Agricultural biotechnology in the Asia-Pacific region
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Biotechnology in crop improvement and production
Biotechnology in animal health and production
Biotechnology in fisheries and aquaculture
Biotechnology in forestry
Policy and institutional arrangements for agricultural biotechnologies in the region
Commercialization and role of the private sector
Regional and international biotechnology programmes in the region
Prospects of agricultural biotechnology in the Asia-Pacific region
FAO Research and Technology Development Division
This paper analyses the current status and future prospects of research and development of modern biotechnology in crops, livestock, forestry and fisheries in Asia and the Pacific Region. Here the region is taken to mean the FAO grouping of 30 countries starting from Iran and Pakistan in the West to Japan in the East and seven of the South Pacific island countries plus Australia and New Zealand.
Although almost all countries in the region have biotechnology programmes, the level of advancement and involvement varies widely. The three developed countries of the region, namely, Australia, Japan and New Zealand, and particularly the first two, are frontline countries in the world in R & D of this new and fast developing field As regards the developing countries, moderate to comprehensive biotechnology R & D programmes are taking place in China, India, Indonesia, Malaysia, Pakistan, the Philippines, the Republic of Korea and Thailand. Of the remaining developing countries, Bangladesh, the Democratic People's Republic of Korea, Iran, Nepal, Sri Lanka and Viet Nam are striving to establish appropriate biotechnology programmes. Several of the Pacific island countries have also developed in vitro culture facilities for taro and other selected crops essentially for micropropagation, freeing of planting materials of viruses and quarantine purposes. Fiji has also made considerable progress in somaclonal selection and in vitro culture of sugar cane.
The regional scenario presented in this paper is essentially a reflection of the situation in the three developed countries and eight developing countries (China, India, Indonesia, Malaysia, Pakistan, the Philippines, the Republic of Korea and Thailand) which are fairly advanced in modern biotechnology. The country situations analysed individually provide an account of diversity programmes and preparedness for research and development of agricultural biotechnology in the region.
Biotechnology in crop improvement and production
In vitro culture for micropropagation of virus-free materials
Vitroplants of potato, sweet potato, Allium species, taro, strawberry, sugar cane, several ornamentals such as chrysanthemum, carnation, Lilium species and gerbera, and fruit plants such as Citrus species, papaya, apple, sweet cherry, pear and grapes freed of or free from viruses are being commercially exploited in several countries in the region, particularly in China, India, Indonesia, Japan, Malaysia, the Philippines, the Republic of Korea and Thailand. Hundreds of thousands of hectares are planted to virus-free vitroplants of potato in the Region with more than doubled productivity, especially in China and the Republic of Korea. For instance, in the Republic of Korea, the widespread use of virus-free planting material produced through in vitro culture techniques increased the national potato yield from 11.9 tonnes/ha in 1980 to 20.3 tonnes/ha in 1986 (Chung, 1989). Subsequently, an in vitro tuberization system was established and became an integral component of the potato seed industry in the country. Production efficiency of the technique is exceptionally high (young, 1993). One of the main problems encountered in this approach is the chance of reinfection during the propagation of virus-free plants. To overcome and manage this problem, simplified diagnostic methods have been developed and are being used to detect viruses and viroids.
In vitro culture for micropropagation
Besides the above, vitroplants are being routinely mass micropropagated for commercial purposes in the case of orchids, other flowers and ornamental plants throughout the region, especially in Southeast Asian countries (Zamora and Barba, 1990), oil palm (Malaysia, India, Indonesia, Papua New Guinea), date palm (Iran and likely to be taken up in India and Pakistan), embryo culture of soft coconuts (Macapuno) (the Philippines and Thailand), cardamom (India; Kumar, 1990), eucalyptus (China and India), Chinese fir (China), rattan (Indonesia, Malaysia, the Philippines), and seed sterile triploid water melons (Japan). Somatic embryogenesis is also being commercially applied. Synthetic seeds of rice and vegetables have been developed by the private sector in Japan. Triploid rubber clones of rubber produced through somatic embryogenesis have outyielded diploid standard clones by about 20 percent in China. The country has also standardized techniques for mass propagation of sugar cane seedlings using embryogenic cell lines and multishoot propagation systems (Fan, 1991).
