Efficiency of Callogenesis and Amenability to Agrobacterium Mediated Transformation of Selected Traditional Sri Lankan 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

ABSTRACT

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.

ACKNOWLEDGEMENT

First of all I thank Dr. B. A. Karunarathne, Dean, Faculty of Applied Sciences, Rajarata University of Sri Lanka for providing opportunity to carry out this project.

I like to express my heartfelt gratitude to my supervisor Dr. T.C. Bamunuarachchige, Head, Department of Biological Sciences, Faculty of Applied Sciences, Rajarata University of Sri Lanka, for invaluable guidance and support to complete this project. Without his assistance this study would not have been successful.

My gratitude also goes to my external supervisor Dr. Gamini Samarasinghe, Research Officer, Rice Research and Development Authority, Bathalegoda for his valuable guidance to make this project a success. And I thank Ms. D. Kekulandara and other staff members at tissue culture laboratory, Rice Research and Development Institute, for their valuable support.

I extend my gratitude to Dr. H. M. V. G. Herath, Senior Lecturer at Faculty of Agriculture, University of Peradeniya and Dr. (Ms.) H. A. M. Wickramasinghe, Senior Lecturer at Faculty of Agriculture, University of Peradeniya for their support. And I like to thank Mr. Udumulla, Technical officer, Plant Molecular Genetics Laboratory, Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya for his kind assistance.

I take this opportunity to thank Dr. G. Anil U. Jayasekera, senior lecturer, Department of Plant Sciences, University of Colombo for his great support.

My special thanks goes to Mr. Imesh De Silva also for his support to make this project success.

I take this opportunity to thank Mr. Ranjan Dissanayake for his great support. I like to thank Mrs. Neelamani Yapa and Dr. (Mrs.) W. M. G. C. K. Mannapperuma for advices and support for making the project a success.

I take this opportunity to thank all other academic staff members who helped in various ways to successfully complete the project.

I wish to express my sincere gratitude to Mr. A. M. B. Prasad, Technical Officer of Botany laboratory and Lab Assistants at the Botany laboratory for their valuable support.

Finally, I thank my friends and family members for their encouragement and support.

LIST OF TABLES

Table 3.1 Different concentration combinations of hormones 24 used for callus induction

Table 4.1 Best hormone combinations for callus initiation of each 30 tested variety

Table 4.2 Best hormone combinations for callus growth of each 31 tested variety

Table 4.3 Mean value of percentage of transformed calli

Table 4.4 Groups of varieties based on transformation efficiencies

LIST OF PLATES

Plate 4.1 Germinated seeds, three days of inoculation

Plate 4.2 Initiated callus after two weeks of inoculation

Plate 4.3 Initiated callus after four weeks of inoculation

Plate 4.4 Callus six weeks after inoculation

Plate 4.5 Callus of Kaluheenaty after six weeks

Plate 4.6 Callus of Kalbalawee after six weeks

Plate 4.7 Transformed callus after GUS assay

Plate 4.8 Transformed callus after GUS assay

Plate 4.9 Callus of Alwee with blue color after GUS assay

Plate 4.10 Callus of Kaluheenaty with blue color after GUS assay

Plate 4.11 Callus of H4 with blue color after GUS assay

LIST OF FIGURES

Figure 4.1 Efficiency of genetic transformation of callus

CHAPTER 1

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)

Agrobacterium mediated transformation, particle gun method, electroporation, chemical treatment and viral mediated transformation are available mechanisms to transfer DNA into plant cells. Among those methods simplest and easiest method is Agrobacterium mediated transformation. Infection mechanism of plant pathogen Agrobacterium tumefaciens is used in this method. Through transfer and integration of T-DNA into host plant genome, it elicits a neoplastic growth in host cells (Tzfira et al., 2002). As a transformation tool this method is more advantageous over other methods. Agrobacterium mediated method leads to relatively uncomplicated insertions with low copy number and minimal rearrangements. It makes this method efficient in gene transfer. Moreover this method is adaptable to different cell types and culture procedures.

