Cloning and expression analysis of rice genes

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Rice is the major staple food of nearly one half of the world's population, and contributes over 20% of the total caloric intake of humans (Bhattacharjee et al., 2002). It is the most important cereal grain food for large part of human population especially in East and South Asia, the middle East, Latin America and the West Indies. Being the second highest in terms of production after maize worldwide, rice is supplying 20% of the daily calories of the total world population (World Rice Statistics,; FAOSTAT,

Rice is the most important food crop of Nepal. It is grown in a diverse environment ranging from low plains having tropical climate to the sub-temperate climate of the mountain region at highest elevation (3050 masl). Nepal has been considered as one of the origin center of rice. In Nepal, rice growing area is 1440 thousand ha with the productivity of 2.56 t/ha. It contributes nearly 20 per cent to the agricultural gross domestic product and provides more than 50 percent of the total calorie requirement of the Nepalese people (

Rice, as many other food crops, is a monocotyledonous crop and can be successfully grown in different environmental conditions such as irrigated or rain-fed low land, deep water, coastal wet land and even on upland under dry condition. It is the fact that rice is being cultivated in all continents of this world except in the extreme northern and southern parts which are always covered by snow. Rice can be cultured in a mixed cropping pattern which helps to encourage biodiversity. Rice in wet land is a good example of home of biodiversity as many kinds of terrestrial and aquatic organisms like fishes, frogs, snails, insects and other various aquatic animals habitat in rice ecosystem which in turn can be a good source of protein and fatty acid. Rice by-products like rice straw, bran have become a major source of food for our livestock which in turn give economic benefits from the livestock production. Rice is a also a good shelter for beneficial insects which are termed as natural enemies of several harmful insect pest in our agro-ecosystem, thus saving environment by decreasing the use of chemical pesticides for harmful pest management. Even, herbivorous fishes in rice field feed on weeds of the rice crops, controlling the harmful weeds to some extent in rice eco-system (

As Arabidopsis, a dicotyledonous model plant, recent advances in molecular studies in rice includes efficient gene transformation, development of highly saturated molecular map, huge number analysis of expressed sequence tags (ESTs). With genome size of 4.3 Mb, rice is a monocot cereal crop which number of analyzed complementary DNAs (cDNA) is almost approaching the number with Arabidopsis. Due to this reason, rice can be considered as Monocotyledonous model plant in the present context (Izawa and Shimamoto, 1996). To study rice genome, around 14,000 molecular markers are already designed (Kurata, 1994) and sequence information of more than 10,000 cDNAs has been already registered in computerized data base (Sasaki, 1994).

Plant diseases and insects; nutrient deficiency; mid and late season water stress; water management and weeds for direct seeded rice are considered as the major constraints in rice cultivation (Kataki et al. 2001). Rice is a good host of several plant pathogenic fungi, bacterias, viruses and nematodes. Production of rice prior to harvesting can be lost up to 50% by insects (34%), diseases (9.9) and weeds (10.8%) (Cramer, 1976). Soil borne diseases, especially diseases caused by plant parasitic nematodes (PPNs), are major bottlenecks to crop production and productivity in the intensive cropping systems with high inputs such as the rice and wheat based cropping systems (Sharma & Rahaman 1998). Sasser and Freckman (1987) estimated the approximate loss of rice production due to plant parasitic nematodes up to 10%. Among more than 35 genera and 130 species nematodes associated with rice (Gerber et al., 1987), rice root nematode, Hrishmaniella oryzae, has been proved one of the most damaging plant parasitic nematodes in all rice growing zones of the world. This nematode has wide range of major crops as its host such as rice, cotton, sugarcane and maize and has high damage level in relatively lower population densities (Southey, 1972). Hirschmaniella spp., especially H. oryzae, is omnipresent in flooded rice eco-system. They cause significant damage in rice and their proper management during rice cultivation gives significant yield increase. As irrigated rice covers about 72% of total rice production, H. oryzae, being predominant in flooded condition and also being abundant in rainfed lowland and deep water rice eco-system, is proved to be the rice parasitic nematode having greatest potential on economic impact (Prot and Rahaman, 1994). Hirschmanniella spp. do not produce specific aboveground symptoms. They can cause yellowing of the plant, reduction of tillers number, and cause delay in flowering. Fortuner (1974) found that H. oryzae can reduce yield by 23% when adequate fertilizers are applied and by 42% when there is no fertilizer application under experimental plot condition.

Naturally, plants have elaborate defense mechanisms to protect themselves from many kinds of pathogens, including fungi, bacteria, viruses, insects and nematodes. Defense responses following the gene-for-gene hypothesis are triggered in plants when the plant resistance (R) gene recognizes, directly or indirectly, a specific pathogen effector molecule often governed by a pathogen avirulence (avr) gene (Jones and Dangl, 2006). If any of the members of this gene pair is inactivated and become absent, it results in susceptibility of the host to the pathogen. Till date more than 40 R genes have been isolated from several plant species, and most of them exhibit highly conserved structures, despite differences between the types of pathogens that are recognized (Takahashi et al., 2010).

