Critical Reviews in Plant Sciences, 26:65–103, 2007 Copyright c Taylor & Francis Group, LLC ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352680701252809 Advances in Transgenic Rice Biotechnology Hitesh Kathuria, Jitender Giri, Himani Tyagi, and Akhilesh K. Tyagi Interdisciplinary Center for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi–110021, India Referee: Dr. Z. T. Li, Mid-Florida Research & Education Center, University of Florida/IFAS, Apopka, FL 32703 Table of Contents I. INTRODUCTION ............................................................................................................................................. 67 II. TRANSFORMATION TECHNOLOGY ............................................................................................................ 67 A. Agrobacterium-mediated Gene Transfer ........................................................................................................ 67 B. Particle Bombardment .................................................................................................................................. 68 C. Other Methods ............................................................................................................................................ 68 III. RICE FUNCTIONAL GENOMICS ................................................................................................................... 68 IV. ANALYSIS OF REGULATORY ELEMENTS ................................................................................................... 69 V. ENHANCEMENT OF STRESS TOLERANCE ................................................................................................. 70 A. Biotic Stress Tolerance ................................................................................................................................. 70 1. Insect Resistance ............................................................................................................................... 70 2. Bacterial Disease Resistance ............................................................................................................... 82 3. Fungal Disease Resistance .................................................................................................................. 83 4. Viral Resistance ................................................................................................................................. 83 B. Abiotic Stress Tolerance ............................................................................................................................... 83 C. Herbicide Tolerance ..................................................................................................................................... 85 VI. IMPROVEMENT OF GRAIN QUALITY ......................................................................................................... 86 A. Nutritional Enhancement .............................................................................................................................. 86 B. Alteration of Starch Content ......................................................................................................................... 87 VII. YIELD IMPROVEMENT ................................................................................................................................. 87 VIII. OTHER APPLICATIONS ................................................................................................................................. 87 A. Control of Plant Development ....................................................................................................................... 87 B. Production of Novel Compounds .................................................................................................................. 89 C. Technological Innovations ............................................................................................................................ 89 IX. TRANSGENE SILENCING .............................................................................................................................. 89 X. ENVIRONMENTAL AND BIOSAFETY ASPECTS .......................................................................................... 90 XI. AGRIBUSINESS AND THE FUTURE OF TRANSGENIC RICE ..................................................................... 91 Address correspondence to Akhilesh K. Tyagi, Interdisciplinary Center for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India. E-mail: [email protected]65
Hitesh Kathuria, Jitender Giri, Himani Tyagi, and Akhilesh K. TyagiInterdisciplinary Center for Plant Genomics and Department of Plant Molecular Biology,University of Delhi South Campus, New Delhi–110021, India
Referee: Dr. Z. T. Li, Mid-Florida Research & Education Center, University of Florida/IFAS, Apopka, FL 32703
Table of Contents
I. INTRODUCTION .............................................................................................................................................67
II. TRANSFORMATION TECHNOLOGY ............................................................................................................67A. Agrobacterium-mediated Gene Transfer ........................................................................................................67B. Particle Bombardment ..................................................................................................................................68C. Other Methods ............................................................................................................................................68
III. RICE FUNCTIONAL GENOMICS ...................................................................................................................68
IV. ANALYSIS OF REGULATORY ELEMENTS ...................................................................................................69
V. ENHANCEMENT OF STRESS TOLERANCE .................................................................................................70A. Biotic Stress Tolerance .................................................................................................................................70
B. Abiotic Stress Tolerance ...............................................................................................................................83C. Herbicide Tolerance .....................................................................................................................................85
VI. IMPROVEMENT OF GRAIN QUALITY .........................................................................................................86A. Nutritional Enhancement ..............................................................................................................................86B. Alteration of Starch Content .........................................................................................................................87
VII. YIELD IMPROVEMENT .................................................................................................................................87
VIII. OTHER APPLICATIONS .................................................................................................................................87A. Control of Plant Development .......................................................................................................................87B. Production of Novel Compounds ..................................................................................................................89C. Technological Innovations ............................................................................................................................89
IX. TRANSGENE SILENCING ..............................................................................................................................89
X. ENVIRONMENTAL AND BIOSAFETY ASPECTS ..........................................................................................90
XI. AGRIBUSINESS AND THE FUTURE OF TRANSGENIC RICE .....................................................................91
Address correspondence to Akhilesh K. Tyagi, Interdisciplinary Center for Plant Genomics and Department of Plant Molecular Biology,University of Delhi South Campus, New Delhi 110021, India. E-mail: [email protected]
Rice is the most amenable crop plant for genetic manipulationamongst monocots due to its small genome size, enriched geneticmap, availability of entire genome sequence, and relative ease oftransformation. Improvement in agronomic traits of rice is boundto affect a sizeable population since it is a primary source of sus-tenance. Recent advances like use of ‘clean gene’ technology ormatrix attachment regions would help improve rice transforma-tion. Function of several novel genes and their promoters has beenanalyzed in transgenic rice. Significant progress has been made inintroducing traits like herbicide, biotic stress and abiotic stress tol-erance. Attempts also have been made to enhance nutritional char-acteristics of the grain and yield. Identification of genes controllinggrowth and development can be used to modify plant architectureand heading period. Transgenic rice can serve as a biofactory forthe production of molecules of pharmaceutical and industrial util-ity. The drive to apply transgenic rice for public good as well ascommercial gains has fueled research to an all time high. Success-ful field trials and biosafety of transgenic rice have been reported.This would act as a catalyst for greater acceptance of geneticallymodified food crops. The lessons learnt from rice can be extended toother cereals thereby opening new opportunities and possibilities.
I. INTRODUCTIONRice is one of the most important cereal crops, providing
staple food for nearly one-half of the global population. Morethan 90% of rice is grown and consumed in Asia where about55% of the world’s population lives, reflecting the value of ricein daily human life. Its importance can be estimated by the factthat the year 2004 was declared as International Year of Rice bythe United Nations Food and Agriculture Organization. In directproportion to the predicted rise in the world’s human population,rice consumption and demand will increase over the next severaldecades. As little new land is available to increase rice cultiva-tion, larger yields will be needed to meet the anticipated higherdemand. Rice is among the few food grain crops amenable togenetic transformation and has recently emerged as the modelcereal for the study of genome organization, gene expressionand function as well as the fate of transgenes (Christou, 1994;Tyagi et al., 1999, 2004; Giri and Laxmi, 2000; Upadhyayaet al., 2000; Tyagi and Mohanty, 2000; Datta et al., 2002; Bajajand Mohanty, 2005). The focus of this review is to trace the de-velopments in the field of transgenic rice biotechnology with aspecial emphasis on the analysis of gene/promoter activity andmanipulation of agronomically useful traits.
II. TRANSFORMATION TECHNOLOGYOver the years, several methods of genetic transformation of
rice have been developed. There is, however, an undercurrent offeeling that indica rice is recalcitrant to high efficiency transfor-mation and some genotypes could not be tamed by employingcommon protocols. This entails search for better transformationprotocols particularly for indica rice (Bajaj and Mohanty, 2005).
A. Agrobacterium-mediated Gene TransferRemarkable progress has been made in the development
of efficient systems for Agrobacterium tumefaciens-mediatedtransformation in rice. Raineri et al. (1990) attempted to raiseand regenerate transgenic calli after Agrobacterium-mediatedtransformation. Later, Chan et al. (1992, 1993) showed regen-eration of Agrobacterium-transformed calli from root explantsand immature embryos. Already about 40 different genotypesof indica, japonica and javanica rice have been transformedusing this approach (Shrawat and Lorz, 2006). Several factorswere found to have an impact on the efficiency of Agrobac-terium tumefaciens-mediated transformation, including Ti plas-mid type (Hiei et al., 1994; Cheng et al., 1998), bacterial strainswith broad host range (Hiei et al., 1994, 1997; Aldemita andHodges, 1996; Dong et al., 1996), culture conditions prior toand during inoculation (Aldemita and Hodges, 1996; Mohantyet al., 1999) and activation of T-DNA transfer process by ex-ogenously added acetosyringone (Aldemita and Hodges, 1996;Rashid et al., 1996; Khanna and Raina, 1999). Among vari-ous explants used, scutellum-derived calli are the material ofchoice for efficient transformation in rice (Hiei et al., 1994; Kantet al., 2001). Recently, Toki et al. (2006) reported the successfulregeneration of transgenic plants within a month after the start
of the aseptic culture of mature seeds by using scutellum fromone day precultured seeds as explants, thus avoiding the riskof somaclonal variations. Strong influence on the transforma-tion frequency is exerted by the plant genetic background inindica rice (Aldemita and Hodges, 1996; Mohanty et al., 1999)although the same is not apparent for three different japonicacultivars (Hiei et al., 1994). Using a high throughput method forAgrobacterium-mediated transformation, Terada et al. (2004)obtained 1,000 stable transformants from only 150 seed-derivedcalli in japonica rice. The genotypic influence is often overcomeby modifying the nutrient medium (Rachmawati et al., 2004; Linand Zhang, 2005) or transformation conditions, since the samenutrient medium is not ideal for all the varieties (Ge et al., 2006;Sridevi et al., 2005).
The vector backbone has significant effect on transformationprocess, as dual binary vector (Komari et al., 1996) and addi-tional vir genes (Vain et al., 2004) enhanced transformation effi-ciency. For the effective elimination of the Agrobacterium aftertransformation, Wang et al. (1997) inserted a castor bean CAT1intron into the selectable marker gene for hygromycin resistance,thereby, abolishing the expression of the gene in Agrobacteriumand rendering it susceptible to hygromycin. Several useful traitshave been introduced in rice by using Agrobacterium, includinggenes for abiotic stress tolerance, biotic stress tolerance, her-bicide tolerance, nutritional enhancement and enhanced photo-synthesis (see Sections V and VIII).
B. Particle BombardmentThe microprojectile bombardment or biolistics is also an
equally successful method (Christou et al., 1991; Cao et al.,1992) as it is considered genotype independent and less laborintensive. It has been associated sometimes with some risk due toarrangement of multiple copies of transgenes, particularly in theform of inverted repeats and problem of high copy number ofthe transgene, unlike Agrobacterium-mediated transformation(Hiei et al., 1994; Kumpatla and Hall, 1998; Dai et al., 2001).Despite all these constraints, L. Wu et al. (2002) have shownsuccessful integration, expression and segregation of multiplegenes in transgenic rice using biolistic method. The problem ofrearrangement also has been overcome up to large extent us-ing minimal linear cassette (including promoter, open readingframe and terminator only) to coat microcarriers. Such ‘cleangene’ technology would be of great importance in avoiding un-desirable effects of vector backbone and also provide an alter-native to Agrobacterium-based method (Fu et al., 2000a). Ricetransformed with five different minimal cassettes showed sim-ple integration pattern coupled with high and stable transgeneco-expression over generations (Agrawal et al., 2005). Using abiolistic approach, several transgenes have been introduced intorice calli where they express transiently or get stably integratedand inherited. This approach has been used for investigation onpromoter, stress tolerance, nutritional enhancement, gene ex-pression, plant development and grain yield (see Sections IV–VIII).