At the onset of using Agrobacterium mediated transformation it was limited to few plant types excluding monocotyledonous plants. But it is now possible to transform wide range of plants including many agronomically important plants (Cheng et al., 1997). Soy bean, potato, tomato, pea were initially commercialized transgenic plants. Monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they are outside the host range of Agrobacterium tumefaciens. However all cereal crops are proved to be transformable via Agrobacterium (Hiei et al., 1994). Agrobacterium transformation of monocotyledonous tissues is successful in the presence of acetosyringone. Acetosyringone is the potent inducer of Agrobacterium virulence genes. First successful Agrobacterium transformations of rice were recorded in 1994 (Hiei et al., 1994).

Higher efficiencies of Agrobacterium mediated rice transformations have been recorded. This method has been successfully applied to both japonica and indica rice varieties (Ye et al., 2000; Tran and Huynh, 2006). Agrobacterium mediated transformation is variety dependent. Therefore, each variety should be tested for responses for transformation prior to introduction of genes of interest.

Oryza sativa and Oryza glaberima are the two major cultivated rice species in a global context. Thousands of varieties of those two species have been reported. However hybrid varieties are the most popular among farmers. Twenty wild rice varieties have been reported worldwide. In Sri Lanka the cultivated varieties belong to Oryza sativa indica variety. There are nearly a thousand traditional varieties of O. sativa present in Sri Lanka. Additionally five wild species have been recorded namely O. nivara, O. rufipogon, O. eichingeri, O. rhizomatis and O. granulata.

Heenati, Dahanala, Hodarawalu, Kalu heenati, Kuru wee, Kurulu thuda, Ma wee, Pola el, Rathel, Suwadel are some of these. Most traditional varieties have been recorded for their own nutritional or medicinal characters. Genes responsible for important characters have not been studied extensively. Thus information on amenability for transformation could be a great help in studying expression of genes in these traditional varieties.

1.2. Justification

In current world situation food is fast becoming the major limiting factor for human life. Rice is a major food item in large part of world population. Thus there is a huge need for improvement of rice to accomplish the demand by increasing yield. There are many barriers for improving yield. Extreme environmental conditions and decrease of cultivated land are major problems. To escape from such barriers, rice plant should be improved for performance. For such improvements, molecular tools are an essential requirement. Transgenic rice plants have been developed which can be applied in functional genomics and crop improvement. Drought tolerance, pest resistance, viral resistance and improved nutritional quality are such engineered traits.

Rice is the model plant of cereal crops as its represents a modest genome of 430Mb with more than 30,000 genes. However most of genes are not functionally analyzed yet. Agrobacterium mediated transformation plays a major role in methods of functional genomics. Because this method is used to produce mutants which are used to identify functions of genes. Thus information on transformation of rice varieties is important in functional genomics.

Most traditional varieties consist of so many important genes which are needed to be identified. Thus this research provides a basis for functional analysis of rice genes as well as production of genetically improved transgenic rice varieties.

1.3. Objectives

To provide information on amenability of selected Sri Lankan traditional rice varieties to Agrobacterium mediated transformation.

To provide information on efficient tissue culture protocol for callus induction of Sri Lankan traditional rice varieties.

To provide information on efficiency of Agrobacterium mediated transformation of Sri Lankan traditional rice varieties.

CHAPTER 2

LITERATURE REVIEW

2.1 Rice

Genus Oryza has two cultivated species as O. sativa and O. glaberrima. There are more than 20 wild species distributed throughout the tropics and subtropics. Oryza sativa is identified as Asian rice (O. sativa) and cultivated worldwide, while O. glaberrima is only cultivated in a few countries in West and Central Africa.

Scientific classification of rice:

Kingdom : Plantae

Division : Angiosperms

Class : Monocot

Order : Poales

Family : Poaceae

Genus : Oryza

Species : Oryza sativa

There are three sub species, Oryza sativa japonica, indica and javanica. Japonica and indica are the two cultivated subspecies. Japonica varieties are usually cultivated in dry fields, in temperate East Asia, upland areas of Southeast Asia and high elevations in South Asia. Indica varieties are mainly grown in lowlands as submerged plants, throughout tropical Asia. The grain of japonica varieties are short and sticky while grain of indica varieties are non sticky and long. The grain of javanica varieties is broad and grows well under tropical conditions (Khush, 1997).

It is estimated that about 120000 varieties of rice exist in the world (Khush, 1997). After the establishment of International rice research institute in 1960, rice varietal improvement was intensified and high yielding varieties were developed. These improved varieties are used for in majority of the cultivated land.