To facilitate reforestation and promote social forestry and agroforestry, India has established two tissue culture pilot plants, one at the National Chemical Laboratory, Pune, and the other at the Tata Energy Research Institute, New Delhi (a private organization) with production capacities of five to ten million propagules/seedlings of elite/plus trees of several important species, such as Eucalyptus tereticornis, E. camadulensis, Tectona grandis, Dendrocolamus strictus, Populus deltoides, Bambusa vulgaris and B. tulda (Mascarenhas, 1991). Micropropagation of teak, rattan, eucalyptus, bamboo and other tree species has been adopted in several other countries of the region.
Doubled haploid breeding
The region has played a pioneering role in this field. Indian scientists were first to produce haploids from another culture in the mid-1960s (Guha and Maheshwari, 1964). Haploid induction through anther culture has been used most extensively by Chinese scientists for breeding of not only rice but also of several other crops. The technique was used for: (i) production of new varieties; (ii) production of inbreds for heterosis breeding; (iii) selection of stable resistant lines against biotic and abiotic stresses; and (iv) purification of male sterile lines. One of the most important advantages of the use of haploids is to reduce the time required to develop and release new varieties. For instance, while the conventional pedigree method of breeding and releasing a pure line rice variety requires about 12 years, the haploid method requires only about five years.
Chinese scientists have succeeded in inducing haploids in about 40 different plant species. They were the first to produce haploids through in vitro culture in wheat, maize, sugar cane, soybean, rubber, grapes and apple. In China, several new varieties of rice, wheat, tobacco, and hot and sweet pepper possessing high yield, superior quality, sometimes tolerance to abiotic stresses such as cold, sometimes early maturity, and resistance to diseases have been released through the use of haploids. The most popular of these varieties are Xin-Xin, Hua-Hau-Zao, Zhong-Hua 8, 9, 10 and 11 of rice; Jing-Hua 1, 3 and 5 of wheat; Haihua 1, 3, 19 and 29 of hot and sweet pepper; and Dan-Yu 1, 2 and 3 of tobacco. These varieties occupied about 1.5 million hectares in 1990 (Fan, 1991), of which the rice and wheat varieties accounted for 800 000 and 650 000 ha, respectively.
Other countries have also used doubled haploid breeding methods for crop improvement. In India, rice lines R4 and R10 derived through this method outyielded the check by 15 to 49 percent in trials conducted during 1984-87. The Republic of Korea released two rice varieties derived from anther culture haploid technique and generally screens annually about 6 000 anther-derived rice lines (Chung, 1989). The technique has also been effective in heterosis breeding of Chinese cabbage. In Japan, several successful varieties have been developed through this technique. Among these are rice varieties Joiku N. 394, Hirohikari, Hirohonami, AC No. 1 and Kibinohana, which are tolerant to cold temperature and are good in taste. A broccoli variety, "Three Main", possessing cold resistance and uniform shape, and a cabbage variety, "Orange Queen", with an orange colour, which are quite popular, were developed by the anther culture method (Ito, 1989).
In vitro selection and somaclonal variation
Australian scientists (Larkin and Scowcroft, 1981) were the leaders in the field of somaclonal variation, demonstrating the efficacy of the approach in improvement of wheat, sugar cane and other crops. Somaclonal variants and lines selected through selection pressure exerted during the culture stage have been released as commercial varieties in several countries in the region. For instance, in Japan, the 1991 list of registered crop varieties included two varieties of Lilium and one each of melon, Gerbera, Cymbidium, statice and strawberry derived through somaclonal variation (Nakajima, 1991). In China, somaclonal mutants of rice possessing resistance to bacterial blight and resistance to AEC coupled with higher contents of Iysine, methionine, isoleucine, serine and tyrosine than the parental lines have been produced. Salt tolerant somaclones of rice, wheat and tobacco were also isolated. Short stature and early maturing somaclonal variants of rice have been isolated in several laboratories in China. India and other countries have also produced somaclonal variants and are using them directly as new lines or in breeding programmes. In Fiji, tissue culture techniques have been used extensively for isolating sugar cane clones resistant to Fiji disease of sugar cane. Thailand isolated improved varieties of banana and chrysanthemum using this technique (Singh, 1990).