C. Other MethodsRice protoplasts can be transformed with naked DNA by
treatment with PEG in the presence of divalent cations such ascalcium. Toriyama et al. (1988) and Zhang and Wu (1988) werethe first to recover transgenic rice using PEG technology andthus it is the first technique used in transgenic rice production.Subsequently, this was followed by other workers and Dattaet al. (1990) were the first to recover fertile transgenic plantsfrom indica rice using PEG. Similarly, Zhang et al. (1988) re-ported the recovery of transgenic rice using electroporation andShimamoto et al. (1989) were the first to recover fertile trans-genic plants using electroporation in japonica rice. However,protoplasts are not easy to work with and regeneration of fer-tile plants is problematic. To improve transformation efficiency,Matsuoka et al. (2005) recently have developed a single-cell ma-nipulation supporting robot for high throughput microinjectionof rice protoplasts.
Besides the standard and widely used techniques as men-tioned above, certain claims of genetic transformation have beenmade using novel methods, based on pollen tube pathway (Luoand Wu, 1989), LASER (Guo et al., 1995), imbibition of em-bryo or seeds in the presence of DNA (Yoo and Jung, 1995)and WHISKERSTM (Matsushita et al., 1999). To avoid need oftissue culture and sterile conditions, in planta transformationmethod of Supartana et al. (2005) depends on a needle dippedin Agrobacterium culture to prick the seed’s embryonic portionthat subsequently grows into a plant and sets transgenic seeds.This may be a step forward in developing a highly efficientmethod comparable to floral-dip in Arabidopsis (Clough andBent, 1998).
III. RICE FUNCTIONAL GENOMICSFunctional genomics can be broadly defined as the area of
genome research devoted to identifying the functions of genes.Rice is a model plant for the discovery of gene function in ce-reals. The rice genome contains about 37,544 protein codinggenes and no known function has been inferred for about half ofthem (International Rice Genome Sequencing Project, 2005; Vijet al., 2006). One important aim of functional genomics stud-ies is to isolate agronomically important genes from rice and totest their efficacy in crop improvement. The highly successfulgenetic approaches to gene identification are based on the disrup-tion of a gene function leading to a scorable novel phenotype.Transposon mutagenesis and T-DNA insertions are importanttools to discover gene functions by reverse genetics strategiesinvolving transgenic plants. Reporter genes for β-glucuronidasefrom E. coli, green fluorescent protein from Aequorea victoria,luciferase from Photinus pyralis or luxA and luxB from lumines-cent bacteria Vibrio harveyi and LC, a member of the maize Rgene family of MYC-like transcription factors regulating antho-cyanin biosynthesis, have been used to gain information aboutexpression pattern of target genes in studies involving enhancer,gene or promoter traps (Springer, 2000). Although homologousrecombination is the best way to target or disrupt a gene func-tion, higher plants are considered recalcitrant to gene targeting,
but some success has been reported by expressing recombina-tion proteins and modified transformation vectors (Costaftis andGuiderdoni, 2005).
AC-DS transposons were first introduced by electroporationinto rice and shown to transpose (Izawa et al., 1991; Shimamotoet al., 1993). However, two component AC-DS system can beinactivated in later generations (Izawa et al., 1997). Suitabilityof maize autonomous transposable element AC through succes-sive generations has been studied for functional genomics inrice and it was demonstrated that AC can be effectively usedfor the functional analysis of the rice genome (Enoki et al.,1999; Kohli et al., 2001). A more advanced gene trapping sys-tem was reported by Chin et al. (1999) where they constructedAC and gene trap DS vectors and introduced them in rice usingAgrobacterium-mediated strategy. In the R1 generation, ∼80%of transgenic plants carrying AC showed the mobilization of trapDS thereby confirming that AC/DS-mediated gene trap systemis an efficient mechanism to analyze functions of genes in rice.Later, it was demonstrated that only 30,000 AC plants would berequired to cover knockout mutants in most genes in rice and canbe then used for rice functional genomics program (Greco et al.,2001). Recently, Kolesnik et al. (2004) have also reported theAC/DS gene trap system in rice. They analyzed 2057 DS flankingsequences to find that ∼80% of them are unique and 72% of theinsertions were found in gene rich regions. Although insertionswere interspersed throughout the genome, but the frequency wastwice as expected in chromosome 4 and 7. A hotspot region of40 kbp in chromosome 7 was also found. Their results againshowed the suitability of this system for large-scale transpo-son mutagenesis in rice. Recently, a novel transiently-expressedtransposase-mediated Dissociation (DS) insertional mutagene-sis system has been developed in rice by Upadhyaya et al. (2006)and efficacy of this system in targeted localized insertion muta-genesis in rice has also been shown.
T-DNA insertional mutagenesis is now frequently used in ricefor identifying new genes (Tyagi and Mohanty, 2000). Earlier,22,090 transgenic rice plants with the T-DNA integrated in theirgenome were generated by Jeon et al. (2000a) with the aimof identifying insertional mutants in various genes and for dis-covering new genes. A promoterless β-glucuronidase reportergene with an intron, multiple donor splice sites and acceptorsplice sites towards the right border of the T-DNA contained ina binary vector was used as a gene trap vector for rice transfor-mation. Organ-specific, tissue-specific, or ubiquitous expressionwas observed in 1.6–2.1% of the tested transgenic lines. T-DNAintegration patterns are different in Arabidopsis and rice as T-DNA integrates in almost all regions of Arabidopsis genome,but only in gene rich areas of rice genome. This difference inintegration pattern could possibly be attributed to the fact that inArabidopsis the gene distribution is more uniform as comparedto rice or due to the difference in the amount of repeated se-quences in their genomes (Barakat et al., 2000). Subsequently,preferential integration of the T-DNA tags in gene rich areas hasbeen reported (S. Chen et al., 2003). T-DNA integration pat-tern and efficacy of this system for functional analysis of rice
genome has been studied widely (Kim et al., 2003; Sallaud et al.,2003). Approximately, 200,000 T-DNA insertional lines are cur-rently available in rice carrying gus or GFP reporters for eithergene or enhancer trap (An et al., 2005). This tool of functionalgenomics has led to identification of some useful genes. In ad-dition, a binary vector GAL4-VP16-UAS transactivation systemhas been established in rice for the discovery of gene functions(Liang et al., 2006). Recently, Zhou et al. (2006) identified dwarfmutant glu from T-DNA tagged rice population. The encodedgene, OSGLU1, controlled the plant height, cellulose synthesisand cell elongation. Provided the known genome sequence andefficient transformation system, like in rice, T-DNA insertionalmutagenesis is the best tool to identify the function of new genes.
With the availability of complete genome sequence, largenumber of mutants, introgression libraries, micro-array data, ge-netic markers, and advanced transformation techniques, rice canbe used to identify genes for improved traits and as referencegenome for genomics studies in other cereals (Hirochika et al.,2004; Y. Xu et al., 2005; van Enckevort et al., 2005; Varshneyet al., 2006). The knowledge gained in rice would be helpful inidentifying the genes in other cereals due to synteny among theirgenomes. However, such comparison should be made on well-established co-linear genomic regions (Vij et al., 2006). Someof the recent examples of genes identified through insertionalmutagenesis include, ent-kaurene synthase involved in GA (gib-berellic acid) biosynthesis (Margis-Pinheiro et al., 2005), blastresistance gene, OSPib (H. K. Kim et al., 2005), a gene involvedin tapetum development, UDT (Jung et al., 2005), rice imma-ture pollen 1 gene (RIP1) involved in pollen development (Hanet al., 2006) and a voltage gated channel protein gene, OSCC1(Nakamura et al., 2006a). In other instances, novel alleles ofcertain genes have been identified by molecular mapping. Theirfunction can be confirmed by complementation of phenotype us-ing transgenic plants as reported in case of blast resistance gene,pi-ta (Jia et al., 2000), a gene responsible for plant regenerationin tissue culture, NIR (Nishimura et al., 2005), lateral branchinggene, OSTB1 (Takeda et al., 2003), fertility restorer gene, RF-1 (Komori et al., 2004), salt stress tolerance gene, SKC1 (Renet al., 2005), cytokinin oxidase gene, OSCKX2 (Ashikari et al.,2005), submergence stress tolerance gene, SUB1A (Xu et al.,2006) and seed shattering genes QSH1 and SH4 (Konishi et al.,2006; Li et al., 2006).
IV. ANALYSIS OF REGULATORY ELEMENTSStrong and constitutive promoters are beneficial for high level
expression of selectable marker genes, which is necessary forefficient selection and generation of transgenic plants. So far,CaMV35S, rice ACT1 and maize UBI1 gene promoters have beenused extensively to drive high and constitutive expression oftransgene in rice. In recent past, some novel gene promoters havebeen tested in rice for constitutive expression. Promoter of ricecytochrome c gene was highly active in almost every plant tissue.It was even more active than rice ACT1 gene promoter in leaves,root, embryo and calli (Jang et al., 2002). Alternatives of widelyused CaMV35S gene promoter have been provided in the form
of promoters from Cestrum yellow leaf curling virus (Stavoloneet al., 2003) and from Milk vetch dwarf virus MDV-C8, PMC8(Shirasawa-seo et al., 2005). Matrix attachment region have beenused to enhance transgene expression in rice (Vain et al., 2002).Some novel regulatory sequences also have been shown to per-form the same function. REB activator sequence when fusedwith GLB promoter enhanced its activity by 3.7-fold in trans-genic rice (Yang et al., 2001). Another such element is sugarresponse sequence from rice. The presence of SRS significantlyenhances the expression level of the ACT promoter in severalrice tissues at various developmental stages (Chen et al., 2002).
Constitutively active promoters are not always desirable forplant genetic engineering because overexpression of a trans-gene may compete for energy and building blocks for synthesisof proteins or RNA that are also required for plant growth undernormal conditions. Availability of inducible and tissue/stage-specific promoters provides a way to express the transgene veryspecifically in the target plant. Expression of genes for stress tol-erance could be engineered better by deploying stress-induciblepromoters or their elements. Several such promoters have beenstudied in rice (Table 1). Studies on dehydration stress regulatedtransgene expression by Su et al. (1998) have shown inducedexpression of gus gene when ABRC1 element was coupled withrice ACT promoter. Promoters of rice POXA and POXN werefound to be root-specific and their expression was also enhancedin the leaves on UV and wounding treatment (Ito et al., 2000).Wound inducibility has also been reported in promoter of MPIgene. Use of this promoter to drive the expression of cry1B intransgenic rice showed better performance (Breitler et al., 2001).Wound inducibility and tissue specificity of regulatory elementswas also seen in puroindoline gene promoter in transgenic rice(Evrard et al., 2007). Use of such promoters for transgene ex-pression in rice would provide dual benefits, i.e., inducibility andtissue specificity. Several promoters that are capable of confer-ring tissue or organ specificity have been studied in transgenicrice, i.e., endosperm-specific (Wu et al., 2000; Onodera et al.,2001), root/shoot-specific (Jang et al., 2002; Fukuzawa et al.,2004; Iwamato et al., 2004), seed-specific (Hwang et al., 2001),anther-specific (Luo et al., 2006) and early development stage-specific (Kusano et al., 2005).