2.1.1 Sri Lankan traditional rice varieties

Nearly thousand traditional rice varieties have been recorded in Sri Lanka. Pokuru samba, Pachchaperumal, Pola al, Kaluheenati, Baalamaawee, Behethheenatiya, Madathawalu, Rathkanda al, Suwandel, Mahamaawee are such traditional rice varieties.

2.1.2 Importance of traditional rice varieties

Behethheenati has medicinal qualities suitable for diabetes patients and as an antivenom agent. Hetadasahal is known to prevent constipation. Kaluheenati reduces toxins in body, improve body strength and can act as an anti-venom agent. Kuru wee and Rathkanda el are considered as having qualities of improving immunity. Ma wee consists of qualities suitable for patients having diabetes, tuberculosis, constipation and haemarhoids. Rathel has the property of prevention of stones in bladder and gall bladder. Additionally, they have qualities of stress tolerance and pest resistance (Rao et al., 2002).

2.2 Genetic transformation of plants

Transformation is the insertion, incorporation and expression of foreign genetic material in plant cells. The process of producing transgenic plants involves techniques to incorporate transgene into plant, detection of transformants and regeneration to produce transgenic plants.

2.2.1 Gene transferring methods

A number of mechanisms are available to transfer DNA in to plant cells such as chemical methods, electroporation, particle bombardment, lipofection, micro injection, fiber mediated method, pollen transformation and Agrobacterium mediated method have been used to mediate gene transfer to plant tissues (Ignacimuthu et al., 2000).

2.2.1.1 Agrobacterium mediated method

Agrobacterium mediated transformation method is the most used method for the introduction of foreign genes into plant cells (Dobhal et al., 2010). In this method Agrobacterium tumefaciens is used. Agrobacterium tumefaciens is a soil plant pathogen. It has unique ability to transfer its DNA (T-DNA) into the genome of eukaryotic cells. The transformation results from the production of a single stranded copy (T-strand) of transferred DNA (T-DNA) molecule by the bacterial virulence machinery. Then it is transferred into the host cell followed by integration into the host genome (Citovsky et al., 2007). When it travels targeting the host genome, T- strand is first transported in to host cell cytoplasm via type IV secretion system (Christie, 2004; Lacroix and Citovsky, 2009). T- Strand travels within the host cell as a nucleoprotein complex. Bacterial virulence proteins and host proteins are involved in nucleoprotein complex (Tzfira and Citovsky, 2002).

Wild type of Agrobacterium species have ability to transform and cause crown gall disease in a limited number of dicotyledonous plant species. Ti plasmid is considered to be the major determinant of host range of Agrobacterium (Loper and Kado, 1979). Under laboratory conditions recombinant Agrobacterium species have ability to transform a very broad range of species including monocotyledonous plant species, bacteria, fungi, protozoa and animal cells (Gelvin, 2003 ; Lacroix et al., 2006).

Modified Ti plasmids are used in transformation technique. Ti plasmid must be disarmed to disable phytohormone production gene. At the first stage of Agrobacterium mediated transformations disarmed Ti plasmid derived co-integrative vectors were used. Because of low efficiency of transformation through co-integrative vectors binary vector system was developed (Walden et al., 1997). The possibility of splitting vir region and T-DNA region into two replicons is used in binary vector system (Hoekema et al., 1983; de Framond et al., 1983). In binary vector system two types of plasmids, termed binary vector and Ti helper plasmid are employed (Lee and Gelvin, 2008). Binary vectors include T-DNA left and right border sequences, plant active selectable marker gene, restriction endonuclease cutting site, origin of replication to allow maintenance in E. coli and Agrobacterium and antibiotic resistance gene (Lee and Gelvin, 2008). Binary vector systems in Agrobacterium have been used successfully to transform plants which were thought to be nontransferable by Agrobacterium such as rice, cassava and maize (Walden et al., 1997).

2.2.1.2 Chemical methods

The first successful chemical mediated gene transfer was achieved by utilization of a chemical reagent, DEAE dextran (Vacheri and pagano, 1965). Ever since, different reagents are used to facilitate DNA uptake by cells. The positively charged polymers, such as DEAE – dextran, polybrene, polyethylenimine and dendrimer, complex with negatively charged DNA molecules, forming a polyplex (Selden, 1996). An enhanced ionic attraction between the net positive charge on the polycation-DNA complex and the negative charge on the cell surface enable the DNA binding and entrance into the cell by endocytosis.

