Asian Journal of Plant Sciences

Year: 2007 | Volume: 6 | Issue: 1 | Page No.: 203-213
DOI: 10.3923/ajps.2007.203.213

Prospects of Nitrogen Fixation in Rice

Parvez Sofi and Shafiq Wani

Abstract: Global agriculture relies heavily on fertilizers which are ecologically as well as economically expensive. Nitrogen which is undoubtedly the most important nutrient input required for rice production, is most frequently also a limiting factor. In a world facing acute energy crisis at global level and unpredictable spurs in world crude oil process due to political turmoil’s and lack of dependable alternative energy resources, it is imperative to develop the system of rice production which, without compromising on yield out-put, lowers dependability on chemical N-fertilizers besides being ecologically compatible. Nitrogen fixation in rice seems to be an efficient prospective system that is compatible with principles of resource conservation and ecological security. The dream project of BNF rice was started in 1992 based on expert recommendations which involve improving endophytic associations between rice and N₂ fixing bacteria, engineering of rice plants capable of forming legume like symbiosis and nodules with rhizobia, transforming rice to ensure expression of nitrogenase and protect nitrogenase system from oxygen damage and enhancing N-use efficiency of rice. A large number of diazotropic microorganisms have been found to be associated with rice roots. Among these endophytic diazotropes, Alcaligenes, Azoarcus, Serratia marcesscens and Azorhizobium caulinodons have received major attention. Though most of the aspects of rice-diazotroph interaction and nitrogen fixation have been elucidated both at genetic as well as molecular level, the engineering of an autonomous nitrogen fixing rice plants is undoubtedly a long term endeavor. A large number of endophytic diazotrophs have been found to be associated with rice and factors encouraging bacterial colonization have been characterized but certain critical differences in rice-rhizobial interaction relative to root nodule symbiosis in legumes have to become a reality. It will require a series of genetic manipulation of nodulation genes from plants and nif genes from bacteria to realize the dream of developing a biologically nitrogen fixing rice.

Keywords: Rice, nitrogen fixation, nod, nif and fix

INTRODUCTION

Nitrogen which is undoubtedly the most important nutrient input required for rice production, is most frequently also a limiting factor. Global agriculture relies heavily on fertilizers which are ecologically as well as economically expensive. Currently Nitrogen is being manufactured by Haber-bosch process (Burgess and Newton, 1977) which is largely an energy dependent process. Petroleum is the major energy source for fertilizer sector and thus fertilizers are vulnerable to political uncertainties and economic fluctuations in global petroleum market (Ladha and Reddy, 2000). It has been estimated that fertilizer production for global needs may require fossil fuel energy equivalent to 100 million tones per year. Due to its finite reserves and being a non-renewable energy resource, sustaining current level of fertilizer production will not be practically feasible on long term basis.

Currently the rice production globally consumes about 10 million tones of nitrogenous fertilizers. In order to boost up rice production to about 13 t na^-1, fertilizer inputs will have to be appropriately enhanced, from current level of 220 kg ha^-1 to 400 kg ha^-1 (assuming 50% fertilizer use efficiency). The super rice series cultivars developed by IRRI require higher fertilizer requirements in order to realize their optimum genetic yield potential.

In a world facing acute energy crisis at global level and unpredictable spurs in world crude oil prices due to political turmoil’s and lack of dependable alternative energy resources, it is imperative to develop the system of rice production which, without compromising on yield out-put, lowers dependability on chemical N-fertilizers besides being ecologically compatible. Britto and Kronzuiker (2004) proposed four areas of emphasis in this regard.

Among these four areas, nitrogen fixation in rice seems to be an efficient prospective system that is compatible with principles of resource conservation and ecological security. Nitrogen, even though present as a major component of atmosphere, is however used mainly by lower organisms, such as bacteria, algae etc. these organisms are called as N-eaters or Diazotropes. Among plants, some 12000 have ability to use nitrogen by virtue of symbiotic relationship with such diazotropes. In such a symbiotic association, the soil bacteria (Rhizobium, Bradyrhizobium and Azorhizobium) infect the root hairs or emerging lateral or adventitious roots and induce formation of morphologically defined structures called as nodules. The nodulation occurs mostly in case of plants belonging to Leguminoseae. The only non-leguminous known to naturally form modules with either Rhizobium or Bradyrhizobium belongs to Genus Parasponia. (Cocking et al., 1996). The bacterial colonies infect wounded root tips. The final structure from such infections is a root swollen on either side of central vascular tissue by cortical cells filled with infection threads containing bacteria actively engaged in nitrogen fixation. One of important studies have revealed that some of Rhizobia strains are capable of nodulating both legumes and Parasponia.