The specific promoter activity is controlled by introns in thecase of rice α-tubulin gene family (Jeon et al., 2000b; Fiumeet al., 2004). Regulatory elements also were found in 3’UTR(Choi et al., 2000) and leader sequence (Locatelli et al., 2002).On analysis of regulatory regions of OSCDPK2 in transgenicrice, it was found that intron in leader sequence is necessary forgene expression and some regulatory elements also were foundin 3′ UTR (Morello et al., 2006). Promoter analysis of LTP1 intransgenic rice showed that it could direct gus expression onlyto vascular bundles of mature leaves (Guiderdoni et al., 2002).However, promoters of six different wheat LTP genes showeddiverse activity in transgenic rice (Boutrot et al., 2007). Simi-larly, a putative enhancer region was identified from rice UBQ2promoter when different deletions were analyzed in rice (Wangand Oard, 2003). In fact, transgenic rice also have been used to
characterize barley GIII promoter that showed the presence ofsalicylic acid responsive element (Y. F. Li et al., 2005), suggest-ing the efficacy of transgenic rice system in studying promoterregion of genes from other plants. Promoters from dicot plantsare functional in monocots, although with a lower strength, sug-gesting partial or selective conservation of signaling pathwaysbetween monocot and dicot. HEV gene promoter from rubberplant was found to be functional and to some extent induciblein rice by Pujade-Renaud et al. (2005). Thus, rice can serve asa good system to analyze the regulatory elements of genes fromdiverse taxa.
V. ENHANCEMENT OF STRESS TOLERANCEThe response of a plant to environmental stress is deter-
mined by many factors like genotype, developmental stage, du-ration, severity, periodicity as well as additive/synergistic effectsof multiple stresses. Stresses trigger a wide range of plant re-sponses, from altered gene expression and cellular metabolismto changes in growth rates and crop yields (Shinozaki andShinozaki, 1997; Bajaj and Mohanty, 2005). Failure to com-pensate for a severe stress can result in plant death. Transgenicapproach can help generate tolerance to several stresses influ-encing rice production (Table 2).
A. Biotic Stress Tolerance1. Insect Resistance
Insects attack all parts of the rice plant and not only exertphysiological damage to the plant, but also act as a vector forviral disease. The most important and widely distributed pestspecies are stem borers, leaf folders, plant hoppers and gallmidges (Maclean et al., 2002). Chemical insecticides providea simple way to control insect infestation, but use of agrochem-icals without effective biosafety rules may lead to both environ-mental and health problems (Bajaj and Mohanty, 2005). Thus,genetic engineering would provide an effective and safe way todevelop insect resistance in rice.
Baculoviral insecticides are an effective means of control-ling predating insect populations. Hukuhara et al. (1999) havereported increasing the insect’s virus susceptibility thereby re-ducing the amount of virus needed for successful application.By introducing the gene for virus-enhancing factor in rice, theeffectiveness of baculoviral insecticides against feeding army-worm larvae was enhanced. But, this method has certain practi-cal limitations like the slow mode of action of the baculovirus,permitting the insect to do considerable damage, and narrowspectrum of virus host specificity. Further, manual harvestingand manipulation push the production costs even higher.
Another strategy for insect resistance is the use of plant pro-teinase inhibitor. Earlier, Irie et al. (1996) generated transgenicrice resistant to insect storage pests using hydrolase inhibitors.Transgenic plants overexpressing CC in rice seeds inhibitedSitophilus zeamais gut proteases. Transgenic rice plants showingenhanced levels of resistance to several crop pests were reportedsimultaneously by using genes for CPTI and potato PINII (Duanet al., 1996; Xu et al., 1996b). SKTI was expressed in transgenic
Electroporation pH3-gus-nost Shoot, root and meristematiccells of young leaves showcell-division dependentactivity, whereas matureembryos, anther cell wall,pistil and coleoptile showcell-division independentactivity
Taipei 309 Bombardment p�LTP2-gus-t Pathogen inducible activity Guiderdoni et al.(2002)
Notohikari Ag pOSEM-gus-nost 55 bp OsEM promoterfragment essential forexpression in shoot, radicle,vascular tissues andepidermal layers of scutellumand epiblast in embryo
Miyoshi et al. (2002)
Taipei 309 Ag pOSKN2-eGFP-gus-nost,pOsKN3-eGFP-gus-nost
Promoter mediates the initialdownregulation of geneexpression during lateralorgan formation
Postma-Haarsma et al.(2002)
- Ag pCMP-gus-nost Constitutive gus expression Stavolone et al. (2003)Taipei 309 Bombardment p�UBI2-gus-nost Higher gus expression than the
35S promoterWang and Oard (2003)
Zhonghua 11 Ag pOSBP73-gus-nost Expression localized to root tip,stem node, panicle, immatureseed
J. Chen et al. (2003)
Nipponbare Ag pMT-gus-nost Root and shoot specificexpression but no gus activitywas seen in rice endosperm
Both 5′ and 3′UTR werefound to contain regulatoryelements
Morello et al. (2006)
Taipei 309 Ag pRTS-barnase-t Anther specific expression Luo et al. (2006)Zhongzuo321 Ag p�PINA-gus-nost Wound inducible expression Evrard et al. (2006)Nipponbare Ag pTALTP-gus-nost Diverse promoter activity Boutrot et al. (2006)
rice to confer resistance against rice brown plant hopper (Leeet al., 1999). Recently, Alfonso-Rubi et al. (2003) generatedtransgenic rice plants overexpressing the barley ITRI gene.Transgenic rice showed higher tolerance against the coleopteranrice weevil, S. oryzae, and survival of the rice weevil was de-creased by 28%. Using an alternative approach, Qiu et al. (2001)transformed rice with spider gene, SPI. Most of the transgenicplants had high toxicity to striped stem borer and leaf folder,two major rice insects. The mortality rate ranged from 38% to72% within seven days after infestation.
Lectins are sugar binding proteins that exhibit antimetaboliceffects against insects and are lethal for sap sucking (hemipteran)insects against which Bacillus thuringiensis endotoxins areineffective. These proteins, therefore, provide another strategyfor insect resistance in rice. Previously, rice transformed withGNA had shown resistance to rice brown plant hopper (Sudhakaret al., 1998; Rao et al., 1998). Recently, Sun et al. (2002) alsohave shown enhanced resistance in transgenic rice carryingGNA gene. Therefore, lectins, like GNA, provide an alternative
way to control the small brown plant hopper. The expressionof GNA in rice also conferred resistance against green leafhopper (Nagadhara et al., 2003) and whitebacked plant hopper(Nagadhara et al., 2004). Saha et al. (2006) used another lectingene ASAL to enhance tolerance toward brown plant hopper,and green leaf hopper in transgenic rice. Combination of lectinswith other insecticidal proteins extends the resistance to thevariety of insects. Overexpression of cry and GNA impartedresistance against yellow stem borer, brown plant hopper, greenleaf hopper, and the whitebacked plant hopper (Ramesh et al.,2004) and GNA with SBTI in transgenic rice showed varyingdegrees of resistance against rice leaf folder, Cnaphalocrocismedainalis and brown plant hopper, Nilaparvata lugens (G.Li et al., 2005). Use of another glycoprotein, avidin, hasprovided yet another approach towards stored rice insect pestmanagement (Yoza et al., 2005).
Insecticidal protein encoding cry genes of Bacillusthuringiensis have been exploited for the production of insect-resistant transgenic crops. Fujimoto et al. (1993) were the first to
report insect-resistant rice generated by introduction of modifiedendotoxin gene cry1Ab from Bacillus thuringiensis. Subse-quently, transformation of several rice varieties with variantsof cry has been reported (Tyagi et al., 1999; Table 2). Bringingtrangenic rice, expressing cry gene, into cultivation appears to bea viable option, especially after the success of maize and cottonwith cry genes, to reduce the yield loss caused by lepidopteranpests as this group is responsible for a loss of up to 2–10%of Asia’s annual rice yield (High et al., 2004). Novel codon-optimized cry1Ba showed higher resistance than cry1Aa andcry1Ac towards striped stem borer larvae population and couldconfer complete protection to the plants from the striped stemborer (SSB, Chilo suppressalis) larval attack (Breitler et al.,2000). Highly enhanced pest resistance has been obtained byShu et al. (2000) using synthetic cry1Ab that showed varyingdegrees of broad spectrum resistance against eight lepidopteranpests, both in laboratory and field conditions. Transgenic plantsaccumulated high amounts of cry1Ab protein (1% of total plantprotein) and showed some variation in terms of seedling growthand yield (Shu et al., 2002). Wound-inducible expression ofcry1B conferred high level of protection against striped stemborer, without affecting plant growth and yield (Breitler et al.,2001). Interestingly, cry1Aa transgenic lines were used to de-velop hybrid resistant lines (Wang et al., 2002), thus providingefficient SSB resistance donor lines in rice breeding programs.Khanna and Raina (2002) transformed three elite varieties ofindica rice with a chimeric cry1Ac gene and insect bioassayshave shown obvious toxic effects towards yellow stem borer.Protection against yellow stem borer or striped stem borer isalso evident in some other reports (Husnain et al., 2002; Marfaet al., 2002). On comparison, cry1Ac was more effective towardslepidopteran insects and cry2A was found more effective againstrice leaf folder (Chen et al., 2005). Gene pyramiding of cry1Ac,cry2A and GNA in transgenic rice was more effective against avariety of insects than single gene (Maqbool et al., 2001; Locet al., 2002). A novel approach has been adopted by Mehlo et al.(2005) using fusion protein comprising the N-terminal of cry1Actoxin and a C-terminal of the nontoxic RB. Fusion protein over-expressing plants were more toxic to a larger variety of the insectpests than cry1Ac-expressing plants alone. Their studies revealthat the use of such hybrid toxins is an alternative strategy foreffectively managing insect pests using transgenic rice plants.
A large number of transgenic rice lines have been produced inthe past, but only very few have been field tested. Successful fieldperformance of rice expressing fusion gene derived from cry1Aband cry1Ac (Tu et al., 2000), cry1Ab (Wu G et al., 2002; Ye et al.,2001; 2003), cry1Ac and cry2A (Bashir et al., 2004) and cry1Band cry1Aa (Breitler et al., 2004b) have been reported. However,variation in gene expression during field trials has shown somelimitation of transgenic rice with cry genes which needs to beovercome by further investigation like target specific expression.It has been argued that insect resistant transgenic plants couldbe a sustainable approach when combined with novel and usefulstrategies (Ferry et al., 2006).