The most common chemical used to stimulate DNA uptake is PEG, polyethyleneglycol, which increases the permeability of cell membranes (Gietz and Woods, 2001). Disadvantage of using this method is its unpredictability of integration of transgene. It cannot be used on some plant species and chemical fusion frequency may vary for a given species.

2.2.1.3 Electroporation

In this method an electric field is used to form holes in the plasma membrane allowing DNA to be taken up by the cell. A high mortality rate (25-50% survival) is a disadvantage. When this method is coupled with PEG, highly successful transformation has been achieved on a variety of species and tissue types. However it requires protoplast regeneration which is difficult (Sawahel and Cove., 1992).

2.2.1.4 Particle gun method

This method is also known as micro projectile bombardment or biolistic method. In this method DNA is bound to tiny metal particles. They are fired by gunpowder driven piston at the target cell with a velocity of about 430 meters per second. The bombardment incorporated the DNA into the genome of some of the cells that survived.

A major advantage of micro projectile bombardment is that DNA can be delivered to competent cells that are located within all types of tissues, including calli or organs (Watad et al., 1998).

2.2.1.5 Micro injection

Micro injection involves the introduction of DNA solutions under pressure into plant protoplasts by means of micropipettes. This technique is successful with the development of methods for the immobilization of cells during injection and methods for their subsequent culture (Sawahel and Cove., 1992).

2.2.2 Detection of transformants

For detection, a reporter gene is included in to the gene construct that is to be incorporated into plant genome. A reporter gene encodes an enzyme with an easy assayable activity that is used to report on the transcriptional activity of a gene of interest. The sensitive enzyme activity should not be found in the studying organism. There are two types of reporter systems as scorable reporters and selectabe reporters.

2.2.2.1 Scorable reporters

Expression of scorable reporter gene results in a quantifiable phenotype. It makes the cells containing it to look different from other cells. Those reporters are used for a variety of purposes including confirming transformation, determining transformation efficiency and monitoring gene or protein activity in transgenic plants.

The first attempt of using scorable reporter system was the use of expression of β –galactosidase gene of Escherichia coli. However it failed due to the presence of endogenous activity in higher plants (Helmer et al., 1984). Chloramphenicol acetyl transferases (CAT), fire fly luciferase (LUC), β-glucuronidase (GUS) are scorable reporters which are used to detect transgenic plants.

The gene encoding chloramphenicol acetyl transferase (CAT) was isolated from the Escherichia coli which catalyses the transfer of the acetyl groups from acetyl coenzyme A to chloramphenicol.

Fire fly luciferase (LUC) gene is isolated from Photinus pyralis. Luciferase enzyme confers the organism the ability to glow in the dark. This gene is not destructive to the plant.

GUS (β-glucuronidase) is a predominantly used reporter to study gene expression in plants. The gene is E.coli uid A encoding I2-glucuronidase enzyme, which catalyze hydrolysis of glucuronides. Transformed cells turn blue in the presence of substrate X-gluc. GUS gene is advantageous due to few reasons. There is no detectable background level of glucoronidase activity in higher plants. The enzyme is very stable and can be assayed at any physiological pH with an optimum pH between 5.2 and 8.0. There is wide variety of glucoronides including many chromogenic or flurogenic substrates which allow the histochemical, spectrophotometric and fluorometric measurements of GUS gene expression (Sheeba et al., 2010). The disadvantage associated with the use of GUS assay is the destruction of plant material.

The green fluorescent protein (GFP) is another scorable reporter, which is used in detection of transgenic plants. It was discovered by Shimomura et al. (1962) from jellyfish Aequorea victoria. This reporter gene is used for the study of dynamic process. It exhibits bright green fluorescence when irradiated with blue light. The use of GFP as a reporter molecule has several advantages over the other reporter systems such as β-galactosidase, luciferase, alkaline phosphatase, chloramphenicol acetyltransferase, and GUS. Because GFP does not need exogenous substrate for detection.