Genetic basis of rhizobium-legume interaction: The induction of nodules harbouring nitrogen-fixing bacteria is result of complex interaction between Rhizobia and plant. It involves several sets of genes from both partners in a coordinated expression. The plant genes involved in different aspects of symbiosis are called as Nodulin genes. Nodulin genes are of two types; C-Nodulins which are common to all legumes and S-Nodulins, present in specific species (Lodha and Nainawatie, 1993). These nodulin genes are either constitutive or inducible or repressible. They are numbered in order of size. N-1 being the largest and N-30 (globin) the smallest one.

Almost 30 Nodulin genes have been identified, leghaemoglobin being the most well characterized. Nodulin-35 of Soybean has a uricase activity. A root nodulin of Phaseolus vulgaris is glutamine synthetase. The nodulin from Dolichos biflors, apyrase (Nucleotide phosphohydrolase) was isolated by Etzler et al. (1999). A similar ortholog of such apyrase (Gs-50) was isolated from Glycine soja by Stacey et al. (1999). Nodulin genes affect number and size of nodules, nodule morphogenesis and rate of nitrogen fixation activity. A detailed analysis of nucleotide sequence, mechanism of regulation and function of all nodulin genes is essential for genetic manipulation of biological nitrogen fixation.

A number of bacterial gene sets which control the events leading to nodule formation are collectively called as Sym genes, which consist of nod, nif and fix genes. In fast growing strains of rhizobia, sym genes are present on a large plasmid whereas in slow growing strains they are present on bacterial chromosome.

The nod genes are bacterial genes involved in nodule formation. Based on function various nod genes are:-

The nod genes are also classified on the basis of complementation or non-complementation by their heterologous strains. The nod A, B and C belong to first category and nod D belongs to latter category. The nod D is a regulatory gene which activates other nod genes. A number of such gene sets have been isolated and characterized. One such gene cluster is nod ABCD (Kondorsi, 1984) which controls general nodulation function, such as root hair curling and induction of meristematic cell division (Jacobs et al., 1985). Another set of genes nod EFGH also called as hsn ABCD conditions the host specificity of nodulation (Debelle and Sharma, 1986; Fisher et al., 1987). A number of other nod genes have been identified, some of which act in a manner similar to hsn ABCD genes (Renalier et al., 1987).

Apart from nod D which is expressed constitutively, other nod genes are induced by plant signals (Mulligan and Long, 1985). The analysis of root exudates revealed that certain flavones are potential nod inducers. In R. trifolii and R. melilotus, luteolin has been found to be major inducer (Peters et al., 1986). However, in addition to such inducers, product of nod D is also essential for their expression. Gettfert et al. (1986) found three different versions of nod D viz., D₁, D₂ and D₃ which regulate host-specific nod gene expression, by corresponding to specific root exudates (Gyorgypal et al. 1988). Thus it can be considered that D₁, D₂ and D₃ are allelic forms of nod D.

Another set of Sym genes called as nif genes one highly conserved in various diazotrophs. Various types of nif genes are nif H, D and K, which code for nitrogenase polypeptides.

The use of nif DHK probe of Klebsiella pneumonia and DNA hybridization studies have revealed that nif genes of Rhizobium and Bradyrhizobium have strong homology with Klebsiella. Despite such homology, there are certain differences in structural organization of nif genes in bacterial genome. In case of R. melilotus and R. leguminoserum, they are a part of a single operon while as in case of B. japonicum and R. phaseoli they reside on different operons. The regulatory mechanism of nif gene expression has been extensively studied in R. melitotus. The expression of nif A gene is regulated by ntr gene system (Ow et al., 1985) in case of Klebiella but in Rhizobium ntr has no role. The most important function of nif gene is activation of another gene set called as fix genes. An important aspect of nif genes which is of paramount significance is that nif gene transfer between bacteria has been observed in direct cell contact. The localization and characterization of nif genes on plasmids offers opportunity of transferring such genes to plants in near future. A number of nif genes have been identified and characterized structurally as well as functionally. Table 1 shows various such nif genes and their structural and functional details (Dean and Jacbs, 1992; Galton and Smith, 1993).