(Xoo), along with blast and sheath blight are most importantdiseases of rice and have a worldwide distribution. Bacterialblight and blast have been controlled successfully by resistantvarieties, but due to natural variants and evolution of resistance-breaking strains of these pathogens, continuing release of newresistant varieties is a necessity. A total of 30 endogenous genes,conferring host resistance against different Xoo strains havebeen identified and named as Xa1 to Xa29 (Xiang et al., 2006).Transgenic approach using these or other genes provides a rea-sonable alternative for genetic enhancement towards bacterialresistance.
The most promising endogenous rice gene for bacterial blightresistance identified so far is Xa21 (Song et al., 1995), whichconferred complete protection against bacterial blight. This genehas been transferred into other varieties with desirable unifor-mity of expression in field tests (Tu et al., 1998; Zhai et al., 2004).Another endogenous gene, Xa26, encoding receptor kinase-likeprotein (same as Xa21) has also conferred bacterial blight resis-tance (Sun et al., 2004). In addition to these, a novel approachexploited by Sharma et al. (2000) to confer bacterial leaf blightresistance in rice was to overexpress cecropin B peptide. Ce-cropins are antibacterial peptides having broad spectrum bacte-rial lytic activity against gram negative and gram positive bac-teria, but not against eukaryotic cells. Transgenic plants showedtolerance towards bacterial leaf blight, however, low expressersdid not perform as well as the high expressers. Targeting of pep-tidase sensitive peptides like cecropin to the intracellular spacesby means of signal peptides protects the peptide from degra-dation. Overexpression of OSWRKY71 transcription factor inrice also resulted in enhanced resistance to virulent bacterialpathogens Xanthomonas oryzae pv. Oryzae (Xoo) 13751 sug-gesting its role in rice defense signaling pathways (X. Liu et al.,2006).
Genes from other sources also have been transferred to ricein order to generate bacterial disease resistance, e.g., ferredoxingene from sweet pepper (Tang et al., 2001) and ASTH1 from oat(Iwai et al., 2002). Arabidopsis NPR1 gene also could enhancethe resistance to bacterial blight in transgenic rice. Some con-served proteins interacted with NPR1 in rice, thus suggesting theconservation of defense pathways between rice and Arabidopsis(Chern et al., 2001; 2005). A maize HM1 homologue in rice,OSYK1, could confer resistance against bacterial brown stripe(Hayashi et al., 2005).
OSRAC1 from rice is involved in pathogen resistance againstboth bacterial blight and rice blast disease and alters the expres-sion of D9 gene, a defense-related gene isolated from lesion-mimic mutants of rice resistant to rice blast (Ono et al., 2001).OSRAC1 is also involved in lignin biosynthesis, an importantpolymer in plant defense (Kawasaki et al., 2006). Using a trans-genic approach, Jung et al. (2006) found that OSRACB is a nega-tive regulator of disease resistance in rice. Detailed investigationon these small GTPase genes will certainly help in understanding
disease response in rice and use of a common approach againstbacterial and fungal disease.
3. Fungal Disease ResistanceFungal disease resistance genes have been identified from
rice. Most promising endogenous resistance (R) gene, Pi-ta (cy-toplasmic NBS receptor), is responsible for resistance to fungaldiseases. Substitution of alanine for serine at position 918 wasresponsible for susceptibility to Magnoporthe grisea, causal or-ganism of rice blast disease. Overexpression of dominant Pi-ta allele in a susceptible variety could confer complete resis-tance against blast disease (Bryan et al., 2000). Recently, Quet al. (2006) identified a cluster of six tandemly repeated dis-ease resistance–like genes from rice, of which one (Nbs2-Pi9)encodes a transcription factor and showed broad spectrum resis-tance against different Magnoporthe grisea isolates in transgenicrice. Another gene, OSDR8, is involved in thiamine biosynthe-sis and acts upstream in defense signal transduction pathway(G. Wang et al., 2006). Pathogenesis-related (PR) genes havebeen used widely to address fungal tolerance in plants. Lin et al.(1995) raised transgenic rice expressing ChI11, which showedonly partial protection against sheath blight disease. However,significant resistance against Rhizoctonia solani has been re-ported using the same gene (Sridevi et al., 2003). Another chiti-nase gene (RC7) could confer tolerance against R. solani (causalorganism of rice sheath blight disease) in transgenic rice (Dattaet al., 2001). A gene pyramiding approach used by Datta et al.(2002) involved Xa21, for resistance to bacterial blight, cry genesfor insect resistance and RC7 for sheath blight resistance. A highdegree of tolerance was achieved in transgenic lines against Rhi-zoctonia solani. Rice GNS1 (PR-2) gene has also been used todevelop resistance against Magnoporthe grisea. Transgenic riceshowed enhanced expression of two PR genes, PR-1 and PBZ-1,and resistant-type lesions and stunted plant growth (Nishizawaet al., 2003). However, overexpression of RIR1b gene (a mem-ber of the defense-related gene family WER1) could confer en-hanced tolerance against Magnoporthe grisea without affectingplant growth (Schaffrath et al., 2000). Another endogenous gene,OSYK1, maize HM1 homologue in rice, also confers toleranceagainst Magnoporthe grisea in transgenic rice (Uchimiya et al.,2002). Using a transgenic approach, need of endogenous sali-cylic acid for blast resistance has also been demonstrated (Yanget al., 2004). Recently, dominant resistance gene Pid2 has beenidentified from tolerant rice variety Digu (X.W. Chen et al.,2004). Later this gene was found to be encoding a receptor-likekinase protein and a single amino acid substitution at position441 in transmembrane domain was responsible for disease tol-erance. Overexpression of this gene in transgenic rice couldconfer blast resistance even in a susceptible variety (X. Chenet al., 2006), suggesting importance of this gene in plant breed-ing programs. Several other genes encoding antifungal proteinshave been introduced in rice for fungal resistance (Table 2), withvariable degrees of success and some are effective against bacte-rial as well as fungal disease, e.g., cecropins (Coca et al., 2006).
4. Viral ResistanceInsect-vectored viral diseases cause considerable damage
to the rice plant and drastically reduce the yield of the plant.Hayakawa et al. (1992) raised transgenic rice against rice stripevirus to demonstrate for the first time that CP-mediated resis-tance to virus infection can be extended to cereals and to theviruses transmitted by an insect (plant hopper) vector. To gen-erate virus-resistant transgenic rice, japonica rice was trans-formed with a hammerhead ribozyme, a catalytic molecule withsequence-specific RNA cleavage activity (Han et al., 2000). Theexpression of ribozyme confers resistance to infection by ricedwarf virus. However, silencing of gene had an effect on thedegree of viral resistance in some lines. Rice is also a host toRRSV. Resistance against this virus has been introduced in riceby expressing 39 kDa spike protein of RRSV (Chaogang et al.,2003). Lentini et al. (2003) have reported transformation of sus-ceptible rice cultivar with RHBV nucleocapsid protein gene andmoderate to complete resistance was seen in transgenic lines.RHBV is the causative virus of a major rice viral disease thatcan cause significant damage (up to 50% loss) of the total yield.
Another severe viral disease that affects rice productivity isthe rice tungro disease. The infection is caused by two viruses,RTBV and RTSV. Sivamani et al. (1999) transformed ricewith RTSV coat protein genes, CP1, CP2, and CP3, individ-ually or together, and showed that transgenic plants providedmoderate but significant protection against RTSV. Their studydemonstrated pathogen-derived resistance to infection by RTSV.Replicase-mediated resistance (REP-MR) strategy adopted byHuet et al. (1999) involved the expression of RTSV replicasecoding sequence in sense or antisense orientation. Antisenseplants showed moderate resistance while sense constructs con-fer near immunity (100% resistant plants) to RTSV infection.Therefore, these two approaches, with coat protein or replicase,can be used to achieve viral resistance and such plants can pos-sibly be used for assessing their impact on tungro disease in thefield. Alternatively, host factors involved in RTBV replicationcan be evaluated as possible candidate to confer resistance totungro virus (Dai et al., 2004).
RYMV infection is a serious cause of concern in rice-growingareas of Africa as it is a major reason for economic loss. Resis-tance against RYMV has been achieved by expressing its coatprotein gene in rice (Kouassi et al., 2006). In another study,mutations in the eukaryotic translation initiation factor 4G pro-vided resistance against the RYMV virus in transgenic rice(Albar et al., 2006). Ongoing investigations show that significantprogress could be made towards engineering virus resistancetrait in rice. However, field trials of these lines would provide arealistic assessment to open the way for their commercialization.
B. Abiotic Stress ToleranceIn agricultural systems, abiotic stresses are responsible for
most of the reduction that differentiates yield potential fromharvestable yield (Mittler, 2006). There is a range of external
factors that adversely affects the growth and development of cropplants. These include high temperature, chilling, freezing, water-deficit (drought and salinity), high light intensity, flooding, andexposure to ozone and heavy metals. Most abiotic stresses di-rectly or indirectly lead to the production of free radicals andreactive oxygen species, creating oxidative stress (Mittler et al.,2004). Thus, it is imperative to develop stress-tolerant varieties.Furthermore, with extension of crop cultivation to environmentswhich are not optimal for the growth of crop plants, developmentof stress-tolerant plants is becoming increasingly important.
Transgenic approaches offer new opportunities to improvetolerance to abiotic stresses (Tyagi and Mohanty, 2000; Sahiet al., 2006; Bohnert et al., 2006). Large numbers of reportsare available on engineering abiotic stress tolerance in rice, es-sentially using compounds like compatible solutes, membranetransporters and regulators of signal transduction or transcrip-tion (Table 2). Overproduction of various compatible soluteshas been tested in rice, e.g., glycine betaine (Sakamoto et al.,1998; Mohanty et al., 2002; Sawahel, 2003; Su et al., 2006), tre-halose (Garg et al., 2002; I. C. Jang et al., 2003a), proline (Huret al., 2004; Su and Wu, 2004) and polyamines (Noury et al.,2000; Thu-Hang et al., 2002; Roy and Wu, 2001; Capell et al.,2004), to achieve significant drought, cold, and salt tolerance.Late embryogenesis proteins (LEA) are hydrophilic proteins firstreported from barely. Overexpression of LEA encoding genesfrom barley or wheat in rice could enhance drought tolerance(Xu et al., 1996a; Rohila et al., 2002; Cheng et al., 2002). Trans-genic plants also performed better than wild type plants underprolonged water deficit conditions (Babu et al., 2004).
Like compatible solutes, use of membrane transporters holdsgood promise (Yoshida, 2002). Water channel proteins, aquapor-ins, are members of the major intrinsic protein family which reg-ulate the passive movement of water across membranes. Aqua-porins might have a possible role in providing drought toler-ance to plants as they affect the root water uptake. The overex-pression of RWC3 confers drought tolerance in transgenic rice(Lian et al., 2004). Another promising strategy against salinitystress is the use of Na+ transporters, which transport cytosolicNa+ to vacuole and, thus protect cellular machinery. Na+/H+
antiporters from Artiplex or E.coli confer high salt tolerancein transgenic rice (Ohta et al., 2002). Overexpression of riceNa+/H+ antiporter gene could transport the sodium to vacuoleand provide increased salt tolerance (Fukuda et al., 2004). Thesame antiporter gene from halophyte Suaeda salsa further in-creased salinity tolerance in rice (Zhao et al., 2006). Recentlycloned QTL, SKT1, from rice salt-tolerant variety Nona Bokraalso provides a potential tool for improving salt tolerance notonly in rice but also in other crops (Ren et al., 2005).