2.2.2.2 Selectable reporters

The cells that contain this type of marker gene show the ability to survive under selective conditions. These selective conditions would result in the death of the cells lacking that specific gene. Most commonly used selective agents are antibiotics. Selectable markers are of two types, antibiotic resistance marker and herbicide resistant marker. hpt, nptII, als and bar genes are selectable reporters.

nptII gene is the most commonly used selectable reporter. nptII gene is isolated from E. coli K12 strain. It encodes for aminoglycoside 3` phosphotransferase enzyme which inactivates a range of antibiotics such as kanamycin, neomycin and puromycin. In the first reports of gene transfer to rice, kanamycin was used (Zhang et al. 1988; Zhang and Wu 1988). Kanamycin is an effective selective agent for transformed rice protoplasts. But rice callus shows natural resistance to this antibiotic. Transformed rice callus survive on medium containing levels of kanamycin up to ten times higher than that sufficient to kill many other species (Caplan et al., 1992). Therefore kanamycin is not efficient in detection of transgenic rice. hpt gene is another selective marker which was isolated from E. coli. It encodes for hygromycin phosphotransferase that inactivates the antibiotic, hygromycin. Use of this gene is much effective than use of nptII gene as rice plants haven’t natural resistance for hygromycin (Christou and Ford, 1995).

Bar gene was originally isolated from Streptomyces hygroscopicus, and confers resistance to the herbicide bialaphos (bar). An important advantage of this selection strategy is that rice plants can be sprayed with the selective agent in the in the field. Park et al. (1996) were the first to use bar gene for the selection of rice tissue transformed by Agrobacterium.

2.3 Development of rice transformation system

In the initial years, because of the lack of a good regeneration system and gene delivery methods, protoplast transformation with electroporation or PEG was the method of choice. Toriyama et al. (1988), Zhang and Wu (1988) recovered transgenic rice plants using PEG. In the same year, Zhang et al. (1988) reported recovery of transgenic rice using electroporation. Shimamoto et al. (1989) and Datta et al. (1990) were the first to recover fertile transgenic plants using electroporation and PEG in japonica and indica rice, respectively. Subsequently, these methods have been used widely by different groups for developing transgenic rice. However, regeneration of fertile plants from protoplasts is time consuming, laborious and highly genotype dependent. There were few other problems such as somaclonal variations, multi-copy integration and regeneration of albino plants. Therefore, scientists preferred the other methods of gene delivery.

With the development of particle gun method, it was used successfully for transformation with immature embryos of rice (Christou et al., 1991). The method was further improved by Cao et al. (1992) and Li et al. (1993). Since then, biolistics has been widely used for transformation of japonica, indica and javanica rice. In a significant development, Chen et al. (1998) reported transformation of japonica rice with multiple genes using gene gun method. Tang et al. (1999) reported transformation of rice with four genes by co-transformation using particle gun method. Sudhakar et al. (1998) demonstrated efficient transformation of rice using a portable and inexpensive particle bombardment device termed a particle inflow gun. The biolistic method is claimed to be genotype independent with rice varieties (Christou, 1996); Datta et al., 1999).

Earlier rice was considered to be recalcitrant to Agrobacterium mediated transformation. But during the decade of 1990 successful efforts were recorded. Early attempts to regenerate transgenic calli from Agrobacterium mediated transformation were not successful (Raineri et al., 1990). Subsequently, regeneration was achieved from Agrobacterium infected calli of root explants (Chan et al., 1992) and immature embryos (Chan et al., 1993).

2.3.1 Agrobacterium mediated transformation of rice

In 1994 and 1996 first efficient transformations were recorded (Hiei et al., 1997). Thereafter several rice varieties have been transformed successfully. A large number of morphologically normal, fertile, transgenic rice plants have been obtained from Agrobacterium mediated transformation. The efficiency of rice transformation was similar to that for dicotyledonous plants (Hiei et al., 1994). Many transgenic rice varieties have been improved which contain many important traits which leads to improve quality and quantity of the yield. Pest resistance (Tu et al., 2000), infectious disease resistance, drought tolerance and improved nutritional value are traits which have been used to produce transgenic rice.

2.3.2 Agrobacterium mediated transformation of Sri Lankan rice varieties

Sri Lankan hybrid rice varieties have been successfully transformed by Agrobacterium mediated transformation method. Transformation of BG 94-1 was carried out by Jayasekara et al. (2010). Agrobacterium tumefaciens strain GV 3101 carrying gene construct in pCAMBIA was used in that transformation. The study carried out by Rathnayake and Hettiarachchi (2010) has shown that, Sri Lankan rice variety Bg 250 can be transformed successfully by Agrobacterium mediated transformation.