Somaclonal variation was exploited in a novel way under a joint programme of China and Australia to transfer from Thinopyrum intermedium resistance to Barley Yellow Dwarf Virus (BYDV) in wheat (Triticum aestivum). Single cell callus cultures from F1 embryos rescued were initiated and induced to form plants showing somaclonal variation which were then selected for BYDV resistance. Cytological analysis of the genotypes showing stable resistance revealed that chromosomal rearrangement of the chromosome carrying Thinopyrum introgressed segment had occurred during the tissue culture phase to confer the stability (Brettell et al., 1988).
The Biotechnology Centre at the Indian Agricultural Research Institute (IARI) has standardized the protocols of plant regeneration of Brassica carinata and is isolating somaclonal variants suitable for Indian conditions. Useful somaclonal variants for earliness, plant height, maturity, etc. have been induced in B. juncea and B. napus (Chopra and Sharma, 1991).
In vitro techniques for plants provide systems analogous to the prokaryotic systems where variants can be selected and mutations can efficiently be induced and isolated at cellular level. By applying effective selection pressure on naturally variant or mutagenically treated cell cultures viz. use of saline media for screening salinity resistant/tolerant cell lines or use of pathogen toxins for isolating disease resistant cell lines, the efficacy and efficiency of both induction and identification of useful mutants are increased considerably. Using this technique, Chinese, Indian and Filipino scientists have isolated rice mutants tolerant to higher concentrations of salt. Mutants having higher protein and Iysine content in their seeds were also isolated. Using this approach, pathogen-resistant mutants of tobacco, rice, wheat, barley, sugar cane and maize have been isolated and used in breeding programmes in several countries. In Japan, (Nakajima, 1991), disease-resistant lines of rice, tomato and tobacco were isolated using this approach (Table 1).
Examples of disease-resistant plants successfully selected using tissue culture, Japan
|Crop||Disease||Selection stress||Selection stage|
|Oryza sativa||Rice blast||Tenuazoic acid||Protoplast-derived callus|
|Lycopersicon esculentum||Tomato fusarium wilt||Fusaric acid||Leaf-derived callus|
|Avena sativa||Oat leaf stripe||Victorin||Regenerants from callus and their progeny|
|Nicotiana tabacum||Tobacco mosaic virus||No stress||Shoots regenerated from TMV- infected callus|
|Lycopersicon esculentum||Tomato bacterial wilt||NO stress||Regenerants from callus|
Embryo Culture and Distant Hybridization
The technique is being used in several Asian countries, particularly in the case of orchids, peanuts, cotton, cabbages, citrus and peaches. In India, promising recombinants have been obtained using embryo rescue techniques in distant crosses of cultivated Phaseolus, jute and peanut. In China, using embryo rescue and in vitro culture of a distant hybrid Triticum aestivum x Agropyron elongatum, a new wheat variety, Xiaoyan No. 6, was produced and has been grown widely giving additional wheat production (Fan, 1991). In Japan, three cultivars each of Citrus, Prunus, and Brassica species, and five cultivars of Lilium species were developed in recent years using the embryo rescue technique (Ito, 1989).
Cell fusion and somatic hybridization
Japan and China are particularly deeply involved in this work. During the past ten years or so, as listed by Nakajima (1991), Japanese scientists have reported successful protoplast culture in more than 70 plant species (Table 2). In China, plants regenerated from protoplast have been obtained in about 30 species, including vegetables, medicinal plants, legumes, and other economic crops as well as woody species such as poplar, elm and rubber trees. For the first time, Chinese scientists regenerated plants from monocot Polypogen fugax. With recent reports of success on protoplast culture and regeneration of wheat, it is now possible to have protoclones of most of the major food crops. However, in several cases, the regeneration frequency is low and should be improved. The Republic of Korea had produced cybrid lines of mushrooms which outyielded the best parents and checks by about 100 percent.
Successful regeneration of plants from cell fusion are reported from about 50 interspecific and intergeneric protoplast combinations in the region. Half of these are known from Japan (Table 3). Using cell fusion techniques, scientists have developed novel citrus hybrid varieties, such as "Oretachi" (orange plus trifoliate orange), "Shuvel" (Satsuma mandarin plus navel orange), "Gravel" (grapefruit plus navel orange), "Murrel" (murcott plus navel orange) and "Yuvel" (Yuzu plus navel orange). Using asymmetric cell fusion techniques, Japanese scientists developed Ms-F 224, the first commercial tobacco male sterile line developed in the world bred by cell fusion. Such asymmetric male sterile lines for commercial exploitation of F, hybrids have been also bred in carrots, cabbages and eggplants (Nakajima, 1991).