Genetic engineering for oxidative stress tolerance provides anattractive strategy to achieve tolerance for multiple stresses. En-hanced production of glutathione synthase, an enzyme involvedin ROS metabolism, could enhance the salt tolerance in trans-genic rice (Hoshida et al., 2000). Similarly, drought tolerance oftransgenic rice was improved by overexpressing superoxide dis-
mutase from pea (Wang et al., 2005). Transgenic plants express-ing protoporphyrinogen oxidase also experienced lesser oxida-tive stress than control plants (Jung and Back, 2005). A noveloxidative stress responsive cis-acting element, CORE (coordi-nate regulatory element for antioxidant defense), was identifiedin well-known stress-associated protein gene, OSISAP1, alongwith many other genes encoding proteins of unknown functions(Mukhopadhyay et al., 2004; Tsukamoto et al., 2005; Vij andTyagi, 2006), further giving a hope for improvement of oxidativestress tolerance in rice.
Both biotic and abiotic stresses lead to increase in cytoso-lic concentration of Ca2+ and subsequent Ca2+ signaling acti-vates downstream defense pathways (Knight and Knight, 2001).One of the best studied groups of protein kinases involved inCa2+ signaling under stress is CDPKs (calcium dependent pro-tein kinases). Genes encoding these CDPKs (OSCDPK7, Saijoet al., 2000; OSCDPK13, Abbasi et al., 2004) have been iden-tified from rice and their overexpression in rice improved salt,drought, and cold tolerance. Another calcium dependent protein,calcineurin A, conferred salt stress tolerance in rice, probablyvia enhanced expression of LEA protein genes in transgenicrice (Ma et al., 2005). Overexpression of a MAP kinase gene,OSMAPK5, improved drought, cold, and salt tolerance of rice(Xiong and Yang, 2003). Recently, several genes involved intranscription and translation processes were salt stress respon-sive (Sahi et al., 2006). These genes along with other novelstress-related genes provide the source to engineer stress toler-ance in rice (Cooper et al., 2003; Mukhopadhyay et al., 2004;Vannini et al., 2004).
One major breakthrough in stress biotechnology in the re-cent past was the identification of DREB/CBF regulatory genesfrom Arabidopsis and other plants including rice. Dubouzet et al.(2003) have isolated five DREB genes in rice and these geneswere cold, dehydration, and salt responsive. DREB/CBF fromother plants, e.g., wheat (Shen et al., 2003) and Arabidopsis (S.C. Lee et al., 2004), were introduced in rice. The level of stresstolerance achieved was not as good as in their native plants andtransgenic plants showed growth retardation under normal con-ditions, which may be due to fine tuning of cross talk with het-erologous signal pathways (Oh et al., 2005). Interestingly, con-stitutive overexpression of Arabidopsis DREB1 and ABF3 genein rice could improve abiotic stress tolerance without affect-ing plant growth (Oh et al., 2005). Using the genes for DREB1from rice, OSDREB1A and OSDREB1B, and from Arabidopsisthaliana, DREB1A, DREB1B and DREB1C, Ito et al. (2006)have generated several transgenic rice lines. The transgene ex-pressed under the constitutive UBI promoter showed increasedtolerance to salt, low temperature, and drought conditions, buthad retarded growth under normal conditions. However, stressinducible expression recovered normal vegetative growth, sug-gesting requirement of finely tuned expression of these genes.Further studies are needed to obtain maximum output from sucha sensitive approach. However, constitutive overexpression ofa plant-specific NAC transcription factor gene, SNAC1, in rice
could enhance drought and salt stress tolerance. Interestingly,improved drought tolerance was extended to field conditions, astransgenic plants showed 23–34% higher seed production thancontrol plants under severe drought (Hu et al., 2006).
Damage due to low temperature stress has two importantcomponents, i.e., the temperature and the duration of stress. Coldstress can reduce flower induction, pollen production and germi-nation, and can cause male sterility in sensitive species like rice.The chilling resistance of higher plants is closely correlated withthe levels of unsaturation of fatty acids in phosphatidylglycerol(PG) from chloroplast membranes (Nishida and Murata, 1996).Genetic manipulation of the level of unsaturation of fatty acids inPG has successfully altered the chilling tolerance in rice (Yokoiet al., 1998; Ariizumi et al., 2002; Takesawa et al., 2002).
Heat stress in plants, especially in cultivated agriculturalcrops, is a complicated phenomenon since it often occurs si-multaneously with drought stress and is detrimental to both thevegetative and reproductive stages of rice (Satake and Yoshida,1978). Heat shock proteins are the best candidates for intro-ducing the heat tolerance trait through transgenic approach, asseen in yeast and Arabidopsis (Queitsch et al., 2000). Such pro-teins have also been identified from rice and overexpressionof heat stress responsive factor, SPL7, in mutant backgroundshowed high temperature tolerance as revealed by suppression ofhigh temperature–induced leaf spots development (Yamanouchiet al., 2002). Arabidopsis HSP101 gene when transferred torice could enhance the high temperature tolerance in transgenicplants (Agarwal et al., 2003). Another heat shock protein usedto provide tolerance to high temperature and UV exposure issHSP17.7 (Murakami et al., 2004). A good correlation wasseen between the levels of sHSP17.7 expression and the levelof thermotolerance in transgenic plants. Few reports are avail-able on genetic engineering for heat stress tolerance in rice.However, several heat stress–associated proteins or heat shockfactors (HSF) have been identified in rice, e.g., HSP90 (D. Liuet al., 2006), HSP26 (B. H. Lee et al., 2000), HSF6, HSF12(J. G. Liu et al., 2005). These genes could be potential candi-dates for further improvement of thermotolerance in rice, usingendogenous genes.
Submergence stress is another major constraint to rice culti-vation especially in South East Asia, since most of the cultivarsdie within a week of complete submergence (Xu et al., 2006).The first report on genetic improvement of rice for submergencetolerance was by Quimio et al. (2000), where overexpression ofPDC1 enhanced submergence tolerance in rice. Since this traitis a complex trait, major QTL responsible for submergence tol-erance has been identified on chromosome 9 (Toojinda et al.,2003; Siangliw et al., 2003). This locus, SUB1, comprises threeethylene responsive-factor–like genes, SUB1A, SUB1B, SUB1C.Overexpression of SUB1A in intolerant japonica rice could con-fer submergence tolerance by downregulating SUB1C and up-regulating ADH1, suggesting the key role of SUB1A (Xu et al.,2006). SUB1A reduced the ethylene production and GA respon-siveness, thus causing quiescence of plant growth, an adaptive
strategy for stress tolerance (Fukao et al., 2006). Introduction ofthis gene in other rice varieties would certainly extend the ricecultivation in flood prone areas.
Bioavailability of nutrients to the plant can become a seri-ous form of abiotic stress if soil conditions are unfavorable fortheir uptake despite the abundant presence of these mineral nu-trients. High soil pH is one such widely prevalent problem thataffects iron availability. One strategy to cope with this prob-lem, as demonstrated by Higuchi et al. (2001), is to overexpressthe nicotianamine synthase gene, whose product trimerizes S-adenosylmethionine to form one molecule of nicotianamine, anintermediate in the biosynthetic pathway of the mugineic acidfamily of phytosiderophores. Phytosiderophores are natural ironchelators that graminaceous plants secrete from their roots to sol-ubilize iron in the soil. Takahashi et al. (2001) have engineeredrice with a barley genomic DNA fragment having two NAATgenes, NAAT-A and NAAT-B, encoding for enzymes involvedin the biosynthesis of phytosiderophores, to release increasedlevels of deoxymugineic acid, an iron-solubilizing chelator. Inalkaline soils with low iron availability, high expressers had ahigher leaf chlorophyll content, better height and overall growthin terms of shoot dry weight per pot. Grain yield was also morethan four times that of untransformed controls. In addition toiron, phosphorus absorption also can be a limitation in pH im-balanced soils leading to Pi starvation conditions. Yi et al. (2005)found that OSPTF1 provided tolerance to phosphate starvationin transgenic rice plants. Under Pi limiting conditions, trans-genic plants had a higher tiller number and biomass yield incomparison to wild type plants implying that overexpression ofOSPTF1 had enhanced tolerance to Pi deficiency. Such effortswould be of great importance towards rice cultivation in nutrientpoor soil.
C. Herbicide ToleranceOne of the first genetically modified agronomic traits to be
tested in the field and used commercially was for herbicidetolerance. There has been a rapid development in our under-standing and production of several herbicide resistance trans-genic plants with different target genes available from varioussources, ranging from bacteria to humans (Mullineaux, 1992).In rice, bar was the first gene tested for herbicide (Basta, glufos-inate) tolerance (Christou et al., 1991; Datta et al., 1992; Oardet al., 1996). Two gene families that play major roles in confer-ring tolerance to herbicides are P450 monooxygenase and glu-tathione S-transferase (Ohkawa et al., 1999). Cytochrome P450monooxygenase, a drug-metabolizing enzyme system in plantsand animals, has been widely used to address herbicide toler-ance trait in rice. Inui et al. (2001) used this approach to generatetransgenic rice plants expressing human CYP2C9 and CYP2C19.The transgenic plants showed tolerance to sulfonylurea herbi-cide chlorsulfuron and also showed cross-tolerance to severalother herbicides with differing modes of action like mefenacet(oxyacetamide class), metolachlor (chloroacetoanilide class),
norflurazon (pyridazinone class), and pyributicarb (thiocarba-mate class) in the germination tests performed. A similar strat-egy was used by Kawahigashi et al. (2005a, 2006) to co-express human CYP1A1, CYP2B6 or CYP2C19 in rice. En-hanced herbicide cross-tolerance was due to the additive effectof introduced P450 genes. Transgenic plants carrying humanCYP2B6 gene showed faster degradation of herbicide, meto-lachlor (Ohkawa and Ohkawa, 2002). In addition to human cy-tochrome, Ohkawa’s group also generated Nipponbare trans-genic plants expressing two atypical cytochrome P450, i.e.,CYP2B22 and CYP2C49, from pig (Kawahigashi et al., 2005b).Upon being challenged by varying concentrations of herbicidesat the germination stage, the transgenic rice plants metabolizedthe substrate herbicides more rapidly than nontransgenic controlplants. These transgenic plants can be used in breeding programsaimed at generating herbicide-tolerant crops.
