National Center for Genetic Engineering and Biotechnology

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The National Center for Genetic Engineering and Biotechnology (BIOTEC) was established under the Ministry for Science, Technology and Energy in 1983. When in 1991 Thailand established the National Science and Technology Development Agency, BIOTEC became one of the Agency's centers. It operates autonomously outside the normal framework of civil service and state enterprises. This enables it to operate more effectively to support and transfer technology for the development of industry, agriculture, natural resources, environment, and the socioeconomy.

BIOTEC policy provides the resources for the country to develop the critical mass of researchers necessary to achieve Thailand's national R&D requirements in biotechnology. This is achieved through in-country R&D, the facilitation of transfer of advanced technologies from overseas, human resource development at all levels, institution building, information services, and the development of public understanding of the benefits of biotechnology.

BIOTEC is both a granting and implementing agency. It allocates approximately 70 percent of its R&D budget to several universities and research institutes in Thailand, and 30 percent for in-house research projects. The facilities of national and specialized laboratories are made available for in-house research programs as well as for visiting researchers. The Science and Technology Park, which was completed in early 2001, houses BIOTEC's main laboratories.

Several research programs have been undertaken by a BIOTEC-appointed committee of recognized experts in the field. The major biotechnology programs and activities are described below.

1. Shrimp

Basic knowledge about the major cultivated shrimp species has lagged behind technical innovations that have led to successful intensification of culture, and to ever-increasing world production. Sustaining high production will require innovation to minimize adverse environmental impacts. Biotechnology will play a central role in helping to learn more about shrimp and thereby improve rearing practices. BIOTEC's support will focus on issues dealing with shrimp diseases and with improvement of the seed supply. The disease work has so far emphasized the characterization, diagnosis, and control of serious shrimp pathogens, particularly the viruses responsible for yellow head disease (YHD) and white spot syndrome (WSS). Luminescent bacterial infections have contributed to declining production to a lesser degree. These diseases have become progressively more serious threats to the industry as it has grown and intensified. Indeed, the work supported by BIOTEC on YHD and WSS viruses has been instrumental in substantially reducing losses in Thailand. The losses to YHD (probably exceeding $40 million in 1995) and those to WSS (probably exceeding $500 million in 1996) could have been much more serious without the basic knowledge and the DNA diagnostic probes made available to the industry by Thai researchers. Checking for subclinical WSS virus (WSSV) infections by polymerase chain reaction (PCR) has been a common practice to help farmers screen out WSSV +ve before stocking (Flegel 1997).

The Shrimp Biotechnology Service Laboratory was established at BIOTEC in 1999 to summarize the reference PCR methods for viral disease detection. Laboratory objectives are to serve as the reference laboratory for major shrimp pathogen diagnosis based on molecular techniques, to conduct research, and to provide assistance for molecular detection of various shrimp viruses.

It has been reported that WSSV can be vertically transmitted and become widespread among wild broodstock. In addition to the disease problem, a decline in the growth rate of shrimp produced from currently available wild broodstock has also been observed. Production of specific pathogen free animals and the development of specific pathogen resistant strains are now being used in the United States, Venezuela, and French Polynesia with Penaeus stylirostris and P. vannamei. This could be considered a breakthrough since production of P. vannamei more than doubled during 1992-94. Currently, the most important program involves the do mestication and genetic improvement of P. monodon stocks (Withyachumnarnkul et al. 1998). The project will lead to the development of specific pathogen resistant stocks and improved growth through selective breeding. BIOTEC is also supporting advanced studies on deoxyribo-nucleic acid (DNA) characterization and DNA tagging of the shrimp stocks. These studies are providing the tools that will be important for rapid genetic improvement strategies.

BIOTEC is dedicated to the principle that the players in the shrimp industry should take an active role in planning and financing R&D for their industry. BIOTEC actively promoted the formation in 1996 of an industry consortium, the Shrimp Culture Research and Development Company, dedicated to solving problems common to the shrimp aquaculture industry as a whole. This consortium serves the industry directly and also serves as a bridge to other public and private institutions involved in relevant research, not only in Thailand but throughout the world.