Expression analysis of rice genes under Hirschmaneilla oryzae infection has not been done much till date. Green et al. (2002) worked on analysis of expression pattern of Arabidopsis thaliana tubulin-1 and Zea mays ubiqutin-1 promoters in rice plants in association with the infection of upland rice root nematodes Meloidogyne incognita and Pratylenchus zeae and found that UBI-1 promoter was active at detectable level in most of the root system throughout the growth period of 10 months where 90% of the root tips showed GUS staining throughout the time course. Whereas, TUB-1 promoter used for this study showed lower overall activity in the root system giving 11% reduction in each week in the proportion of main root stained. Changes in gene expression correlated with wound or defense responses have been observed in several plant-nematode interactions especially with root knot nematodes (Gheysen and Fenoll, 2002). General (nonspecific) plant defense genes in tomato plant are upregulated only after12 h of inoculation of tomato roots with root-knot nematodes (Williamson et al., 1994; Williamson and Hussey, 1996). Activated defense genes include peroxidase, chitinase, lipoxygenase, extensin, and proteinase inhibitors.

We have recently found some genes of rice which are suspected to be up-regulated or down-regulated under infection of Hirschmaneilla oryzae. Therefore the main objective of this research to

to see the expression pattern (up-regulation or down-regulation) of certain rice genes under Hirschmaniella oryzae infection

Literature review

Rice (Oryza sativa L.)

Rice is a monocotyledonous crop plant falling in the angiosperm division. The genus of rice, Oryza, contains more than 20 species, out of which only two species are cultivated rice, i.e. Oryza sativa, (Watanabe, 1997) especially cultivated in South-east Asian countries and Japan, and Oryza glaberrima cultivated in West Africa. Rice was originally cultivated in tropical Asian region, since 5000 years BC according to oldest cultivation record, which afterwards extended to temperate regions also (Watanabe, 1997). Rice is the most important staple food in Asia. More than 90% of the total world's rice is produced and consumed itself in Asian region, the region which holds 60% of the total world's population. 35-60% of the caloric requirement of three billion Asians is supplied by rice (Guyer et al., 1998). Bouman et al. (2006) reported rice (Oryza sativa) to be cultivated in about 150 million ha of land worldwide with the average yield of about 3 to 4 tons ha-1 (Padgham et al., 2004).

2.1.1 Taxonomy and Molecular phylogeny

The well known rice genus "Oryza" consists of 22 wild and 2 cultivated species, one of which is Asian rice (O. sativa) and the another is African rice (O. glaberrima) (Semagn et al., 2007).

The genus Oryzae belongs to tribe Oryzeae and family Poaceae ( Hierarchically, Oryza sativa falls in division Angiosperm, class Monocotyledoneae, order Glumiflorae and family Graminae (Nguyen and Tran, 2000). Nomenclature of the species which are closely related to O. sativa have created confusion among rice taxonomists, as they are morphologically very similar and it is difficult to find distingushible morphological character (Vaughan and Morishima, 2003). Time to time, more than hundred names have been proposed for Oryza species including 19 names for Oryza sativa (Oka, 1988; Lu, 2004). A new nomenclature has been proposed by Vaugan et al. (2003) for cultivated and wild rice in Asia: O. sativa sensu lacto subspp. indica and japonica and O. rufipogan sensu lacto subspp. nivara (annual) and rufipogon (perennial).

Figure 1: ML phylogeny of Oryzeae inferred from the concatenated 20 chloroplast fragments under the TVM+I+G model. MP and BI inferences generated the same topology. Numbers near branches are bootstrap percentages of ML and MP, respectively. The branches without numbers indicate 100% bootstrap supports. Stars indicate three successive short interior branches in clade II (source: Tang et al., 2010)

2.1.2 History: Origin, domestication and dispersal

The genus Oryza, to which our cultivated rice belongs, is thought to be originated in the super-continent Gondwanaland around 130 million years ago as a wild grass. During the geographical drift the super continent was broken to form Asia, Africa, America and Australia and Oryzae species probably spread as wild grass thorough out these drifted continents (Chang, 1976). Within crop plants, Asian rice, O. sativa and the African rice O. Glaberrima are thought to be an example of parallel evolution. As shown in figure 2, the Asian common wild rice, O. rufipogon is the wild progenitor of O. sativa and it shows a range of variation from perennial to annual types. Annual types such as O. nivara, were domesticated and later became O. sativa. In a parallel evolutionary path, O. glaberrima was domesticated for annual O. breviligulata which in turn evolved from perennial O. longistaminata (Khush, 1997).