The role of rice glutathione S-transferases in providing her-bicide tolerance has been studied by Deng et al. (2003) usingsilenced GST III subunit in rice suspension culture cells andcalli. GSTs are known to conjugate the herbicide pretilachlorwith glutathione, a reaction induced by safener fenclorim. GSTactivity towards the substrates cinnamic acid, CDNB and preti-lachlor was reduced by as much as 77% in the transgenic plantsharboring the antisense gene as compared to the wild-type. Thiswas consistent with the reduction of accumulation of OSGST IIImRNA in the transformed rice cells. The study highlights theimportance of GSTs in protecting the plants against herbicidestress. Apart from these two approaches, protox gene from Bacil-lus subtilis, known to impart resistance to diphenyl ether herbi-cide oxyfluorfen, also confers herbicide tolerance in transgenicrice (H. J. Lee et al., 2000). Many herbicides inhibit the action ofprotox and resulted accumulation of Proto IX causes oxidativestress. Transgenic rice overexpressing ms-protox in both chloro-plast and mitochondria has been generated (Jung et al., 2004;Jung and Back, 2005). Plants also showed oxidative stress toler-ance probably by limiting the production of ROS. This work ontransgenic technology for herbicide tolerance could be used ingenetic enhancement programmes to transfer this trait to othercereal crops.
VI. IMPROVEMENT OF GRAIN QUALITY
A. Nutritional EnhancementRice is a staple component of the diet for about 3.8 billion
people and its nutritional enhancement would have a huge im-pact over the world population. It is estimated that improved vi-tamin A nutrition could prevent 1 to 2 million childhood deathsper year, since milled rice contains no β-carotene (provitaminA). As a first step in this direction, Burkhardt et al. (1997) trans-formed rice with daffodil phytoene synthase gene. Subsequently,Ye et al. (2000) engineered entire pathways for provitamin Abiosynthesis in the rice endosperm using phytoene synthase(PSY) and lycopene β-cyclase (LCY) genes from daffodil andphytoene desaturase (crt1) from bacterium, Erwinia uredovora.
Thus ‘Golden Rice’ with provitamin A was generated. Later,the entire pathway for β-carotene biosynthesis was reconsti-tuted by using just two structural genes, PSY and crt1. Thus,using only these two genes, the entire provitamin A biosyn-thetic pathway had been activated in other rice varieties (Hoaet al., 2003; Datta et al., 2003). To avoid antibiotic resistancefor selection, mannose-based selection was used (Lucca et al.,2001b). However, requirements for the amount of carotenoidsformed in golden rice has been debated (Zimmerman and Qaim,2004). To increase the β-carotene (provitamin A) content intransgenic rice, a codon optimized crt1 gene under the controlof endosperm-specific promoter has been used. Although bac-terial desaturase production was increased, no effect was seenon carotenoid content (Al-Babili et al., 2006). This problem hasbeen overcome when PSY was targeted. Paine et al. (2005) foundthat maize PSY gene could enhance the carotenoid content up to37 µg/g in ‘golden rice-2’ while testing the PSY gene from differ-ent organisms. This is due to the efficacy of maize PSY gene andstrategy can further improve the provitamin A production in rice(Grusak, 2005). As a part of an international effort, this pathwayis being introduced in rice varieties especially grown in devel-oping country (Potrykus, 2001). Recently, a very high amountsof carotenoids (up to 9.3 µg/g) has been achieved in indica ricevarieties (Datta et al., 2006). Further, barley homogentisic acidgeranylgeranyl transferase gene enhanced the vitamin E contentin transgenic corn (Cahoon et al., 2003). This would providefurther resource to improve content of another vitamin in rice.
Another aspect of nutritional quality that has become a targetfor genetic engineering is the amino acid content of plant foods.One of the examples to increase the nutritional value of rice is tooverexpress α subunit of anthranilate synthase gene, a key en-zyme in the synthesis of tryptophan (Tozawa et al., 2001). Highlysignificant increases in tryptophan level in transgenic lines hasbeen reported (Morino et al., 2005), thereby indicating that mu-tated gene has the potential for increasing tryptophan levels inother transgenic crops as well. Further, expression in rice of atransgene encoding a feedback insensitive alpha subunit of riceanthranilate synthase results in the accumulation of tryptophanin calli, leaves, and seeds (Wakasa et al., 2006). Similarly, engi-neering for a sulfur-rich seed storage protein has improved themethionine and cysteine content in rice endosperm (Lee et al.,2003). A protein with balanced amino acid composition likeAMA1 also can be used to improve nutritional quality in rice(Chakraborty et al., 2000). Levels of α-linolenic acid (a polyun-saturated fatty acid beneficial for health) have been increased inrice bran oil by expressing soybean microsomal omega-3fattyacid desaturase gene. Transgenic rice showed high amounts ofα-linolenic acid in seed oil (Anai et al., 2003).
Iron is a necessary nutrient for both infants and adult humanbeings. Human lactoferrin (HLF) is a major iron binding gly-coprotein in breast milk. Transgenic rice accumulating HLF inseed provided a novel means for nutrient supplement for infants(Nandi et al., 2002), as recombinant human lactoferrin couldmaintain the biological activity in transgenic rice seeds (Suzuki
et al., 2003). Iron content in seed also has been enhanced byexpressing FERRITIN gene in endosperm (Goto et al., 1999;Vasconcelos et al., 2003). A two-fold increase in iron content ofrice seed carrying FERRITIN from Phaseolus vulgaris has beenreported (Lucca et al., 2001a). However, an attempt to increasethe bioavailability of iron by expressing a thermotolerant phytasefrom Aspergillus fumigatus and cysteine rich metallothionine-like protein into rice, met with limited success (Lucca et al.,2001a). Similarily, calcium content of the rice grain was manip-ulated with H+/Ca2+ transporter gene in order to increase thenutritional value of rice and thus, help reduce the incidence ofosteoporosis (K. M. Kim et al., 2005).
B. Alteration of Starch ContentStarch, made up of amylose (linear α-1, 4-polyglucans) and
amylopectin (α-1, 6-branched polyglucans), is the main storagecarbohydrate in rice and has a high nutritional value (Nakamuraet al., 2002). In vivo modification of starch using genetic engi-neering holds potential for both enhancing nutritional qualitiesand obviating post-harvest modifications often necessary for uti-lization of this complex carbohydrate (W. S. Kim et al., 2005).In a novel attempt, wheat puroindoline genes PINA and PINBwere introduced in rice to modify the grain texture. Transgenicgrains were very soft in texture and referred as “Soft Rice.”This altered grain texture has numerous advantages in the foodindustry enabling new food qualities, food uses and other value-added end products. Further, puroindolines have a strong in vivoantimicrobial property (Krishnamurthy and Giroux, 2001). WXencoding granule bound starch synthase has been particularlytargeted for altering the properties of starch in rice (Shimadaet al., 1993; Terada et al., 2000). This amylose synthase gene hastwo functional alleles in rice, WXa in indica and WXb in japon-ica subspecies (Hirano et al., 1998). Introduction of WXa genein japonica background resulted in 6–11% increase in starch(Itoh et al., 2003). Alteration in amylopectin branching also hasbeen targeted to change the physiological properties of starchby silencing the isoamylase 1 gene or overexpressing E. coliglycogen-branching enzyme gene in transgenic rice (Fujita et al.,2003; W. S. Kim et al., 2005).
VII. YIELD IMPROVEMENTFood shortage is one of the most serious problems in the
world and a decline in rice yield has been reported (Peng et al.,2004). Thus, increase in grain yield either by breeding or trans-genic technology needs to be given immediate attention. Likemany agronomic important traits, grain yield is also a complextrait and shows continuous phenotypic variation (Ashikari et al.,2005). Significant efforts have been made in this direction inthe recent past. Earlier, enhanced activity of ADP-glucose py-rophosphorylase in transgenic rice had been shown to increaseseed weight per plant (Smidansky et al., 2003). Some work alsohas been done on transferring of C4 plant genes in C3 plantslike rice to enhance photosynthetic efficiency and yield per se
(Matsuoka et al., 1993, 1994; Ku et al., 1999; Nomura et al.,2000; Fukayama et al., 2001). Although, these efforts hold greatpromise, they have met with limited success so far. Light signaltransduction pathways are also being targeted for genetic ma-nipulation of yield in crops (Kong et al., 2004). Interestingly,Arabidopsis phytochrome A gene could increase the seed yieldby 6–21% in transgenic rice (Garg et al., 2006).
Phytohormones regulate various aspects of plant growth anddevelopment. Recently, a link between phytohormones (GA, cy-tokinin, brassinosteroids) metabolism and grain yield has beenestablished in rice (Sakamoto, 2006). Overaccumulation of cy-tokinin and reduced sensitivity or production of GA and brassi-nosteroids showed high grain yield in mutant rice. Short varietiesthat respond abnormally to GA are agronomically important;such dwarf transgenic rice has been produced by overexpressingArabidopsis gibberellin insensitive gene (GAI), but yield vari-ation was not demonstrated (Peng et al., 1999). Later, Sasakiet al. (2002) identified gene underlying SD1 locus, responsiblefor dwarfism and high grain yield, as GA oxidase (GA20ox-2).Gibberellin 2-oxidase gene overexpression resulted in semi-dwarf but high grain yielding plants (Sakamoto et al., 2003).Similarly, cytochrome P450 monooxygenase, involved in GAcatabolism, controlled seed development in rice (Tanabe et al.,2005; Zhu et al., 2006). A recent study by Sakamoto et al. (2006)has shown that manipulation of brassinosteroid synthesis alsooffers a promising approach in improving rice grain yield. Theyhave identified a brassinosteroid-deficient mutant that showederect leaves and increased number of panicles (∼17–20% highergrain yield than control plants). Underlying gene encodes forC-22 hydrolase and catalyses the rate limiting step of brassinos-teroid synthesis. Transgenic rice has been raised with partiallyexpressing brassinosteroid receptor gene, OSBR1. Transgenicplants showed erect leaf phenotype and about 30% higher grainyield than wild-type plants (Morinaka et al., 2006). A majorbreakthrough in efforts in increase in grain yield came withthe cloning of a grain number QTL, gn1, accounting for 21%more grain per panicle from rice variety Habataki. The under-lying gene encodes for cytokinin oxidase/dehydrogenase thatdegrades the cytokinin in inflorescence meristem. The defectiveallele of OSCKX2 showed enhanced accumulation of cytokininand increase in grain yield in transgenic lines (Ashikari et al.,2005). Identification of this gene along with other yield deter-minants (genes involved in phytohormones metabolism) holdsgood promise to improve grain yield.