In China, somatic hybrids between Nicotiana tabacum and N. rustica, N. tabacum and N. glauca were used for developing new commercial varieties of tobacco (Fan, 1991). Chinese scientists have also pioneered the pollen tube gene introduction technique for cotton and rice breeding. In this technique, after self-pollination, the desired exogenous DNA is introduced to the embryonic cells. Seeds which develop from such transformed embryos result into transformed plants, thus avoiding the need for protoplast fusion technique. Genes responsible for disease resistance and other traits have been transformed successfully in rice and cotton.
But this technique has low repeatability. In fact, the entire cell fusion technique has not been as successful in producing somatic hybrids as initially expected. With the availability of Agrobacterium-mediated and biolistics-based methods of gene transfer, the thrust on the protoplast fusion approach has somewhat slackened, although the asymmetric fusion method has special appeal for production and diversification of cytoplasmic male sterility and manipulation of other cytoplasmically controlled systems.
Successful protoplast culture systems in Japan
|Food crops||Lycopersicon esculentum|
|Glysine max||L. Chilese 1/|
|Ipomoea batatas||L. glandulosum 1/|
|I. trifida 1/||L. peruvianum 1/|
|I. triloba 1/||L. pimpinellifolium 1/|
|I. trichocarpa 1/||Petasites japonicus|
|Oryza sativa||Raphanus sativus|
|O. rufipogon 1/||Solanum melongena 1/|
|Solanum tuberosum||S. Integuriforium 1/|
|S. bulbocastanum 1/||Zingiber officinale|
|S. nigrum 1/|
|S. pinnatiscutum 1/||Ornamentals - Flower plants|
|Vigna angularis||Angelonia salicariefolia|
|Forages and turfs||Cyclamen persicum|
|Agrostis alba||Dianthus chinensis|
|A. stolonifera||Dianthus japonicus|
|Festuca arundinacea||Dianthus caryophyllus|
|Lotus corniculatus||Eustoma grandiflorum|
|Medicago sativa||Gentiana triflora var. japonica|
|Panicum maximum||Limonium perezeii|
|Paspalum dilatatum||Lilium xformolongi|
|Trifolium repens||Matthiola incana|
|Zea mays||Pelargonium crispum|
|Zoysia japonica||P. odoratissiumum|
|Allium cepa||Fruit and forest trees|
|Allium chinense||Actinidia chinensis|
|Allium sativum||Citrus limon|
|Asparagus officinalis||C. reticulata|
|Brassica campestris||C. sinensis|
|B. oleracea||C. unshiu|
|B. napus||C. yuko|
|Sinapis arvensis 1/||Eucalyptus saligna|
|Cucumis melo||Populus alba|
|Daucus carota||P. sieboldii|
|Lactuca sativa||P. charkowiense x caudina|
|L. serriola 1/||Vitis vinifera|
|Erigeron philadelphicus 1/||Broussonetia kazinoki|
Source: Nakajima, 1991.
1/ Wild relatives.
Regenerated plants from cell fusion in Japan
|Oryza sativa + Echinochloa oryzicola|
|Oryza sativa + O. Brachyantha|
|Oryza sativa + O. Eichingeri|
|Oryza sativa + O. Officinalis|
|Oryza sativa + O. perrieri|
|Brassica campestris + B. Oleracea|
|Brassica oleracea + Raphanus sativa 1/|
|Brassica oleracea + Moricandia arvensis|
|Brassica oleracea + Sinapis turgida|
|Brassica napus + B. Nigra|
|Brassica napus + Raphanus sativa 1/|
|Daucus carota + D. Cupillifolius|
|Lactuca sativa + L. Virosa|
|Lycopersicon esculentum + Solanum muricatum|
|Solanum melangena + S. integrifolium|
|Ornamentals - Flower plants|
|Petunia x hybrida + P. parodii|
|Fruit and forest trees|
|Citrus sinensis + C. Paradisi|
|Citrus sinensis + C. Unshiu|
|Citrus sinensis + C. sinensis x tangerina|
|Citrus sinensis + C. sinensis x Poncirus trifoliata|
|Citrus sinensis + Poncirus trifoliata|
|Nicotiana tabacum + N. Africana 1/|
|Nicotiana tabacum + N. Debneyl 1/|
|Nicotiana tabacum + N. Rependa|
|Nicotiana tabacum + Salpiglossis sinuata|
Source: Nakajima, 1991.