Figure 2: Evolutionary pathway of 2 cultivated rice species (source: Khush, 1997)

According to Porteres (1956), the African cultivar, O. glaberrima, was originated in Niger river delta. The swampy basin of the upper Niger river is considered as the primary center of diversity for O. glaberrima and there are two secondary centers for the same species to the southwest near the Guinean Coast. The primary center was thought to be formed around 1500 BC, while the secondary centers were formed 500 years later.

Probably, the domestication of wild rices has been started since 9000 years ago. during Neothermal age about 10 000-15 000 years ago, natural development of annual rice species at different elevations in East India, Northern Southeast Asia and West China occurred due to changeable climatic factors like alternating periods of drought and variations in temperature (Whyte, 1972). Domestication in Asia could have occurred independently and concurrently extending from the plains below the eastern foothills of the Himalayas in India towards upper Myanmar, Northern Thailand, Laos and Vietnam, to South China (Chang, 1976; Ramiah, 1937; Roschevitz, 1931). In this Asian arc, rice was grown as a form of shifting cultivation by clearing the forest areas. The crop was probably grown by direct seeding and without standing water. China was the first country which started the process of soil puddling and transplanting seedlings, the system which made rice become truly domesticated. In Southeast Asia, in the early period, rice used to be produced under dryland conditions in the uplands and it is recent record that the people started cultivating in lowland, thus, covering vast river deltas. Linguistic evidence also provide reasonable proof that the early origin of cultivated rice in this Asian arc. In several regional languages, the general terms for rice and food or for rice and agriculture are synonymous which shows that the concept of agriculture originated as the domestication of rice started. At the very beginning, Welhelm G. Solheim II in 1966 discovered the most convincing archeological evidence for domestication of rice in Southeast Asia (Solheim, 1972). 14C and thermoluminescence test of the imprints of grain and husk discovered in pottery sherds grain and husks of O. sativa at Non Nok Tha in the Korat area of Thailand gave the dating to at least 4000 B.C older prints. Ancient India is one of the oldest regions for the cultivation of O. sativa. The oldest grain samples of rice excavated at Mohenjodaro now in Pakistan (before in India) date back to about 2500 B.C. (Andrus and Mohammed, 1958). Sharma and Manda (1980) reported that the oldest carbonized grains found in India date back to about 6750 B.C. According to Chang (1976), the oldest remains of cultivated rice date back to 500 BC whereas carbonized rice grains found in Tongxieng County of Zhejiang province were identified as 7040 years old. Similarly, the second oldest of 6960 years old was obtained from Hemdu relic in Yuyao county of the same province.

The dispersal of rice from the Himalayan foot hills of ancient India began towards Western and Northern plain region of India, Afghanistan, Iran and Sri Lanka. It has been known that the rice was major food crop in Srilanka as long as 1000 BC. In Greece and neighboring countries of the Mediterranean, rice was introduced by members of Alexander the Great when they were returning to India in 324 BC. However, in Europe rice was established much later than the 15th or 16th century. From India rice also spread to Madagascar and East Africa and then to countries of West Africa (Khush, 1997). Evidence shows that O. rufipogon was dispersed from Pakistan to China and Indonesia (Oka, 1988).

Japonica rice, which is domesticated as tropical Japonica in the northern part of Southeast Asia and South China also being cultivated as temperate japonica as it, later, moved towards Korea from China and from Korea to Japan at the beginning of the 1st century. Both indica and japonica were transferred to Malaysia, Indonesia and Philippines from mainland Southeast Asia. Afterwards they were distributed from the Philippines to Taiwan. Tropical japonica was introduced by migrating Malays to Madagascar from Indonesia during the 5th or 6th century. At the same time, it also migrated to West African countries from Indonesia through the Portuguese priests. Due to this reason, all of the upland varieties in West African countries are tropical japonica. The Portuguese also brought the tropical japonica and indica to Brazil while Spanish people were the one to carry them to the other Latin American countries. That's why, in Latin American countries, all of the cultivated upland varieties are tropical japonica and lowland varieties are indica rice. In USA, the first record of rice is from 1685, and it was probably introduced from Madagascar while slaves were imported by USA from that area. (Khush, 1997).

2.1.3 Importance of rice Dietary supply

Figure 3: Share of rice of total calories consumed (sources: FAO and World Bank, 2010)

Rice is said to be staple food of world's poor. Rice is the most important food crop of the developing world and a staple food of the half of the world's population. More than 3.5 billion people throughout the world depend on rice for more than 20 % of their daily calorie requirement. In many asian countries, per capita consumption of rice can be higher than 100 kg per annum. For about 520 million asian people, most of which are very poor, rice provides more than 50% of their calorie requirement. In sub-Saharan Africa, the people, before a decade, rarely ate rice, but these days, they have started consuming rice daily and per capita consumption has doubled since 1970 to 27%. Average per capita consumption of rice in South America is 45 kg and more than 70 kg has been reached in Caribbean ( Production and economic importance

China was the highest rice producer in 2007 with the production of 186454,000 tons which was 29.29% of the total world rice production and India was in the 2nd position with the production of 143534,000 tons. Indonesia and Bangladesh were third and the fourth highest producers, producing 55039,000 and 42904,000 tons respectively followed by Vietnam, Thailand, Myanmar and Philippines, (WRS, 2009). According to USDA (2009) report, China, India, Indonesia and Bangladesh produce two-third of the global rice production. Rice is also produced in North & South America and Africa but in very less quantity compared to Asian countries.