The other class of sym genes which govern the ability of rhizobia to fix nitrogen are called as fix genes. The activity of fix genes is regulated by nif genes. Various fix genes such as fix A, B and C have been characterized. Different fix genes share stringer homology in rhizobia than Bradyrhizobium. Even their genetic organization determine the late nodule developmental activities (Kondorosi et al., 1989), by expression of late nodulin genes. Verma et al. (1988) proposed that there may be several such late nodulin genes which may require different bacterial signals for expression. In an earlier study, it has been found that fix genes may also control the expression of various nif genes (Putnoky et al., 1988) using several fix mutants, (Putnoky et al., 1988) found that fix mutants induced nodules but do not express nif structural genes probably by impairing expression of nif A regulatory gene.

Biologically nitrogen fixing rice (bnf-rice): Plants take nitrogen mostly in form of nitrate and ammonia. By far, no green plant has been found to be capable of obtaining diatomic nitrogen directly from atmosphere. Thus biological nitrogen fixing rice is a non-natural existence. But, if brought into existence by genetic manipulations, it would amplify the potential for nitrogen supply to rice plant as the fixed nitrogen would be available to plant directly with no loss (Ladha and Reddy, 2000). However, things are always easier said than done. It will require assembly of complex enzyme and provision of appropriate physiological conditions in the absence of environment normally provided by a prokaryotic cell (Dixon et al., 2000). Thus putting all the essential units of such a complex biochemical system in appropriate order is both a challenge as well as opportunity to develop rice with inherent capacity to fix atmospheric nitrogen. This is all the more important because it has been estimated that if only half of N₂ applied to lowland rice could be obtained from biologically fixed nitrogen, it would save about 7.6 million tones of oil annually. Ladha et al. (1997) proposed two possible approaches to this end.

The dream project of BNF rice was started in 1992 based on expert recommendations at the workshop on the feasibility of nitrogen-fixing capability in rice organized by IRRI. Four major approaches recommended at workshop were:-

A number of institutions are working independently or in a collaborative framework to achieve the goal of identifying and improving endophytic diazotrophic associations in rice and determining the predisposition of rice for developing an endosymbiotic relationship with rhizobia (Table 2).

Identification of endophyte diazotrops in association with rice: Plant root system offers an excellent micro habitat for bacterial growth. A number of endophytic diazotrops have been found to be associated with different crops such as sugarcane (Acetobacter diazotrophicus and Herbaspirillum) and Kallar grass (Azoarcus). Other studies revealed that a large number of N₂-fixing bacteria were present in root interiors of certain grasses (Remhold-Hurcek and Fendrik, 1986; Olivares et al., 1996). These studied encouraged the research community looking for development of efficient rice-endophyte system for conferring in plant ability to fix atmosphere nitrogen. Initially the techniques for accurate enumeration and isolation of putative endophytes were standardized (Stoltzfus et al., 1997). This was followed by isolation and characterization, by PCR based techniques, of various prospective endophytes from various wild as well as cultivated rice cultivars (Barraquo et al., 1997). Various prospective diazotrophic endophytes isolated from rice are:

Among these endophytic diazotropes, Alcaligenes, Azoarcus, Serratia marcesscens and Azorhizobium caulinodons have received major attention. They are aggressive colonizers. Alcaligenes are seed-borne endophytes, whereas Serratia appears to infect rice grown in soil. Azoarcus is a promising prospect because they have been found to be capable to nitrogenase gene expression in gnotobiotic culture inside rice. In this regard Azoarcus sp. BH72 which is an endophyte of Kaller grass (Leptochloa fusca) has been studied as a model endophyte because of its ability to invade rice. Strong evidence of presence of microcolonies of Azoarcus containing nif H expressing bacteria was reported in japonica cultivar Nipponbare. The bacteria colonized Aerenchyma tissue of rice roots, with strong evidence of nitrogenase gene expression using a reporter strain of Azoarcus carrying nif H: gfp fusion. An important finding was that bacteria were located between cell walls and not inside living cells (Apoplastic expression). Euen et al. (2000) also reported Azoarcus in root apoplasts but did not find any symbiotic organs. In modern cultivars; however Klebsiella, Sphigomonas, Azorhizobium and Burkholderia predominated.