2. Cassava and Starch

About 70 percent of the 16 million tons (t) of cassava roots produced in 1998 was used in the production of pellets and chips; the remaining 30 percent was used mainly to produce flour and starch. A production shortage in 1997-98 prompted the Thailand Tapioca Development Institute (TTDI) and Kasetsart University to develop a new strain with a higher yield, Kasetsart 50. It has an average root yield of 26.4 t/ha and a starch content of 26.7 percent compared with 13.75 t/ha and 18 percent starch content for the best strain available until its release.

The tapioca starch industry is one of the largest in Thailand. In 1998, tapioca starch was worth about $120 million. About 40 percent of starch was used domestically to produce modified starch, sweetener, and monosodium glutamate. Most of the remaining 60 percent was exported. Efficient production, low production costs, and the development of value-added products are vital to the starch industry and the farming sector (total of 1.3 million ha planted to cassava). The program on starch and cassava products was established to provide R&D support and funding. The program is funded jointly by BIOTEC and TTDI to carry out R&D in three core activities: processing, diversification, and characterization.

a. Processing Efficiency

The short-term project aims to improve the processing efficiency of starch production, in particular to minimize water and energy consumption. Wastewater discharge varies from 13 to 50 cubic meters/ton (m3/t) of starch produced, with an average of 20 m3. A benchmark on water use is a priority for the Thai starch industry.

Biotechnology can play an important role in waste utilization. Solid waste (after starch extraction) still contains 50 percent starch (dry weight) and has been used as animal feed. Tapioca, however, is not suitable for the production of feed requiring high protein content. Attempts have been made for protein enrichment using various microorganisms such as As-pergillus and Rhizopus. Nevertheless, the economic feasibility is still in doubt and further technological development is needed. In contrast, turning wastewater into energy through high-rate anaerobic digestion is promising. Though the technology is proven, an adaptation to such high-strength wastewater and low buffering capacity is required to ensure stability of the system. In comparison with the upflow anerobic sludge bed reactor, the fixed bed is easier to control and operate. R&D, however, is focused on increasing loading efficiency. Based on calculations, methane generated from anaerobic treatment of starch wastewater from 60 factories would be approximately 630 million m3 annually. This could be substituted for fuel oil used in drying, saving energy costs of about $4 million annually. There is also the environmental cost of large land areas required for conventional evaporation pond systems. In addition to native starch, production of modified starch is increasing, leaving an excessive amount of sulfate in wastewater. This may interfere with the anaerobic digestion intended for energy production. A number of papers have been published recently on the interactions between the sulfate reducing bacteria and the methanogenic bacteria. Molecular diagnosis has been developed and applied for the mixed culture system. A better understanding of these anaerobic microbes could lead to the biological removal of sulfate, which is the main problem of various industries.

b. Product Diversification

Product diversification is part of the second core research activity. The European Union has set a quota for imported tapioca pellets. As a result, production of biodegradable plastic from cassava starch is being investigated. Increasing use of cassava as a raw material for fermentation products, such as amino acids and organic acids, must proceed, expanding the development of value-added products. To reduce costs of production, however, research is oriented toward the production of good quality cassava chips as a starting material to replace the starch.

c. Starch Structure and Properties

Basic research on cassava starch structure and properties will add to our knowledge and help increase the use of cassava starch. The Cassava and Starch Technology Unit, a specialized BIOTEC laboratory established in 1995 at Kasetsart University, has been studying the physi-cochemical properties of cassava. The unit is well equipped, and provides regular service and training on instrumental analysis of starch properties to the private sector and government agencies.