2.4 Suitable rice explants for genetic transformation

Various tissue types of rice, namely, shoot apices and segments of roots from young seedlings, scutella, immature embryos, calli induced from young roots and scutella, and cells in suspension cultures induced from scutella can be used for transformation. But scutellum derived callus of rice was the most amenable explant for Agrobacterium mediated transformation (Hiei et al., 1994)

2.4.1 Callus induction and regeneration media for rice

In rice a variation exists for response to callus induction, plant regeneration and transformation between indica and japonica types of rice (Hiei et al., 1997). Tissue culture in japonica type has been well studied compared to indica types (Lin and Quifa, 2005; Kumari et al., 2007). Tissue culture of indica type was studied and protocols were optimized recently. Callus induction and regeneration in rice tissue culture depends on a number of factors, such as the genotype of the donor plant, the type and physiological status of the explant, the composition and concentration of the basal salt, and the organic components and plant growth regulators in the culture medium (Ge et al., 2006).

Most reports have recommended 2, 4-D (1.5 – 2.0 mg/L) for callus induction.MS basal medium with combination of 2, 4-D 2 mg/l, kinetin 0.5 mg/l, NAA 3 mg/l, PAA 25 mg/l, and phytic acid 1 mg/l proved to be the most beneficial for callus induction of indica rice in the study conducted by Ge et al. (2006). Kyozuka and Shimamoto (1991) have found that MS basal medium supplemented with glycine 2.0mg/l, sucrose 20g/l, 2, 4-D 2.0 mg/l can be applied for callus derivation from most of japonica and some indica types. Saharan et al. (2004) have reported that best medium for callus induction of indica type is MS basal medium supplemented with 2, 4-D 5 mg/l, Sucrose 30g/l. Rathnayake and Hettiarachchi (2010) have shown that maximum callus induction frequency for Sri Lankan variety Bg250 was obtained on modified N6B5 medium containing 2, 4 D (2mg/L), BAP (1 mg/L) and NAA (1 mg/L). The study conducted by Amarasinghe (2009) has shown that mature seed derived callus initiation and proliferation of nine Sri Lankan traditional varieties can be obtained in N6 basal medium supplemented with proline 1000 mg/l, 2, 4-D 2 mg/l and 3% (w/v) sucrose.

2.5 Applications of Agrobacterium mediated transformation of rice

Rice is the staple food for more than half of the world population. It has become a model monocot plant for genetic and functional genomics studies. In recent years efforts have been taken to improve important agronomical traits through biotechnological techniques (Hao et al., 2009). Transformation has become an important tool for breeding improvement and gene function studies in rice (Ge et al., 2006).

2.5.1 Transformation of rice for crop improvement

A number of economically important genes have been transferred to japonica as well as indica rice. Insect resistance using Bt genes, insect resistance using proteinase inhibitors and lectins, resistance against viruses, herbicide resistance, resistance against fungal pathogens, resistance against bacterial diseases, engineering of rice for abiotic stress tolerance and improved nutritional quality of seeds are few modified traits in rice.

Fujimoto et al. (1993) were the first to engineer japonica rice through electroporation with modified δ-endotoxin gene (cry) from Bacillus thuringiensis. After that different rice varieties were engineered with cry gene expression. Cheng et al. (1998) obtained a large number of transgenic rice plants of different varieties engineered with cry IA(b) and cry IA(c) genes, by Agrobacterium mediated transformation.

For insect resistance, proteinase inhibitors are important as they are part of the plant’s natural defense system against insects. Insect resistance has been developed in rice by using variety of genes encoding pectinase inhibitors. Resistance to stripe stem borer and pink stem borer was developed by potato proteinase inhibitor II (Duan et al., 1996). Cowpea trypsin inhibitor gene is another successful report of insect resistant transgenic rice (Zhen et al., 1999). Successful transgenic virus resistant rice was developed by introducing coat protein (Cp) gene of rice stripe virus (Hayakawa et al., 1992). Capsid protein genes derived from the brome mosaic virus (Huntley and Hall, 1996), and outer coat protein gene of rice dwarf virus (Zheng et al., 1997) are another two succeeded efforts for viral resistant transgenic rice. Zhou et al. (2012) produced transgenic rice plans with the resistance to rice stripe disease, caused by rice stripe virus. They used an RNAi construct containing coat protein gene and disease specific protein gene sequences from the virus.