Another putative endophyte which holds good promise is Serratia macrcesscens. At IRRI various modern varieties were screened and about six different isolates of Serratia were identifies (Hurek et al., 2000). However no N₂-fixing capability in terms of Acetylene reduction assay was detected. An advantage of using Serratia marcesscens is that it can be a potential bio-control agent owing to its chitinase activity (Mc Inroy and Kloepper, 1995) and can also induce systemic resistance in plants (Press et al., 1997).

It has been found that Accrenchyma of rice roots may act as a suitable microenvironment for diazotropic endophytes for introgenase gene expression. The collaborative studies have provided evidences of a large number of diazotrophic and non-diazotrophic bacteria which are capable of colonizing rice seedlings after germination. Thus presently we have a workable pool of bacteria which can be further studied in terms of endophytic interactions with rice which is essential for beneficial rice-endophyte association for in plant N₂-fixing capability in rice. In this regard IRRI and collaborating centers are pursuing following research initiatives to explore and evolve strategies for identification and improving rice-endophytied diazotroph association.

Improving genetic predisposition of rice for symbiotic nitrogen fixation: In symbiotic association between legume and soil bacteria, the rhizobia infect plants via root hairs or cracks caused by emerging lateral roots. This is followed by formation of morphologically defined structures called nodules. In these nodules the bacteria fix nitrogen which becomes directly available to plants. In our endeavor to produce BNF rice, we have to ensure that the diazotropic endophytes develop such association with rice plant where by they can develop reports defined structures. There have been many reports about such nodule like structures at a low frequency (0.1-0.2%) upon inoculation of rhizobia to normal roots (Bender et al., 1990; Jing et al., 1992; Li et al., 1991; De Bruijin et al., 1995) or enzyme treated roots in presence of PEG and CaCl₂ (Al-Mallah et al., 1989). Reddy et al. (1997) studied the interaction between various rhizobial strains and rice and found that:-

Cocking et al. (1996) inoculated rice seedlings treated with enzyme cellulase and pectolyase (which dissolve cell wall) with Azorhizobium caulinodans (ORS571) and both the varieties Lemont and IR42 developed N₂-fixing activity as evidenced by acetylene reduction assay. Reddy et al. (1998) stated that predisposition of rice is essential for symbiosis. They found that like legumes, certain exudates from rice roots induce transcription of nod genes of Rhizobium sp. NGR 234 but neither observed any root deformation not true nodule formation. they also reported that certain genes in rice c DNA were similar to legume Early Nodulation genes (ENOD 93 and ENOD 40) and are important for early developments for nodule formation. The legume ENOD 12 gene can be expressed in response to rhizobial Nod factors in rice (Reddy et al., 1998). Rice genome was found to posses varied levels of homology to ENOD 40 (Kouchi et al., 1999). Recent results with ENOD 12 transgenic plants have shown that rice not only possesses the mechanism to recognize rhizobial Nod signals but also the signal transduction chain to activate ENOD gene transcription and more so the expression is restricted to vascular bundles as in legumes. Thus it can be assumed that ENOD-40 genes in both legumes and rice have a similar regularly mechanism.

An important finding in rhizobia-rice interaction was identification of the specific flavonoi compound. Ladha et al. (2003) found that Naringenin (A flavone) acts as a signal molecule for colonization of rice by Azorhizobium caulinodans ORS571. Similarly an apyrase (Nucleotide phosphohydrolase) was isolated from Dolichos bifloris and has the ability to bind to rhizobial Nod signals (Etzler et al., 1999). Another ortholog of apyrase (GS50) was isolated from soybean (Stacey et al., 1999). These proteins can be prospective receptors of Nod signals. In fact Dey et al. (2002) introduced a plasmid containing GS50 apyrase driven by CaMV35S promoter into rice cells by biolistic method of Taipei-309 which stably integrated and expressed this gene.

Rice as such does not enter into symbiotic association with rhizobia but is able to interact symbiotically with certain mycrorhizal fungi (Khan and Belik, 1995). Two important evidences have been gathered to safely conclude that rice can be engineered to trigger rhizobial nodulation. One of the evidence was provided by Albrecht et al. (1998), who found genetic links between processes of nodule formation and arbuscular mycorrhizae. Another study with nodulation mutants of pea revealed that the early nodulation genes in pea (ENOD 2, ENOD 11, ENOD 12 and ENOD 40) controlling early events of nodule development, also govern the early events of mycorrinzal development (Van Rhijn et al., 1997). Now since rice is able to associate symbiotically with mycorrhizae and in both cases ENOD genes play a key role, it can be inferred that at least some, if not all, genetic mechanisms do exist and function in rice which are instrumental in initiating nodule development, but rice is unlikely to possess all such genetic framework. It is now our endeavour to gather the missing links and complement the existing genetic factors, so that rice acquires such unique traits that are necessary for rhizobial nodulation. This is an essential consideration while working for developing rice cultivars with in plant capacity of N₂-fixation in association with endophytic diazotrophs.