In global market, only 4% of the total rice produced is traded worldwide. Thailand is the top most exporter of rice trading 6 to 7 million ton annually followed by US which export about 40% of its total rice production annually. Major importers of rice are Iran, Iraq and Saudi Arabia followed by Bangladesh, Cote D' Ivoire, North and South Korea, Mexico, Nigeria and Senegal. In Africa, the demand of rice each year is increasing by 2% and the people here consume 25 % of the total imported rice globally (Khush, 1997). Importance in molecular studies

Rice can be a good model among monocot plants as its complete genome sequence is already known (IRGSP, 2005). Now, the obtained complete genome sequences of both japonica and indica rice (Phillips et al., 2007) facilitates the study regarding gene expression, gene constitution and genomic structure. This high-quality map-based sequence is now available worldwide in several public databases, such as GenBank, DDBJ and EMBL (Vij et al., 2006). The genome size of rice is relatively small (420 Mb) compared to other known species, such as wheat (1500 Mb) which favors rice to be more preferred in the plant research community as it is much easier to make faster advances with comparatively small genome (Paterson et al., 2005).

Besides genome sequences, a lot of ESTs have been sequenced and currently more than 250 thousands rice ESTs are available in REDB (Rice EST DataBase) ( According to Kikuchi et al. (2003), over 28000 full length cDNA clones from japonica rice are available in Gene Bank. According to Hiei & Komari (2006), Agrobacterium-mediated transformation can be performed efficiently in rice which also adds more importance in rice as a monocot model plant for the field of molecular biotechnology.

2.1.4 Rice ecology

Rice has adapted itself in wide range of climatic condition, which is being cultivated from the hot deserts of Australia and Egypt, to the cool Himalayan foothills of Nepal (Bhagat, 2003).

According to Rutger (1981), for the rice plant to grow optimally, it requires pH of 5.0-7.5, although it can tolerate a pH of 4.3-8.7 (Duke 1973). Coronel (1980) did not find any adverse effect of acidic soil with pH of 3.5-5.0 on rice root growth in a nutrient culture study in the Philippines. As most of the rice plants are rainfed type, and they are cultivated during the wet season so that the necessity of water is fulfilled naturally. If cultivated during dry season, this rice need frequent irrigation, otherwise. In the tropics, monsoon dependant rice cultivation in prevailing due to the scarcity of irrigation water (Datta, 1981). According to Quayyum and Vergara (1993), relative humidity of 80% is best for rice during seedling stage. Temperature has significant effect on the growth and development of the rice plant. The average temperature for the rice plant ranges between 21°C to 37°C (AIC, 1996).

2.1.5 Rice by water management

Rice can be successfully grown in diverse water environments from aerobic upland rice to deep water rice. Types of rice according to water management can be grouped as irrigated rice, upland rice, rain fed lowland rice, deepwater rice, tidal wetland rice, and aerobic rice. Water table in lowland irrigated rice is generally maintained at 5-10 cm of depth while in rain fed lowlands and uplands condition, the rice field is flooded by rain at least for a part of the cropping season to water depths that exceed even 100 cm for some days. For both irrigated and rain fed lowland, rice seedlings are prepared in nursery bed and then the seedlings are transplanted in puddled field. Deepwater rice varieties can withstand the submerged condition of more than 50 cm of water table for at least a month (Catling, 1992). Rice can be grown in submerged conditions as it contains aerenchymatous cells (internal air channels) which can elongate rapidly when partially covered by floodwater. This biological character of rice enables them to grow above the flood waters avoiding drowning (Kende et al., 1998). Aerobic rice cultivation refers to the new system of rice cultivation done in the nonflooded and unsaturated aerobic soil environment similar as the wheat and maize cultivation (Bouman et al. 2006). Under this system, potentially high yielding varieties are dry seeded in the fields and limited supplementary irrigation is provided when according to the necessity.