Christou (1996) and Datta et al. (1999) engineered several rice cultivars to express the bar gene which serves the dual purpose of selectable marker gene as well as conferring resistance to the herbicide, phosphinothricin .

Improvement of the nutritional quality of rice is an important consideration as rice is the major food for most of the world population. Shimada et al. (1993) reported about production of transgenic rice plants with antisense construct of rice waxy gene coding for granule-bound starch synthase for reduction of amylose content of grain starch. Zheng et al. ( 1995) obtained transgenic rice with the gene for seed storage protein β -phaseolin of the common bean.

Another most important transgenic effort is incorporation of genes responsible for β carotene synthesis pathway. Rice plants possess carotenoids in photosynthetic tissues but not in the endosperm, the edible part. There are many records of transgenic plants having genes involved in synthesis of β carotene in endosperm. Two carotenogenic pathway genes, phytoene synthase (psy) and phytoene desaturase (crt I) were introduced in rice using Agrobacterium mediated transformation (Datta et al., 2006).

2.5.2 Transformation of rice for functional genomics

The goal of functional genomics is to complement the genomic sequence by assigning useful biological information to every gene. Through this, we can aim to improve our understanding of how the different biological molecules contained within the cell interact to make the organism viable. The main target is the elucidation of all molecular, cellular, and physiological functions of each gene product.

Rice has become a model for monocot plants because of efficiency in transformation, its small (430 Mb) genome and the economical importance of this crop (Jeon and An, 2001). Its gene content is comparable to that of other grass plants, such as wheat, maize, barley, rye, and sorghum (Gale and Devos, 1998). Because of the conservation of gene sequences among cereals, the structural and functional analyses of rice have broad practical implications for these other economically important crops. Thus, genomic information for rice is widely applicable when developing products and technologies in both the rice and other cereal crops. Thus functional genomics is important in crop development.

Functional genomics allows analysis of the function of genes at genomic scale. Knocking out or over expressing the gene permits the gene sequence to be linked to a phenotype. There are reverse genetic approaches including homologous recombination (reviewed in Hanin and Paszkowski, 2003), antisense or RNAi suppression (Chuang and Meyerowitz, 2000), and insertional mutagenesis (Feldmann, 1991; Jeon et al., 2000). There are several types of chemical, physical and biological methods for creating mutants, the most widely used are ethyl methane sulfonate treatment, fast neutron irradiation, T-DNA and transposon insertion. T-DNA or transposon insertion is effective than other methods as the inserted element is known (Bouchez and Hofte, 1998 ). Because rice is easy to transform, Agrobacterium mediated T-DNA has been used successfully to produce mutants. This technique is efficient for identifying knockout mutants as well as for both promoter and activation tagging (An et al., 2005).

Homologous recombination (Hanin and Paszkowski, 2003) and viral-induced gene silencing (Robertson, 2004) are also effective techniques. In both of these methods Agrobacterium mediated transformation is used. Through homologous recombination an endogenous gene can be modified into a designed sequence.

RNA interference is another tool that is used to suppress genes in study of gene function. There are two types of RNA molecules involved in RNA interference. They are dsRNA and siRNA. dsRNA act as trigger of RNA breakdown and siRNA is involved in degradation of target mRNA in the final step of RNA interference pathway (Hannon, 2002).

Bi et al. (2009) discovered the function of early nodulin gene OsENOD93-1 by over expression of that gene in transgenic plants.

In the study conducted Jiang et al. (2012) biological functions of an Oryza sativa ribosome-inactivating protein gene 18 (OSRIP18) was investigated by over expressing OSRIP18 gene in transgenic rice plants. They suggested that the function of that gene is upregulation of stress dependant genes.

Yusuke et al. (2006) revealed the function of OsDREB gene of rice genome by over expressing it in transgenic rice plants. They discovered that the proteins encoded by these genes are involved in plant stress tolerance. Therefore, this gene is quite useful in improvement of stress tolerance to environmental stresses in various kinds of transgenic plants including rice.

Thus Agrobacterium mediated transformation of traditional rice varieties plays an important role in identification of function of rice genes.

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