Engineering rice for inherent n₂-fixing capacity: Engineering rice to develop nitrogen fixing capacity will be one of the most crucial contributions of biotechnology to agriculture. The advances in transformation of rice, characterization of various genes from both plant (nodulin genes) and diazotropic endophyte (nod, nif and fix genes), identification of various rice specific promoters that can efficiently drive the foreign genes have made the dream, of having a rice plant with inherent ability to fix atmosphere nitrogen, not only reasonable but also realizable. Parakaran (1997) proposed two approaches for engineering rice plant.

Transformation of rice leaf: The transfer of nitrogen fixing (nif) genes from Klebsiella pneumoniae to Escherichia coli by conjugation and localization of nif genes on bacterial plasmids raised hopes of transferring them to eukoarytoes as well (Schlegal, 1993). However, there are certain genetic and physiological considerations which have, most obviously, put breaks on progress in engineering rice plant as one would expect given the rapid advances that have been made in efficient transformation of rice. These considerations are :

These considerations have been thrust areas of research towards constructing a BNF rice. Dixon et al. (1997) proposed that plastids can be a suitable location for targeting nif genes because chloroplast genes are expressed in a prokaryotic like fashion and polycistronic m-RNA’s are translated. The cluster of nif genes may be introduced simultaneously because now techniques are available where many genes can be transferred into rice (Kohli et al., 1998; Agarwal et al., 2001). The process of photosynthesis and nitrogen fixation can be made to co-exist provided a mechanism to protect nitrogenase from oxygen damage is put in place. A possible mechanism can be temporal separation of oxygen-evolving process (photosynthesis) and oxygen-sensitive process (Nitrogen fixation) by restricting nitrogenase synthesis to dark period and supported by ATP and reductant to be provided by breakdown of endogenous glucan. The light period ensures photosynthesis and replenishment of glucan, (Dixon et al., 2000). However, the ATP and reductant available during dark period in plastids may not suffice the requirements of nitrogen fixation process. Besides, there is no efficient system of O₂ removal for protection of nitrogenase. A proposed mechanism of removal of oxygen is that nitrogenase itself could help remove oxygen. The nitrogenase Fe-protein would be oxidised without loss of activity (Thornely and Ashlay, 1989). However, this leads to problem of cleavage of a major by product i.e., hydrogen peroxide which would need another set of enzymes to be put in system in chloroplasts such as ascorbate peroxidase, monodehydro abcorbate reductase and dehydroascorbate reductase. Photo-respiration has also been found to prevent oxygen damage to nitrogenase but this mechanism eats up much of reductant produced during photosynthesis by photosystem-I.

Ribbe et al. (1997) studied the N₂ fixation in Streptomyces thermoantotrophicus and observed that the nitrogenase was tolerant to superoxide produced from oxygen. The process involves molybednum dinitrogenase and a manganese superoxide oxidoreductase. Such a system if put in chloroplasts can allow expression of active nitrogenase under oxic conditions. However, this system has problems of dependence on superoxide stress. Even if such a conditional expression is corrected by disabling superoxide dismutase, it will again lead to the same problem of increased levels of hydrogen peroxide. Besides such a system is not practically efficient as it has only 10% of activity of conventional nitrogenases at 25°C.

Transformation of rice roots: There are two aspects of root transformation of rice plant. One of the possible cases is to allow expression of nif genes in plastids of non-photosynthetic cells of roots, thus overcoming problems of separating photosynthesis from nitrogen fixation and since the roots of flooded rice are usually under anaerobic conditions. Oxygen damage to the nitrogenase is taken care of. But there are few practical limitations of this approach.