2.1.6 Advances in rice thru breeding and molecular biotechnology

Vitamin A deficiency is one of the major outcomes of malnutrition. Worldwide, nearly 100 to 140 million children are vitamin A-deficient and an estimated 250 to 500 thousand vitamin A-deficient children become blind every year. Though rice is an important source of food energy and calories for 50% of the total world population predominantly in developing countries, milled rice is deficient in many essential micronutrients like iron, zinc, vitamin E and vitamin A (Vasconcelos et al., 2003 and Tan et al., 2005). This could be one of the main reasons for high prevalence of vitamin A deficiency (VAD) in developing countries. Though rice plants possess carotenoids in photosynthetic tissues, carotenoids lacks in the edible part of rice, endosperm. Genes responsible for two key enzymes in b-carotene (provitamin A) biosynthesis pathway, phytoene synthase (psy) and phytoene desaturase (crtI) were isolated and characterized from daffodil (Narcissus pseudonarcissus) and the plant pathogenic bacteria (Erwinia uredovora) respectively (Misawa et al., 1990; Misawa et al., 1993). Ye et al. in 2000 successfully demontrated genetic engineering of the metabolic pathway for biosynthesis of b-carotene (1.6 mg/g total carotenoids) in the endosperm of japonica-type rice cultivar. Later, the success was made in indica-type cultivated rice cultivars too (Datta et al., 2003; Hoa et al., 2003). After this achievement, the concept of genetic engineering-based nutritional enhancement of rice began with the high expectation to contribute to a sustained reduction of vitamin A deficiency (VAD) in developing countries. Recently scientists are further trying to increase the total carotenoids content and also to add other deficient micro-nutrients like iron and zinc.

After the development of transgenic plants, consumers and environmentalists have arose issue over the use of antibiotic selectable marker gene for development of transgenic plants, although strong evidence against the antibiotic markers have not been found yet. Considering the public concern, Datta et al.( 2006) have developed transgenic rice using a nonantibiotic positechTM selection system with phosphomannose isomerase (pmi) as an alternative to antibiotic resistance or herbicide tolerance marker system for selection. They have introduced two key genes, psy and crtI, of carotenogenic pathway in two indica-type rice cultivars, BR29 (a popular high-yielding variety of Bangladesh) and IR64 (important IRRI-bred line popularly grown in Asia), effectively to synthesize b-carotene in the target endosperm tissue.

Lucca et al. (2006) reported that successes in increasing iron root absorption by transgenic approaches improved the plants' ability to cope with iron-deficient conditions but there are no evidence reported till now for increased micronutrient content in the edible part. Therefore, combining high iron traits or rice with golden rice could lead to a highly effective, cheap and simple contribution to the relief of major health problems. Researches for combining high iron and Zinc rice with Golden rice is in progress which can be a great achievement in combating the malnutrition problems at greater levels (Khalekuzzaman et al., 2006).

Aromatic rice has been of great economic value these days. The biochemical basis of aroma was identified as 2-acetyl-l-pyrroline. The compound is found to be present in raw grain as well as in plant. In addition to 2-acetyl-l-pyrroline, there are about 100 other volatile compounds which include 13 hydrocarbons, 14 acids, 13 alcohols, 16 aldehydes, 14 ketones, 8 esters, 5 phenols and some other compounds, which are associated with the aroma development in rice (Singh et al., 2000). Several varieties of Basmati lines have been developed as aromatic rice by using various biotechnological tools. In Basmati rices, genetic transformation was obtained using Agrobacterium, electroporation and protoplast transformation systems (Chowdhury et al., 1997). Burikam and Attathom (1997) used particle bombardment mediated transformation system to transfer Δ1pyrroline-karboxylate synthetase (P5CS) gene in rice cv. KDML-105. Inez et al. (1997) used Agrobacterium tumifaciens containing a versatile binary vector (pCAMBIA 1301) to develop aromatic rice cv. Rajalele. Some selectable marker and reporter genes used were bur, npt II/hpt II and GUS. Several important genes like chitinase, Bt, bacterial blight resistance (Xa-21), rice tungro resistance (RTBV CP gene, RTSV polymerase in sense and antisense orientation), drought tolerance (P5CS) and submerged tolerance (adh and pdc) have been introduced into non-aromatic and aromatic rices (Abrigo and Datta 1996; Burikam and Attathom, 1997; Datta et al., 1997; Fauquet et al., 1997; Gill et al., 1997; Li et al., 1997).

Africa Rice Center (WARDA) has developed a new rice variety especially for African continent naming NERICA which is an acronym for New Rice for Africa. The range of NERICA varieties were developed from a cross between an upland O. sativa tropical japonica variety, WAB 56-104, as the recipient parent, and an O. glaberrima variety, CG14, as the donor parent ().The most popular NERICA varieties have obtained the best traits of both parents: high yield, which could be derived from the O. sativa parent and the ability to grow well in harsh environments from the O. glaberrima parent (Jones et al., 1997; The NERICA varieties are hoped for high contribution in improving the productivity, profitability, and sustainability of rice farming in sub-Sahara Africa.