1/ CMS transfer by cybrids.
In vitro culture for secondary metabolites
Cultured plant cells retain their metabolic potential and can be used efficiently for producing useful secondary metabolites of commerce such as pharmaceuticals, dyes, food additives, sweeteners, flavours and taste enhancers, essential oils and aromatic products. Herbal medicines are popular in China, India and other Asian countries and are gaining importance in the West. Chinese scientists have been using tissue culture since the late 1970s for the production of ginseng sapanins from Panax ginseng. Diasgenin, a female contraceptive produced from in vitro cultured Dioscorea spp and a male contraceptive based on gossypol from tissue-cultured Gossypium spp are under extensive trials in China. In addition, tissue culture technique has been successfully used in the cultivation of Scopolia acutangula, Artemisia annua, and Rauwolfia yunnanesis. India is also using this technique for production of diasgenin and other medicinal and aromatic products.
Cell cultures can also be used as factories for bioconversion of intermediate compounds into more valuable products. Shikonin, an expensive compound obtained from the roots of Lithospermum erythrorhizon, has been used by the Japanese traditionally as a vegetable dye and in cosmetics and toiletries. However, due to overexploitation and other human activities, the plant has become almost extinct in Japan. To reduce their dependence on the import of this plant material, Japanese scientists have developed a tissue culture method for the commercial production of shikonin. Another example where tissue culture production of an industrial compound has reached commercial level is berberine from Coptis juponica (Fujita, 1990). In tissue cultures the yield of high value compounds can be enhanced by feeding the cells with the intermediate compounds of their biosynthetic pathways (biotransformation), manipulation of culture conditions and selecting high yielding cell lines. With further refinements of techniques the bioreactor and fermenter based production of secondary metabolites could be rendered highly cost-effective and time-saving.
In vitro conservation of germplasm
Several plant species in the region, including a few commercial species, produce recalcitrant seeds and it is thus difficult to conserve them through seeds. Furthermore, some species are shy seed bearers and even fail to produce seeds. In addition, living collections of clonally propagated perennial crops face the problems of maintenance of heterozygous and heterogeneous populations, the long life cycle and large space required, and a high possibility of exposure to the threats of pests and diseases and other biotic and abiotic stresses. To circumvent these difficulties, in vitro conservation of vegetatively propagated and recalcitrant seed-producing species is being increasingly adopted as a complement to other methods of conservation. For short- to mediumterm storage (working and active collections), the slow growth method is used whereas for long-term storage (base collections) cryopreservation is the method adopted.
There is therefore a good case in the region for in vitro storage of several of the important plant species, and the countries involved are in fact building such facilities. Orchid germplasm is routinely maintained in vitro in India, the Philippines and Thailand. With assistance from the Department of Biotechnology, the National Bureau of Plant Genetic Resources (NBPGR), in India has established extensive in vitro storage facilities and is already storing germplasm collections of certain root and tuber crop species as well as a few fruit species. The bulk of the Indian potato collections at Central Potato Research Institute, Shimla, are conserved through tissue culture. China is also developing comprehensive facilities for in vitro conservation.
The Southeast Asian Banana Germplasm Bank, currently maintained as a living collection in Davao, the Philippines, has already been put under in vitro storage. The country is also maintaining some of its desired soft coconut germplasm through embryo culture. Malaysia is in the process of duplicating some of its ex situ living collections of oil palm and rubber as in vitro collections. Several of the Pacific Island countries are maintaining their taro collections in vitro because of the danger of the accessions being lost to viral and other diseases under field conditions. Thus, there is wide scope for using biotechnology for conservation and utilization of tropical plant germplasm (Sastrapradja and Sastrapradja, 1990; Withers, 1990; Singh, 1991).