3. Rice

Rice yields in Thailand are low. One of the major constraints is blast disease, especially in high-quality rice cultivars such as the aromatic Khao Dawk Mali. In northern Thailand, about 200,000 ha of rice were affected by blast in 1993, causing serious economic loss and resulting in government intervention of about $10 million to assist disease-struck farmers. Another $1.2 million was spent on fungicides (Disthaporn 1994). Breeding higher resistance levels to blast in Thai rice has been attempted. Limiting factors, however, are lack of insight and information on resistance genes, and the complex structure of the pathogen populations. Genetic analysis provides an efficient tool to identify useful resistance genes in the host while analyzing the race composition of the pathogen population. Recent research applying molecular genetic methods (DNA fingerprinting of a blast isolate collection at Ubon Ratchathani Rice Research Station, and mapping of host resistance genes by the DNA Fingerprinting Unit at Kamphaengsaen campus of Kasetsart University) are providing baseline data on the interaction between rice and blast. The project is working on three closely related areas as follows:

(i) Establishment of a suitable differential cultivar series; identification of resistance genes conferring complete and partial resistance to blast disease in rice.

(ii) Pathotype and molecular genetic characterization of the blast pathogen population in Thailand. So far, more than 500 monospore isolates have been deposited with the BIOTEC specialized culture collection.

(iii) The special case of fertile isolates; the potential of using Thai isolates of Magnaporthe grisea for the development of a molecular diagnostic tool for pathogen race analysis. The degree of fertility can be assessed from the timing and number of perithecia that develop. BIOTEC has the capacity to test the mating type of about 80 isolates per month.

This project is a nationwide, network-type collaboration combining molecular genetics and classical approaches to help scientists breed rice cultivars with improved blast resistance.

BIOTEC provided $1.5 million in 1999 to fund the Rice Genome Project Thailand. On behalf of Thailand, BIOTEC has joined the International Collaboration for Sequencing the Rice Genome (ICSRG) by sequencing 1 megabyte annually of chromosome 9 for the next 5 years. BIOTEC is expected to provide about $3.7 million to cover this work. Chromosome 9 was selected based on previous extensive work on the fine genetic and physical maps surrounding the submergence tolerance quantitative trait loci (QTL), the prospect of gene richness, and the small chromosome size. Joining ICSRG will allow Thai scientists to directly access the rest of the genome sequence made available by the other collaborating members. Gene discovery from wild rice germplasm will be undertaken in parallel to efficiently use the genome sequence data. The project will bring Thailand into the international scientific arena, incorporate state-of-the-art technology, and improve Thailand's competitive edge in the international rice market.

4. Dairy

In 1997, Thai milk consumption was 12 liters/person/year. Milk production is still insufficient to meet local demand, and Thailand has to import more than 50 percent (worth $305 million) of the dairy products consumed in the country. An additional 130,000 cows are needed to meet the national demand.

Reproductive efficiency is a primary determinant of dairy herd production profitability. Milk yield (10 kilograms/day) is still far below the average (30 kilograms/day) of most developing countries. It is therefore important to promote an increase in dairy production through science and technology. The major programs are breeding and feeding. The lack of proper management is another major contributing factor to an underproductive dairy industry.

Traditional breeding practices in Thailand have been too slow to meet national requirements. And importing pregnant heifers or young quality-bred calves from abroad is too costly. Cutting-edge technologies such as embryo transfer, in vitro fertilization, embryo sexing, and semen sexing have been studied by Thai scientists for more than 10 years. Nevertheless, the technologies have not yet been adopted. Technology transfer and training of Thai researchers at the leading laboratories or companies are now under discussion. The goal is to increase production of high-quality heifer calves at the lowest cost.

5. Genetic Engineering

By the mid 1970s, with biotechnology centered on genetic engineering and molecular biology, Thailand was ready to adopt the new tools and apply them to various practical problems, first in the biomedical field and later in agriculture and other areas. A few specific examples will be given here to highlight the application of molecular biology and genetic engineering to agricultural development. Efforts in agricultural biotechnology and genetic engineering have been focused on three main areas: plant transformation, DNA fingerprinting, and molecular diagnosis of plant and animal diseases. The first area should lead to the production of transgenic plants with superior properties including resistance to diseases and insect pests, and tolerance for abiotic stresses.

a. Plant Transformation

The Plant Genetic Engineering Unit, the specialized laboratory of BIOTEC at Kasetsart University, Kamphaengsaen, was established in 1985 to work on plant biotechnology and genetic engineering. A transgenic tomato plant carrying the coat protein gene of tomato yellow leaf curl virus was first developed to control this serious virus disease of tomato (Attathom et al. 1990). The same approach was taken to develop transgenic papaya resistant to papaya ringspot virus and pepper resistant to chili vein-banding mottle virus (Chaopongpang et al. 1996; Phaosang et al.