The problem of water scarcity in the productivity of Asia's irrigated rice systems is increasing year by year. It has been estimated that 2 million ha of Asia's irrigated dry-season rice and 13 million ha of its irrigated wet-season rice may experience ''physical water scarcity'' by 2025, and most of the irrigated dry-season rice producing land out of 22 million ha in South and Southeast Asia may suffer ''economic water scarcity'' (Tuong and Bouman, 2003). To mitigate this problem, scientists are developing aerobic rice cultivars with high yield capacity which can be cultivated like an irrigated upland crop, such as wheat or maize. It is possible to achieve high yields under irrigated but aerobic soil conditions by generating new varieties of ''aerobic rice'' that combine the drought-resistant characteristics of upland varieties with the high-yielding characteristics of lowland varieties (Lafitte et al., 2002). Some successes have been achieved in China, where breeders have produced aerobic rice varieties with the productivity of 6-7 t ha-1 which are now being intensively cultivated by farmers on some 190,000 ha, in irrigated lowlands where water is scarce and in favorable rainfed uplands (Wang Huaqi et al., 2002).

2.1.7 Biotic constraints in rice

The major biotic factors affecting rice yields are insect pests, diseases, and nematodes. Rice stem borer is one of the major pests of rice and found to be more destructive in tropical regions of Asia, the Mediterranean regions and the Middle East. Brown plant hopper (BPH), which is also virus transmitter, leaf roller, armyworms, rice ear cutting caterpillar and rice bug are other destructive insect pests of rice plant. Rats, as a rodent, are also major problems in rice production (IRRI, 2009). Rice has been attacked by more than 70 diseases caused by fungi, bacteria, virus and nematodes (Ou, 1985). Each year, huge amount of rice production is being destroyed fungal diseases which would otherwise be sufficient to feed 60 million people (Barman and Chattoo, 2005). ). Rice blast caused by Magnaporthe grisea, sheath blight caused by Rhizoctonia solani and brown spot caused by Bipolaris oryzae are the three major fungal diseases of rice. Rice sheath blight disease (Rhizoctonia solani) occurs throughout the rice-growing areas in subtropical, tropical and temperate countries due to its wider host range and adaptive capability. Normally, this disease causes 20-25% yield reduction but can cause 50% yield loss in case of susceptible cultivars. Yield losses of 50 to 85%, in rice, due to blast pathogen Magnaporthe grisea has been recorded in the Philippines which can cause even upto 100% yield losses under favorable environment (IRRI, 2009). Bipolaris oryzae, the causal micro-organism of Brown spot disease in rice is cosmopolition and is found in all rice-growing countries worldwide including Asia, Africa and America. This disease can cause a loss of 50% to 90% of rice production. Bakanae (Gibberella fujikuroi) is another widely distributed fungal disease in all rice growing areas and affects both upland and lowland rice causing up to 20% yield loss (IRRI, 2009). Among bacterial diseases associated with rice, bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae, and bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola, are the most important ones (Datta, 1981). Ou (1985) reported the yield loss of 20-50% by bacterial blight while Wang et al. (2007) reported that bacterial leaf streak may cause 30% of yield loss in rice. Tungro, grassy stunt and yellow dwarf are the most important viral diseases (Datta, 1981). More than 35 genera and 130 species nematodes associated with rice (Gerber et al., 1987).

2.1.8 Nematode problems in different rice eco-system

Plant parasitic nematodes are microscopic hidden worms attacking many economically important crop plants which can be a major constraint to obtain high yields of rice. They are often unnoticed as most of them are root parasitic. Except a foliar nematode Aphelenchoides besseyi, the "white tip" nematode that occurs in most rice environments (Bridge et al., 1990), the rice plant parasitic nematodes are not homogeneous across all rice ecosystems. Yield losses due to A. besseyi range from 0 to 70% and vary with variety, year, and country (Port and Rahaman, 1994). Meloidogyne spp. and Pratylenchus spp. are the major rice parasitic nematodes in upland. M. incognita, M. javanica, and M. arenaria occur in most of the upland rice-growing areas in Africa (Luc and de Guiran,1960; Babatola, 1980; Fortuner, 1981) and South America (Bridge et al., 1990). Yield losses of 16-32% were reported with M. graininicola in India (Biswas and Rao 1971, Rao and Biswas 1973). Pratylenchus indicus and P. zeae are two major root lesion nematodes found in upland rice ecosystem (Bridge et al., 1990). In an experiment, significant yield losses (34%) have been observed with P. indicus under low initial number (30 per seedling) of nematodes (Prasad and Rao, 1978). 7 species of genus Hirschmaniella viz. H. belli, H. gracilis, H. imamuri, H. mexicana, H. mucronata, H. oryzae, and H. spinicaudata (Bridge et al., 1990) damage rice plant among which H. oryzae is the most prevailing one in flooded rice ecosystem (Prot et al., 1994; CUC and Prot, 1992). Beside, A. besseyi, few nematodes like Meloidogyne graminicola and Ditylenchus angustus can cause damage to rice plant in deep water rice ecosystem. D. angustus, is the causal agent of ufra disease in Bangladesh, India, Myanmar, and Vietnam (Ou, 1985) which can cause few to 100% yield loss in rice.