Besides the developed countries, several developing Asian countries are using monoclonal antibody techniques for identification of pathogens and for indexing materials. In China, more than 50 kinds of hybridoma strains have been constructed which secreted various kinds of monoclonal antibodies to viruses, bacteria, cancer, etc. India is applying monoclonal antibody techniques for the diagnosis and epidemiology of tungro virus of rice and other important viruses. The Central Potato Research Institute, Shimla, India, has been using monoclonal antibodies, cDNA and enzyme-linked immunosorbent assays (ELISA) for detecting and characterizing viruses of potatoes. Several other developing countries have also been using this technique coupled with ELISA tests to detect peanut viruses. Thailand has been using cDNA probes for detecting Tomato Yellow Leaf Curl Virus (TYLCV), Papaya Ringspot Virus (PRY) and mycoplasma causing sugar cane white leaf (Attathom, 1993).
Recombinant DNA technology and transgenics
As regards the developed countries of the region, Australia and Japan have comprehensive programmes on recombinant DNA technology and production of transgenics for commercial exploitation. Australia was the first country in the world to have engineered and released a micro-organism for biological control of crown gall in plants. Australia's first field trial of transgenic plants took place in 1991, involving transgenic potato varieties resistant to leafroll virus, developed by the Commonwealth Scientific and Industrial Research Organization (CSIRO) Division of Plant Industry. During 1988-91, several laboratories in Japan, using electroporation or Ti or Ri, reported successful production of transgenics in Oryza saliva, Citrus sinensis, Cucumis melo, Lactuca saliva, Solanum tuberosum, Nicotiana tabacum, Morus alba, Actinidia chinensis, Atropa belledona. Brassica oleracea, Lycopersicon esculentum, Vigna angularis and Vitis vinifera (Nakajima, 1991). For effective gene expression, promoter regions/genes have been identified for tissue-, age- and pathogen-specific expression of the genes under transfer. Stable and useful transgenics for protein quality and for viral resistance have been produced in rice, potato, tomato, melon and tobacco (Table 4).
Transgenic plants of agricultural crops produced using useful genes, Japan
|Crops||Genes transferred||Transformation system|
|Oryza sativa||Rice glutelin||E.P.|
|RSV coat protein||E.P.|
|Bar (bialaphos resistance)||E.P.|
|Solanum tuberosum||a -amylase||Ti|
|Lycopersicon esculentum||TMV coat protein||Ti|
|Cucumis melo||CMV coat protein||Ti|
|Nicotiana tabacum||cDNA of mild TMV strain||Ti|
|ttr (wildfire disease resistance)||Ti|
Source: Nakajima, 1991.
Molecular studies, gene tagging and mapping studies have been intensified in several laboratories in Japan. In 1991, a rice genome project, on the lines of the famous human genome project, was initiated. Restriction Fragment Length Polymorphs (RFLPs), (Yang et al., 1990; Kawase et al., 1990), YAC clones or cosomid library and Several Sequence-Tagged sites (STSs) approaches are being deployed for the purpose. Cloned chromosome segments including useful genes are being analysed and characterized at molecular level. For identification and characterization of rice somatic chromosomes in in situ hybridization, imaging methods have been developed (Fukui and Ijima, 1991). Several countries in the region are using RFLP, RAPD and other techniques for gene tagging and creation of genome maps not only of major food crops but of topical industrial and fruit crops such as rubber, oil palm, durian and mangosteen. This approach is likely to increase the interaction between biotechnologists and plant breeders.
In China, protocols for production of transgenics for disease resistance in rice, tobacco, soybean and Brassica sp. have been standardized and transgenics are at various stages of development (Fan, 1991). Chinese scientists have attained considerable success in developing oilseed rape lines resistant to turnip mosaic virus through genetic engineering means. In India, transgenics are at various stages of production in cotton, rice, jute, Vigna aconitifolia and Brassica sp. (Bhatia, 1993). In the Republic of Korea, transformants were produced in tobacco, rice and tomato and work has been intensified for identification and isolation of useful genes, especially for disease resistance (Chung, 1991). The country is developing improved vectors for increasing the efficiency of transformants production. Malaysia is engaged in developing transgenics rubber and oilpalm, besides rice (Zakri, 1991). In Pakistan, biotechnological manipulation of protein content and quality and resistance to Ascochyta blight in chickpea has been pursued in the last few years (Riazuddin, 1991). Thailand, using the coat protein approach, has developed tomato and papaya lines resistant to TYLCV and PRV, respectively. Moreover, a mild strain of PRV has been developed for the mass preimmunization programme (Attathom, 1993).
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