1996). Sri Somrong 60, a Thai cotton variety, was successfully transformed with cryIA[b] gene expressing a toxin from Bacillus thuringiensis (Bt). Development of transgenic rice varieties has been supported by the Rice Biotechnology Program launched by BIOTEC and the Rockefeller Foundation. An example is the transformation of Khaw Dawk Mali 105, an aromatic Thai rice with delta 1 pyrroline-5-carboxylate synthetase for salinity and drought tolerance. Most transgenic plants are now being tested in the greenhouse in accordance with the Biosafety Guidelines (Attathom and Sriwatanapongse 1994, Attathom et al. 1996). Field testing of transgenic plants developed in Thailand will begin in 2000.

b. DNA Fingerprinting

Using DNA fingerprinting and PCR, scientists can identify organisms and genes, and make genetic maps. DNA probes and specific gene sequences have made possible molecular methods for diagnosis of plant and animal diseases. Molecular mapping of genes in rice involving submergence tolerance, rice blast, aroma, cooking quality, and fertility restoration were accomplished using three mapping populations. A backcross breeding program for the improvement of Jasmin rice was initiated. In the first stage, resistance to bacterial leaf blight, submergence tolerance, resistance to brown planthopper and gall midge, and photoperiod insen-sitivity were main areas of focus. Restriction fragment length polymorphism markers were an important limiting factor for high throughput and cost effectiveness. The PCR marker for Xa21 gene is the most reliable for marker-assisted backcrossing in rice.

c. Molecular Diagnosis

Tomato production in the tropics and subtropics faces serious constraints due to bacterial wilt (BW), a disease caused by the bacterial pathogen recently reclassified as Ralstonia solanacearum (previously Pseudomonas solacearum). In Thailand, an endemic outbreak of BW in tomato, potato, pepper, ginger, and peanut occurs each year, causing a yield loss of approximately 50-90 percent depending on growing conditions. BW-resistant varieties cannot easily be developed due to the nature of the quantitatively inherited resistance that involves several genes. Marker-assisted selection (a breeding method of selecting individuals based on markers linked to target genes), in addition to phenotypic measurement, is essential and useful only for enhanced resistance to diseases. At this time, three putative QTLs corresponding to BW resistance have been found using amplified fragment length polymorphism markers. Once markers closely linked to BW-related QTLs are well established, they can be used for marker-assisted breeding for enhanced resistance to BW in tomato. A tomato consortium has been set up to extend public-private collaboration.

BIOTEC has set up the DNA Fingerprinting Service Unit at Kasetsart University. The unit has provided services to public and private concerns for more than 2 years. The main services are DNA fingerprinting and DNA diagnosis.

6. Biocontrol

In 1996, Thailand imported 38,000 t of chemicals, mainly insecticides and herbicides. The global trend of going organic is an opportunity for Thai farmers to supply fresh organic produce, especially fruit and vegetables, to the world. Over the past decade, developmental work on biocontrol in Thailand has continued to receive active support from BIOTEC and the Thailand Research Fund. Two companies are now commercially producing Trichoderma to control Sclerotium rolfsii, and Chaetomium to control soil fungi such as Phytophthora (Yuthavong 1999). BIOTEC and the Department of Agriculture have set up a pilot-scale production facility to produce nuclear polyhedrosis virus (NPV), Bt, and Bacillus sphericus. NPV is widely used to control Spodoptera moth in grapes. Bt produced locally has gained popularity over the last few years. The capacities of pilot plants at Mahidol University and King Mongkut's University of Technology, Thonburi, are fully taken up with Bt production. Commercial production may begin soon. A project at Mahidol University to transfer the chitinase gene into Bacillus thuringiensis subsp. israelensis has received support from BIOTEC.

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