2.2 Rice root nematode: Hirschmaniella oryzae

About half, out of 24 known species of Hrishmaniella, are parasitic to rice and worldwide, worldwide and H. oryzae is the most commonly found plant-parasitic nematode on irrigated rice especially in areas where rice has been cultivated, for a long, with continuous irrigation facility and when the plants are grown under constantly flooded condition (Bridge et al., 2005). H. oryzae can adapted very efficiently to the constant flooded conditions in which irrigated rice is often being grown in the lowlands (Fortuner & Merny, 1979). It is one of the few plant-parasitic nematode species that can easily survive under anaerobic conditions (Babatola, 1981).

2.2.1 Systematic position of Hirschmaniella oryzae

Phylum: Nematoda

Class: Chromadorea

Subclass: Chromadoria

Order: Rhabditida

Suborder: Tylenchina

Infraorder: Tylenchomorpha

Superfamily: Tylenchoidae

Family: Pratylenchidae

Subfamily: Hirschmanniellinae

Genus: Hirschmanniella

Species: Hirschmanniella oryzae

(Source: Duncan & Moens, 2006)

2.2.3 Morphology of H. oryzae

The species was described by de Man (1880) from a moist meadow at Wassenaar near Den Haag, The Netherlands. More detailed data on morphological characters of this species were particularly given by Hirschmann (1955a, b), who based her observations on specimens collected in southern Germany. Sher (1968) provided a brief description from his studies of specimens from the type locality, from a second locality in The Netherlands, and from Texas, USA, and Brzeski (1998) added data supplementing previous descriptions.

Fig. 4 Hirschmanniella oryzae. (A)Whole female. (B) Female tail. (C) Female terminus.b (D,E) Variation in gubernaculum shape. (F) Male anterior end. (G) Male tail (after Sher, 1968). (Source: OEPP/EPPO Bulletin, 2009)

According to Bridge & Starr (2007), the size of this nematode varies from 1.1-4 mm and is slender in shape with annulated, anteriorly flattened or hemispherical labial region which is continuous with the body contour. The stylet is strong with well developed basal knob and its length varies from 15-46 μm. The oesophageal glands are elongated and are ventrally overlapped with the intestine. Ebsary & Anderson (1982) gave the description of genital organs of both male and female of this species. According to him, female genital system is didelphic with two equal well developed branches and the vulva is present at mid-body. Tail of this nematode is conoid or pointed with mucron. There is no sexual diamorphism in adults. The male bursa does not cover the whole length of the tail.

2.2.3 Life cycle of H. oryzae

It is migratory endo-parasite of root, the larva and adult of which always enter roots from root tip, moves freely in the air channel between radial lamellae of the parenchyma and in older roots, may be found anywhere between the region of base and tip and sometimes even found in coleoptiles but not in the lower part of the leaf sheath (Buangsuwon et al., 1971). After few days of entry, female starts laying eggs which hatch within 4-5 days after being laid inside the root. Under favorable environmental condition this nematode can complete life cycle in about 30 days (Mathur and Prasad, 1974). Karakas (2004) reported that the nematode completes its life cycle from L2 stage to next L2 stage in 33 days at 28â-¦C. Each stage of juveniles changed after molting and molting stages M2, M3 and M4 lasted for 2, 3 and 6 days respectively to develop adult male or female. Mode of reproduction for this nematode is sexual (Southey, 1972; Karakas, 2004).

2.2.4 Symptoms in rice plant on H. oryzae infection

Hirschmanniella spp. do not produce specific aboveground symptoms. Their infection in root may result in yellowing of the plant, reduction of tiller number, and delay in flowering. H. oryzae does not cause specific symptoms in infested plants (Kawashima and Fujinuma, 1965)

Vander Vecht and Bergman (Verma and Singh, 1989) observed the nematode penetrating into the roots of healthy rice plants, feeding on parenchymatous tissues and multiply in there which leads finally to the discoloration of cortex. Reduction in total sugar, decrease in aminoacids and liberation of phenols are some metabolic changes that have been recorded in the rice plants infected by Hrishmaniella oryzae (Rao et al., 1986). The number of panicles (Yamsonrat, 1967) and grain weight (Venkitesan et al., 1979) were affected in rice due to H. oryzae infection. In Japan, decrease in tillering induced by H. oryzae is more important in soils with low rH, resulting in a disease called "Akiochi" and yield losses are more significant in these soils. (Kawashima, 1964). Infested seedlings undergo growth retardation, height and weight of the plant are decreased and, at the same time, the browning of the roots is higher at high initial densities (Kawashima & Fujinuma, 1965). The same authors reported that, nematodes interfere with the physiology of the roots, decreasing their oxidizing capacity and inducing their coloration by iron oxide. H. oryzae affected rice plant by causing decreased tillering and root weight in India (Mathur & Prasad, 1972). In the U.S.A., this root parasitic nematode caused decay of the tip of primary roots in the layer of the soil with a high rH (Hollis, 1967). Hirschmanniella spp. stunted rice in Thailand (Buangsuwon et al., 1971).