Another aspect of root transformation is to enact mechanism for development of nodule like structure which provide a competition free habitat for endophytic diazotrophs. Even though a number of diaziotrophs have been found to be associated with rice. There are certain obvious dissimilarities between the rice-diazotroph association and the root-nodule symbiosis in legumes. Engineering rice roots will have to mainly focus on effecting more intimate association between rice and the rhizobia. This will mainly rely on manipulation of nod genes and nodulin genes. the factors in legumes which ensure rhizobial infection and colonization (Flavones) will have to be put in place in rice root system. Such a system of rice root transformation should ensure expression of unique traits in rice which improve their pre-disposition to rhizobia and consequently development of nodules. Various such factors such as flavonoids (Luteolin, Naringenin) and certain enzymes like apyrase have been identified which act as receptors for Nod signals and have been successfully expressed in rice. The genes governing such traits or factors can be candidate genes for introduction into rice preferably driven by root specific promoters to favorably engineer rice to develop nodule like structure and eventually the rice plant having its own mechanism of nitrogen fixation. This is indeed a long term endeavour. Dey et al. (2002) stated that rice does possess some if not all of such traits (Nodulins) found in legumes that are important for nodulation. If this hypothesis is correct then reconstituting the nod signal recognition in rice could allow this plant to interact more intimately with rhizobia. This line of thought gives more support to idea put forth by Mylona et al. (1995) and Carol et al. (1996) that legume plants acquired the ability to form symbiotic nitrogen fixing nodules by recruiting genes that have common functions in all plants. Now it is indeed a daunting challenge whether the genetic engineering can re-recruit the counterparts of these genes in rice to form functional nodules.

Benefits of BNF-rice: BNF-rice if developed can render following benefits to Agriculture especially in resource poor developing countries (Parakaran, 1997).

Saves energy: The process of nitrogen fixation only requires 16 ATP’s which is much less than the Haber-Bosch process used for chemical fixation of nitrogen. The Haber-Bosch process i.e

requires high pressure (100-200 atm) and high temperature (400-600°C). thus such high energy inputs can be saved if BNF rice is developed.

Saves money: It has been estimated that BNF rice can save almost 20 billion dollars annually (US agency for International development, 1994).

Saves environment: BNF rice is environment friendly technology. In chemical Nitrogen fixation carbon dioxide (CO₂) is released into environment. Carbon dioxide is a potential green house gas and causes global warming with severe implications on climatic patters of world. Similarly another toxic gas N₂O (Nitrous oxide) is released during denitrification bacteria (Galton and Smith, 1993).

Saves labour and soil: If rice is made to have in plant capacity to fix nitrogen for itself, a large amount of labour incurred in spreading fertilizer can be saved. Moreover, at the end of the season, BNF rice stubbles can be put into the soil to act as bio-fertilizers.

More productive: Engineering rice BNF system with either improved association with the rhizobia or by nif gene transfer has been found to be more productive Table 3 compares the potential and feasibility of various systems in rice aimed at nitrogen fixation (Papademerion, 2003).

Saves hunger: Rice feeds almost half of worlds populations. The increased pressure to increase rice production in view of ever increasing population means more requirement of nitrogen fertilizers. If BNF system can be put in place in rice, it will surely save millions of people from hunger given the declining energy resources of the world.

CONCLUSIONS

Since the inception of its idea, the development of BNF-rice not only poses a ardous challenge but a tremendous opportunity to bio-technologists and molecular biologists to realize this dream. Though most of the aspects of rice-diazotroph interaction and nitrogen fixation have been elucidated both at genetic as well as molecular level, the engineering of an autonomous nitrogen fixing rice plants is undoubtedly a long term endeavour. A large number of endophytic diazotrophs have been found to be associated with rice and factors encouraging bacterial colonization have been characterized but certain critical differences in rice-rhizobial interaction relative to root nodule symbiosis in legumes have to be worked out to make the rice-bacteria interaction so intimate that nodulation becomes a reality. The research towards developing a BNF rice has made remarkable progress in terms of elucidation of genetic and molecular mechanisms underlying this intricate biological process but the fact is that every newer insight takes us to newer complexities, hitherto unknown. The continued efforts in a collaborative framework will surely lead us to success provided the difficulties faced are overcome by consistent and problem oriented research. Some of the major problems identified in developing a BNF-rice include: large number of nif genes that need to be engineered into a rice plant, unpredictability of expression in a enkaryotic genetic background and lack of knowledge about differential oxygen concentrations in different plant tissues to identify most appropriate location for targeting nif genes. Thus as Dixon et al. (2000) stated, “In attempting to engineer nitrogen fixing plants we are taking a huge leap into unknown. Each step along the way may lead us to new difficulties or bring remarkable surprise”. The surprise may well be a working prototype of a BNF-rice which may be able to compliment if not totally replace the N₂-supply of rice plant.

REFERENCES