VIII. OTHER APPLICATIONS
A. Control of Plant DevelopmentAny variation in flower development is directly related with
grain yield in cereals, thus much attention has been paid in un-derstanding the molecular mechanism of flower developmentusing transgenic approach. MADS box transcription factor, OS-MADS1, has been found to control the development of lemmaand palea (Prasad et al., 2001) by affecting the expression of
another MADS box gene, OSMADS16 (Z. X. Chen et al., 2006).Knockdown of OSMADS1 showed altered lemma and palea dif-ferentiation, while overexpression could induce lemma-like dif-ferentiation in glumes, suggesting differential role of OsMADS1in lemma and palea development (Prasad et al., 2005). The ABCmodel of flower development is widely accepted; however, func-tions of C class of genes are not well understood. Recently, usingRNAi approach and T-DNA insertion lines in rice, Yamaguchiet al. (2006) described the function of two C class genes, OS-MADS3 and OSMADS58. OSMADS3 was involved in stamenidentity and lodicules number, whereas OSMADS58 was relatedto carpel and floral meristem development. The study suggesedthat these two duplicated genes have sub-functionalized to per-form different roles in distinct whorls. A novel MADS box geneOSMADS22 altered spikelet development probably due to in-determinate cell fate in spikelet meristem in transgenic rice(Sentuko et al., 2005). The ectopic expression of rice AGA-MOUS ortholog, OSMADS3, caused the homeotic transforma-tion of lodicules into stamens (Kyozuka and Shimamoto, 2002).Their findings confirm the fact that lodicule is an equivalent ofa dicot petal and that the ABC model can be applied to riceat least for organ specification in lodicules and stamens. Genesnot coding for MADS domain also are involved in determin-ing flower architecture, e.g., OSFOR1 regulated the number offloral organs (Jang et al., 2003), whereas OSYABI controlled de-velopment and maintenance of stamens and carpels (Jang et al.,2004).
Knowledge of factors controlling the flowering time and tran-sition of shoot to floral meristem provides an attractive area tomanipulate plant architecture in order improve the agronomictraits. Overexpression of floral control gene LEAFY in Ara-bidopsis is sufficient for the transformation of lateral shoots intoflowers and causes early flowering (Weigel and Nilsson, 1995).However, in rice, reduction in flowering time was accompaniedby decrease in yield and alteration in panicle morphology (Heet al., 2000). Further, overexpression of rice homolog of termi-nal flower 1 (TFL1)/centroradialis like genes, RCN1 and RCN2,caused a delay in transition to reproductive phase, accompaniedby more branched and denser panicle (Nakagawa et al., 2002).Overexpression of OSGI, an ortholog of Arabidopsis GIGAN-TEA, delayed the flowering in transgenic rice. Some degree ofconservation in molecular mechanism of flowering in rice andArabidopsis was also suggested (Hayama et al., 2003). Thiswas also supported by recent report of M.L. Xu et al. (2005),where ectopic expression of Arabidopsis flowering promotingfactor 1 in rice showed reduced flowering time. These reportssuggested a certain degree of conservation between monocotsand dicots with respect to the molecular mechanisms controllingphase transition from shoot to floral meristem.
Plant architecture related studies would be helpful in under-standing the molecular mechanism of this complex trait andknowledge gained in rice can be applied to other cereals crops.OSTB1, teosinte branched 1 homologue in rice, has been foundto be a negative regulator for lateral branching in rice (Takeda
et al., 2003), whereas a rice homologue of pinhead/zwille gene,called OSPNH1, is involved in leaf developmental processes inrice (Nishimura et al., 2002). Similarly, other genes involvedin plant growth and development have been characterized intransgenic rice, e.g. MONOCULM 1 (Li et al., 2003), histoneacetylase (Jang IC et al., 2003b) and expansins (Choi et al.,2003). Interestingly, suppression of a rice gene with a SAP-likedomain showed overall plant growth inhibition (Chen J et al.,2003). Brassinosteroids are required by plants for normal growthand development. Silencing of brassinolide-enhanced gene, OS-BLE1, resulted in repressed growth and the gene is possibly in-volved in brassinolide mediated growth cascades in rice (Yanget al., 2003; Yang and Komatsu, 2004). Recently, OSBLE3 wascharacterized in rice by Yang G et al. (2006). Growth retarda-tion and reduced internode cell length was observed suggest-ing that OSBLE3 was involved in cell elongation in rice (Yanget al., 2006). On the other hand, silencing of a pentatricopep-tide protein gene OSPPR1 in rice resulted in chlorophyll defi-ciency and impaired chloroplast development probably due toan effect on the processing of plastid RNAs (Gothandam et al.,2005).
MicroRNAs have emerged as the new agent in controllingseveral aspect of plant development. Dicer like proteins are ho-mologs of dicer protein in plants and essential for biogenesisof miRNAs. B. Liu et al. (2005) have raised transgenic rice ex-pressing RNAi construct of OSDCL1 and silenced lines showedseveral developmental defects suggesting an important role inrice development. A transcription factor involved in ethylenesignal transduction OSEIL1 was overexpressed in rice plantsresulting in short root, coiled primary root, and slightly shortshoots as well as elevated response to exogenous ethylene, sug-gesting that it may be a positive regulator of ethylene responsein rice (Mao et al., 2006).
The molecular mechanisms that control development of rootsystem in monocots are not clearly understood. Only a smallnumber of mutants related to root formation are available inmonocots. Recently, genes involved in auxin-mediated root de-velopment have been identified in rice. The most promisinggene, OSRAA1 (root architecture associate-1), is induced byauxin in root meristem and inhibits growth of primary root inoverexpressing lines. This is also accompanied by increase inIAA level and a positive feedback regulation of OSRAA1 to IAAmetabolism may be involved in rice root development (Ge et al.,2004). IAA (AUX/IAA) is a negative regulator of auxin signal-ing. The IAA gene, OSIAA3, has been shown to affect root de-velopment in rice (Nakamura et al., 2006b). Cytoplasmic malesterility (CMS) is a widespread plant reproductive feature thatprovides a useful tool to exploit heterosis in crops. Expressionof an abnormal mitochondrial open reading frame orf79 in CMSlines results in a cytotoxic peptide that causes gametophytic malesterility (Z. Wang et al., 2006). On the same theme, RF-1 geneof rice has also been tested for restoration of fertility in malesterile line of rice by raising transgenic plants (Komori et al.,2004).
B. Production of Novel CompoundsGenetic transformation of rice has been used for producing
novel compounds. The plant system provides several advantagesover animal or prokaryotic system, e.g., more safety for humanuse, post-translational modification of expressed protein, lessexpensive extraction and purification (Yasuda et al., 2005). En-dosperm targeted production of recombinant compounds in ricefurther provides the benefit of storage in the form of seed. Single-chain Fv antibody (ScFvT84.66) against a well-characterizedtumor-associated marker antigen, carcinoembryonic antigen,had been produced in rice and was stable in transgenic dryseeds for at least five months at room temperature (Stoger et al.,2000). Transgenic rice system has been successfully used forthe production of industrial valuable enzyme transglutaminase(Claparols et al., 2004), N(hydroxycinnamoyl)transferase (S.M. Jang et al., 2004), lupin acid phosphatase (Hamada et al.,2004), human lysozyme (Hennegan et al., 2005), glycogen-likepeptide for type II diabetes treatment (Yasuda et al., 2005), aller-gen specific T-cell epitope (Takagi et al., 2005), human granulo-cyte colony stimulating factor, hG-CSF (Hong et al., 2006), andlinoleic isomers for reducing fat and hypertension in animals(Kohno-Murase et al., 2006). More than a billion people in theworld suffer from hypertension. Foods rich in antihypertensiveactivity might hold the promise of reducing the disease. L. Yanget al. (2006) developed transgenic rice plants expressing the an-tihypertensive peptide RPLKPW fused with rice glutelin storageprotein in seeds. Recombinant protein accounted for about 10%of total seed protein in transgenic seeds.
Rice cell suspension culture systems also provide an alterna-tive to animal/bacterial cell lines for the production of recombi-nant compounds for human use as risk of contamination is verylow. Thus, Huang et al. (2002) transformed rice calli and cellsuspension cultures using a codon-optimized synthetic gene forhuman lysozyme. The recombinant human lysozyme showedequally potent bactericidal activity as the native one. It has beensuggested that problems encountered in drug formulation pro-duced in microbial or mammalian systems could be overcomeby the use of plant systems, as they exhibit different patterns ofprotein glycosylation and N-linked glycan processing. Humaninterferon-gamma produced in transgenic rice cell suspensionculture showed the same biological properties as the commer-cial product. This was evaluated by testing anti-dengue virusactivity of these proteins in human cell lines (T. L. Chen et al.,2004). Glycosylation pattern of human lactoferrin in transgenicrice expressing its cDNA was found more mammal-like as com-pared to maize and other plant systems (Fujiyama et al., 2004),demonstrating the potential of rice as a promising system forthe production of heterologous proteins. Similarly, recombinanthuman α-1-antitrypsin produced in rice cell lines could be con-veniently collected in a cell compartment within the membranebioreactor, 5 to 6 days after being induced (Terashima et al.,1999; McDonald et al., 2005). Recently, Gu et al. (2006) re-ported the expression the Helicobacter pylori urease subunit Bgene in rice and provided a basis for further studies on the po-
tential of transgenic rice for delivery of edible vaccines againstHelicobacter pylori.
C. Technological InnovationsSeveral technological innovations have been helpful to im-
prove potential and ease of utilization of transgenic rice. A highlevel of targeted expression of transgene(s) within the trans-genic plant is highly desirable depending on the requirement.Nguyen et al. (2004) have reported that the application of thebacteriophage T7 RNA polymerase directed gene expressionsystem in transgenic rice and it was three-to-five-fold betterthan CaMV35S gene promoter. In another approach, MAR fromtobacco, TM2, enhanced reporter gene expression in transgenicrice when used with tissue specific or constitutive promoter (Xueet al., 2005). Estimation of transgene copy number is an impor-tant exercise and L. Yang et al. (2005) have developed a simplereal-time quantitative PCR-based method to estimate transgenecopy number in transgenic rice. This method showed almostsimilar results as obtained with Southern hybridization data.Real-time PCR for determination of copy number in transgenicrice has also been used by Prior et al. (2006). Recently, siRNAmediated post-transcriptional gene silencing (PTGS) has beenestablished as the method of choice for functional analysis. Tanget al. (2004) have used this technique to silence the function oftargeted gene in transgenic rice cells. They studied silencing ofGFP by using two different siRNA designed from coding regionof GFP.
IX. TRANSGENE SILENCINGFor any transgenic crop to be useful, it is important that
the transgene is expressed stably through generations. Severalfactors contribute to the variation in transgene expression, i.e.,integration site, transgene copy number, transgenic locus con-figuration, epigenetic silencing mechanisms, flanking matrix at-tachment regions, and even the stages of plant tissue (Kumpatlaand Hall, 1998; Morino et al., 2004; G. Yang et al., 2005). In-activation of transgene present in multiple copies and those re-lated to homologous endogenous sequences is well established(Finnegan and McElroy, 1994). In most cases of homology-dependent gene silencing, inactivation was due to epigeneticmodification of DNA (methylation) which once established wasfound to be faithfully transmitted to subsequent generations andhence, considered to be stable during inheritance (Flavell, 1994).However, reversal of silenced state has been reported (Fu et al.,2000b). Kumpatla and Hall (1998) have provided evidence forretention of the reactivated state, renewal of silencing and stablemaintenance of the re-inactivated condition in transgenic rice.Another valuable observation was made by Kloti et al. (2002)wherein they have shown spreading of silencing among differenttissues through generations. Methylation pattern was also ex-tended from promoter to coding region and reversed on applica-tion of 5-azacytidine. However, silencing is not always reversedby 5-azacytidine suggesting the role of post-transcriptional gene
silencing (Wang and Waterhouse, 2000). PTGS in transgenicrice for endogenous gene has also been reported (Kusaba et al.,2003; Morino et al., 2004).