2.3 Rice transformation system

Initially, protoplast transformation with electroporation or PEG was the method of rice transformation. Toriyama et al. (1988) and Zhang and Wu (1988) recovered transgenic rice 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. Later this technique became less popular as it is time consuming, laborious and highly genotype-dependent. Somaclonal variations, multi-copy integration and regeneration of albino plants are other problems related to this technique (Tyagi and Mohanty, 2000).

2.3.1 Particle gun mediated gene delivery

Microprojectile bombardment technique using particle gun or biolistics was used successfully for transformation in immature embryos of rice (Christou et al., 1991). After Cao et al. (1992) and Li et al. (1993) further improved this technology; this method has been widely used for transformation of japonica rice. Further, many scientist used this method in transformation of indica and javanica rice (Tyagi and Mohanty, 2000). Chen et al. (1998) reported transformation of japonica rice with multiple genes using biolistics. The group bombarded rice tissue with 14 different pUC-based plasmids and out of total transformed plants, 17% of had more than nine target genes and 85% contained more than two target genes. The growth behaviour and morphology of these plants were normal. During this work, integration of multiple transgenes, interestingly, occurred at single or two loci which have derived hope for engineering of novel biosynthetic pathways in rice. Tang et al. (1999) reported transformation of rice with four genes by co-transformation using biolistics. Two out of the four genes used were economically important, viz., Xa 21 and GNA, responsible for providing resistance against bacterial blight and sap-sucking insects, respectively. Molecular analysis confirmed that over 70% of the transgenic plants recovered contained all four genes. The majority of the transgenic plants showed expression of these genes. By the biolistics method more than 70 rice varieties already transformed and this method is claimed to be genotype-independent and transformation frequency as high as in dicots has been reported in some cases (Tyagi and Mohanty, 2000). It should, however, be noted that different workers have reported variable frequency of transformation. This method has been successfully used to transfer a number of economically important genes in rice (Oard et al., 1996)

2.3.2 Agrobacterium mediated rice transformation

The first reliable Agrobacterium mediated transformation in rice was achieved in 1994 (Hiei et al., 1994) who produced large number of fertile and morphologically normal transgenic plants from japonica rice. This group demonstrated successful integration of foreign DNA in rice chromosome through A. tumefaciens though there was controversy earlier in the ability of Agrobacterium in transformation of monocotyledonous plants as they are not natural host of this bacterium. Hiei et al., 1994 reported that addition of acetosyringine to the media and temperature of 22â-¦C - 28â-¦C was compulsory during co-cultivation phase. GUS expression in the tissue immediately after infection gave good selection of preferred tissues for co-cultivation. Among the 2 strains and 2 vectors used, they found strain LBA4404 with vector pTOK233 was more effective in transformation. This method took 3 to 4 months to produce transgenic rice plants from the beginning of tissue culture. Similarly, Rashid et al. (1996) described the production technique of transgenic indica rice. Toki (1997) also followed the same work and methodology of Rashid et al. (1996) on japonica rice expect with some modification by adding casamino acids and proline in the medium for callus induction and in the medium used for selecting transgenic calli and all cultures were stored under 30â-¦C except during co-culture period. This improved culture method shortened the period of tissue culture, ie., differentiation period, minimizing the somaclonal variation and transgenic plants were regenerated from callus within 2 months. The author also developed new binary vector "pSMABuba" for rice transformation. Agrobacterim tumefaciens strain EHA101 with vector pSMABuba has been found more effective in rice transformation.

Figure 5: Schematic representation of Agrobacterium mediated transformation in rice (source: Toki, 1997)

Hoque et al. (2005) developed an efficient Agrobacterium mediated transformation method in Bangladeshi indica rice. Among mature and immature embryos used, immature embryos gave higher frequency in transgenic plant production though calluses derived from3-week old matured embryo was excellent as starting material. For recalcitrant Bangladeshi genotypes such as BR22, super-binary vector (pTOK233) was generally more effective than the binary vector (pC1301-Xa21mSS). The authors also found acetosyringone (200 µM) essential for co-cultivation period.

Toki et al. (2006) reported the scutellum tissue from day 1 pre-cultured seed in the media containing 2 mg l-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) were competent for Agrobacterium-mediated rice transformation. This early infection of rice with Agrobacterium also enhanced the efficient selection of transformed calli due to which the transformation procedure was rather quick and transgenic plantlets were produced within a month. Earlier, it has been reported that 2 to 3 weeks old rice callus derived from scutellum tissue of matured seeds is competent for Agrobacterium-mediated rice transformation (Hiei et al., 1994).