on explant suitability, media composition, and genotypic influences on transformation success.
CONCLUSIONS AND SUGGESTIONS: Summarizes the key findings regarding genotype-specific responses and suggests further research into plant regeneration and functional gene analysis.
Keywords
Agrobacterium tumefaciens, Callus induction, Genetic transformation, Sri Lankan rice, Tissue culture, 2,4-D, Kinetin, Functional genomics, Transgenic plants, GUS analysis, Genotype dependence, Crop improvement, Indica rice, Plant biotechnology.
Frequently Asked Questions
What is the primary focus of this research?
The research focuses on assessing the amenability of twelve traditional Sri Lankan rice varieties to Agrobacterium-mediated genetic transformation and optimizing callus induction protocols for these specific genotypes.
Which rice varieties were included in the study?
The study utilized ten traditional varieties (Dahanala, Kaluheenaty, Kalubalawee, Madael, Gonabaru, Kahatawalu, Kaharamana, Alwee, Wannidahanala, Kottiyaran) along with two improved varieties (BG380 and H4).
What is the primary research goal?
The primary goal is to establish efficient tissue culture and transformation protocols that enable functional genomics research in traditional Sri Lankan rice varieties.
What methodology was employed for transformation?
The study used Agrobacterium tumefaciens strain GV3101 harboring the pBI121 vector, utilizing the GUS reporter gene system to detect successful transformation.
What did the results indicate regarding callus induction?
Results showed that callus induction is genotype-dependent, and that 2,4-D is essential for initiation, with optimal hormone concentrations varying significantly among the tested rice varieties.
What are the key themes addressed in the work?
Key themes include plant tissue culture optimization, Agrobacterium-mediated gene transfer, the importance of traditional rice varieties, and the application of transgenic technologies for agronomic trait improvement.
Why are traditional rice varieties important according to the text?
Traditional varieties are noted for harboring important genes related to medicinal and nutritional value, as well as drought and pest resistance, which are not yet fully understood.
Was the transformation efficiency consistent across all varieties?
No, the study observed significant variation in transient transformation efficiency, with rates ranging from approximately 45% to 100% depending on the specific rice genotype.
- Citar trabajo
- Krishanthi Bandara (Autor), 2012, Efficiency of Callogenesis and Amenability to Agrobacterium Mediated Transformation of Selected Traditional Sri Lankan Rice Varieties, Múnich, GRIN Verlag, https://www.grin.com/document/367829