To overcome gene silencing, use of the backboneless T-DNAwas beneficial in rice (Fu et al., 2000a). Interestingly, the in-tegration pattern found in linear DNA transformed plants wassimple with low copy number integrations and low frequencyof transgene rearrangements. The advantage of linear, minimalDNA bombardments was evident as transgenic plants did notshow silencing of the transgene up to R4 generation. Gene si-lencing also can be reduced by modifying the transgene expres-sion cassette. MAR reduced or eliminated transgene silencing asthey increased gene expression and also reduced variation be-tween different transformation events (Spiker and Thompson,1996). Gene silencing was reduced up to 50% when a novelMAR (TM2) from tobacco was used with both tissue-specificand constitutive promoters in transgenic rice (Xue et al., 2005).In combination with the above, integration on selected loci maybe used in the future as an approach to avoid gene silencing(Tzfira and White, 2005).
X. ENVIRONMENTAL AND BIOSAFETY ASPECTSEnvironmental release and biosafety have been subjects of
debate regarding transgenic plants, especially for food crop likerice (Bajaj and Mohanty, 2005). A large number of transgenicrice generated to show insect, bacterial, and herbicide resistancehave been tested in the field with a view to evaluate protection,influence on yield, environmental impact, health and gene flow(Oard et al., 2000; Tu et al., 2000; Ye et al., 2001, 2003; Bashiret al., 2004; Huang et al., 2005). Although several selectablemarkers are available in the form of antibiotic and herbicideresistance genes, demand for marker-free transgenic plants ishigh. Currently, there are two main strategies to achieve thegoal of marker-free transgenic plants. The first is to excise orsegregate marker genes from the host genome after regenerationof transgenic plants and the second is based on the strategycalled marker-free transformation. Site-specific excision of atransgenic DNA sequence containing the marker gene by theuse of a chemically regulated cre/lox recombination system waspossible (Zuo et al., 2001; Sreekala et al., 2005). This systemwas further modified by using a membrane translocater proteinto deliver the enzymatically active cre protein into the plant cell,thus avoiding the regulated expression of cre (Cao et al., 2006).The approach was simple and efficient in generating marker-free transgenic rice. FLP/FRT recombination system providesanother way to remove the marker gene (Radhakrishnan andSrivastava, 2005), but this system was not as efficient as cre/loxsystem. AC/DS transposon system also has been used to obtainmarker-free transgenic rice (Jin et al., 2003), however, besidesthe laborious sexual crossing exercise, it was also less efficient.
Another successful and simple method used in marker-free transgenic rice production is ‘Twin T-DNA’ binary vector(Komari et al., 1996) and its modifications (Breitler et al., 2004a;
Parkhi et al., 2005). Such vectors carry two T-DNA: one carryingmarker gene and other with gene of interest. Co-transformationfollowed by transgene segregation would result in marker-freetransgenic plants. This technique was further improved by usinga double right border sequence, one on either side of the markergene followed by gene of interest and one copy of the T-DNAleft border sequence (Lu et al., 2001). Endo et al. (2002) haveused multi-autonomous transformation (MAT) vector, makinguse of Agrobacterium oncogenes as a positive selection marker.MAT vector was constructed for rice transformation by fusingchimeric ipt (isopentyl transferase) and GFP genes with 35Spromoter and site-specific recombination R/RS system for theirremoval after transformation along with gusA, nptII and aphIVgenes outside of R/RS cassette. This system was very efficientfor generation of marker-free transgenic rice, as it does not need aselecting agent and sexual crossing. A novel approach adoptedby Vain et al. (2003) makes use of a dual binary vector sys-tem, pGreen/pSoup. pGreen is a small Ti binary vector that canonly replicate in the presence of pSoup in the same bacterialstrain. Co-transformation with both the vectors, one carrying thetransgene and other carrying the selectable marker, followed bysegregation in subsequent generation will produce marker-freeplants.
The ‘clean gene’ approach, which involves only minimumlinear cassette (promoter gene and terminator) to transform ricecalli, has provided another attractive strategy to generate marker-free plants (Fu et al., 2000a; Agarwal et al., 2005). Recently,Nam-Hai Chua and colleagues reported the use of regenera-tion promoting genes as a substitute for the traditional antibioticmarker genes. By appropriate manipulation of a regeneration-promoting gene, only transformed cells can regenerate in theabsence of key growth regulators (Zuo et al., 2002). Betaine alde-hyde dehydrogenase gene from spinach (Daniell et al., 2001) andphosphomannose isomerase (PMI) from E. coli have been usedas positive selection in a rice transformation system. In such sys-tem, only transformants would show preferential growth. PMIgene has been exploited in the production of golden rice (Luccaet al., 2001b) and its biosafety has also been assessed (Privalleet al., 2001). Recently, a QTL responsible for plant regenerationin tissue culture system was found to encode a ferredoxin-nitritereductase (NIR) gene in rice. This gene degrades the nitrite inthe medium and thus, provides the very tempting strategy to usenitrite as a selecting agent and NIR as a marker gene (Nishimuraet al., 2005). It is further advantageous since NIR is a rice en-dogenous gene. In search of identifying new selectable markers,Ochiai-Fukuda et al. (2006) have shown that blasticidin (an an-tibiotic that is used for control of blast disease in rice) degradinggene can be used as a visible selectable marker when combinedwith green fluorescent protein and transgenic rice plants couldbe regenerated within 51 days after the microprojectile bom-bardment.
The risk assessment and safety issues related to geneticallymodified food are of paramount importance (Kuiper et al.,2001). The safety/allergenicity of the introduced trait, toxicity,
occurrence and implications of any unintended side effects, aswell as influence of introduced trait on the properties of food andits processed forms are some parameters commonly discussed.Rats fed with rice carrying either soybean glycinin (Mommaet al., 2000) or phosphinothricin acetyltransferase (Y. Wanget al., 2000) gene showed no pathological or histopathologicalabnormalities in liver, kidney, or blood, suggesting the biosafetyof such transgenic food (Y. Li et al., 2004). Rice expressingcry showed similar grain quality and nutrient composition asthe wild type plants and, after cooking, the cry1Ab protein wasundetectable suggesting that the protein is heat labile, gets de-graded upon cooking, and cannot retain its toxicity even forinsects (D. X. Wu et al., 2002). Accumulation of cry in rhizo-spheric soil of transgenic plants is also considered a matter ofconcern. However, a recent report by Wang et al. (2006) showedthe absence of cry1A toxins from five kinds of rhizospheric soilof transgenic rice. Notwithstanding such reports of promise, itshould not be ignored that the rhizosphere itself may harbornatural cry-containing bacteria.
XI. AGRIBUSINESS AND THE FUTURE OFTRANSGENIC RICE
Rice is one of the three important cereals and it is producedannually at worldwide levels of more than 600 million tons.Unlike the other major cereals, more than 90% of rice is con-sumed by humans. Approximately half of the world’s popula-tion derives a significant proportion of their caloric intake fromrice consumption. Application of molecular techniques to riceimprovement will help to achieve better yields. A continuouseffort to isolate and identify novel useful genes and promotersis necessary. Transgene technology in particular is likely to be-come a powerful tool for this purpose. Eventually, the largestchallenge will be to combine these genes and promoters in asystematic and logical way to create novel transgenic rice linesto help maximize plant vigor, yield, and stress tolerance. It isexpected that misinterpretations and misunderstandings of theregulatory process and of the potential of GM crops would notbe allowed to block a technology that is already delivering realbenefits today and promises important benefits for sustainableagriculture in the future. At the same time a robust analysis andregulatory system including post-release surveillance and riskaversion packages should be in place to remove any fear of theend users of technology.
The World Health Organization estimates that the earth’s pop-ulation will reach 9 billion by 2050. The vast majority of thisincrease will occur in the developing countries of South EastAsia and Sub-Saharan Africa. To meet the challenge of foodsupply over the next 50 years, we must push the production offood on essentially the same area of land in the face of decreasingwater supplies and with respect to the environment. This entailsdevelopment of rice cultivars with a higher yield potential (Penget al., 1999). A review of the global status of transgenic crops in2005 (www.isaaa.org) shows that transgenic crops grow in 20
countries and the global area of transgenic crops was 90 millionhectares, representing an increase of 11% over 2004. Driven bythe economics of rice production and declining domestic riceproduction, China has accelerated its drive to commercializetransgenic rice technology, thereby becoming the first countryin the world to adopt GM rice into the mainstream. Their focus isprimarily on producing high yielding transgenic varieties that areinsect pest resistant. Several transgenic varieties that are tolerantto either lepidopteran pests, bacterial blight, rice blast fungus,drought, or salt stress have been in field trials since 1998 (Jiaet al., 2004). It is being assumed that once countries like China,where rice is an integral part of the society, accept transgenicrice then it would trigger a cascade effect of acceptance of GMfoods not only in China but also in other developing countrieswhere rice is a staple food. One of the recent claims of fieldtrials accompanied with assessment of biosafety by Huang et al.(2005), which might help in greater acceptance of transgenicrice, is of transgenic rice varieties GM Xianyou 63 and GM-II-Youming 86 that require only 20% of the amount of pesticidesrequired by untransformed controls and have a 6% increase inyield in comparison to the wild-type rice plants. Such transgenicplants reduce the risk of health hazards caused by pesticides, areenvironmentally friendly and help increase the net income forthe farmer. China has projected that the country will have a netgain of four billion dollars from its cry expressing rice varietiesalone in the year 2010 and Iran has already grown 4,000 hectaresof transgenic rice in 2005 (www.isaaa.org).
It is extremely important to realize that harvestable yieldshave reached a plateau phase from plants generated by con-ventional breeding. It is desirable to create superior transgenicrice plants that can grow in compromised environments and havehigher yields with decreasing arable land availability. Gene pyra-miding or multigene engineering using genes involved in variousagronomic traits is a powerful approach to obtain superior ricevarieties (Ashikari and Matsuoka, 2006). This approach has al-ready been used to confer resistance against a broad range ofdifferent rice pests and pathogens using genes like cry, Xa, chti-nase and GNA (Maqbool et al., 2001; Datta et al., 2002). A be-ginning has already been made and large-scale multi-locationalfield trials are being conducted to determine the viability of thelab-proven products that various groups have reported in rice.It is hoped that the ultimate benefit of such mammoth exerciseswould reach the farmer and the consumer. At the same time,evaluation of gene function, creation of new alleles and theirfield level testing via transgenic approach should be considereda regular activity for creating options to help achieve sustainablerice production in the future.
ACKNOWLEDGMENTSWe thank Dr. P. Christou, ICREA, Universitat de Lleida,
Spain and Dr. S. Datta, Calcutta University, India, for criti-cal reading of the manuscript and useful comments. Our re-search work is funded by the Department of Biotechnology,
Government of India., H.K. and J.G. acknowledge the Councilof Scientific and Industrial Research, New Delhi, for the awardof Research Fellowship.
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