Introduction of agronomically useful genes in rice

been less successful in monocots, but in the last few years significant progress has been made in this direction in rice [41 – 45]. Early attempts to regenerate transgenic calli from Agrobacterium-mediated transformation were not successful [46]. Subsequently, regeneration was achieved from Agrobacterium-infected calli of root explants [47] as well as immature embryos [48]. However, scientists were not convinced about the effectiveness of Agrobacterium as a vector for rice transformation. In a significant development, Hiei et al. [42] reported transformation of japonica rice using Agrobacterium. They constructed some unique vectors called ‘super-binary’ vectors which have additional 6ir genes in the binary plasmid itself. This modification led to achievement of high transformation efficiency in japonica rice. Scutella-derived calli and Agrobacterium tum- efaciens LBA4404 pTOK233 were found to be the most suitable explant and effective strain, respectively. Several necessary requirements for successful transformation, such as the use of acetosyringone and a temperature of 22 – 28°C during co-cultivation, were also pointed out. Molecular and genetic analyses of a large number of transgenic plants up to R2 generation together with sequence analysis of T-DNA junctions in rice were provided. Subsequently, transformation of japonica rice by Agrobacterium was reported by other groups [49 – 52]. Aldemita and Hodges [53] obtained transgenic indica as well as japonica rice using immature embryos. Rashid et al. [54], Mohanty et al. [55], and Khanna and Raina [56] reported successful transformation of elite indica varieties with Agrobacterium at high efficiency. Molecular, genetic as well as biochemical analyses of transgenic plants up to R2 progeny was reported [55]. In addition, transformation of javanica rice has also been reported [57]. Using isolated shoot apices as explant for co-cultivation, Park et al. [58] reported generation of transgenic rice plants by Agrobacterium. Besides, Toki [59] has reported a new binary vector pSMABuba for rice transformation. In another significant development, Komari et al. [60] designed some unique plasmids that carry two separate T-DNA segments, one carrying the non-selectable marker gene gus and the other carrying the selectable gene hph in the same plasmid. These vectors were employed for generation of marker-free transgenic plants. The frequency of co-transformation with the two T-DNA was found to be greater than 47 reflecting the effectiveness of the system. The integration and segregation of T-DNAs were confirmed by molecular analysis. Notwithstanding the recent advances made in the area of Agrobacterium-mediated transformation of rice [61], there are already a few reports available where Agrobacterium has been used to produce transgenic rice with economically important genes [50,62 – 66].

3. Introduction of agronomically useful genes in rice

Considerable success has been achieved by plant breeders in developing improved rice varieties and it is imperative to argue that the success achieved with transformation techniques will supplement plant breeding programmes [3]. This becomes im- portant in view of the need to raise rice production from 560 to 850 million tons by 2025 to support additional rice consumers [1] and to manage the loss due to various biotic and abiotic stresses, such as insect pests, salinity, low temperature, water logging and drought [67]. Remarkable progress has been made since the recovery of the first transgenic rice plants just a decade ago. A number of economically important genes have been transferred to japonica as well as indica rice and scientists are now looking forward towards pyramiding of genes in rice. 3 . 1 . Insect resistance using Bt genes Although the cost associated with management practices and chemical control of insects ap- proaches 10 billion, still there is a 20 – 30 loss of crop across the globe [68]. Special attention has been paid to genetic engineering of rice for insect resistance since it has not been possible to find genes for sufficient levels of resistance against many of them [69]. Chemical insecticides remain the only way to control insect pests in rice cultiva- tion, which are under serious public debate be- cause of food safety concerns and environmental pollution. An alternative strategy is the applica- tion of genetic engineering for production of proteins with insecticidal activity by the rice plant. Fujimoto et al. [70] were the first to engineer japonica rice through electroporation with modified d-endotoxin gene cry from Bacillus thuringiensis. It was found that the R2 generation of transgenic rice was more resistant to insects than wild type plants. Later, Wu¨nn et al. [71] obtained transgenic indica rice cultivar IR58 ex- pressing a synthetic cryIA b gene driven by 35 S promoter through particle bombardment. Insect bioassays revealed effective control of two of the most destructive pests of rice in Asia, the yellow stem borer YSB and the striped stem borer SSB. Interestingly, feeding inhibition of the two leaf folder species Cnaphalocrous medianalis and Marasima patanis was also observed. In order to achieve high expression, Nayak et al. [72] recon- structed cryIA c gene and transformed indica rice cultivar IR64 with the synthetic gene. Insect bioassays revealed the resistant nature of trans- genic plants to the damage caused by YSB, al- though the level of expression was low. In the following years, various synthetic and modified cryIA b driven by constitutive 35 S, Ubi 1 , Act 1 as well as tissue-specific Pepc, maize pith-specific promoters have been used to achieve desirable level of resistance [20,73 – 75] against SSB and YSB. Datta et al. [20] reported that a large num- ber of transgenic plants caused 100 mortality of YSB larvae. The authors suggested the use of tissue-specific promoters for minimizing the ex- pression of Bt protein in edible parts. In a signifi- cant development, Cheng et al. [62] obtained a large number of transgenic rice plants of different varieties engineered with cryIA b and cryIA c genes, which have been codon optimized, by Agrobacterium-mediated transformation. South- ern, Northern and Western analyses up to R1 generation were performed to show the integra- tion, inheritance and expression of the transgenes. Use of the Ubiquitin promoter led to high level expression up to 3 of total soluble protein of transgene product. The toxic nature of the trans- genic plants to SSB and YSB was revealed by insect bioassays. Recently, the need to revise the management strategy for cry-dependent resistance has arisen as reports regarding development of insect resistance against a single cry gene were written. Develop- ment of insect resistance for cry 1 A a , cry 1 A b , cryIA c and cry 1 F has been reported. By per- forming an elaborate series of genetic crosses, Tabashnik et al. [76] proved that just one autoso- mal gene in diamond back moth confers cross-re- sistance against the four cry genes mentioned above. Keeping this in view, Maqbool et al. [77] transformed rice with a novel cry 2 A gene by parti- cle bombardment. Molecular and biochemical analyses confirmed transmission of the gene to R2 progeny. In one plant line, the protein was ex- pressed to the level of 5 of total leaf protein. In other plants, the expression level was between 0.01 and 1. Insect bioassays revealed the effectiveness of this gene in providing resistance against YSB and rice leaf folder, two of the major pests of rice. In another strategy for improvement of rice, Alam et al. [78] transformed an IRRI maintainer line IR68899B, which is used for the production of hybrid rice with cryIA b gene. Southern and Western blot analyses confirmed the integration, expression and transmission of the transgene to R2 progeny. Insect bioassays revealed enhanced resistance against YSB in the transgenic plants. The effectiveness of gene pyramiding to obtain durable resistance against insect pests has also been reported recently by Christou’s group [79,80]. They obtained transgenic indica rice plants with insecticidal genes cryIAc, cry 2 A and the snow- drop lectin Gna gene. These triple transgenic rice plants R0 and R1 showed significantly higher resistance to insect pests. Keeping in view that at present 8 billion is annually spent on insecticides worldwide out of which nearly 2.7 can be re- placed by Bt technology applications, Bt research in rice assumes great importance. 3 . 2 . Insect resistance using proteinase inhibitors and lectins For insect resistance, genes encoding plant proteinase inhibitors are of particular interest as they are part of the plant’s natural defence system against insect predation [81]. Hosoyama et al. [82] obtained transgenic rice through electroporation harbouring a chimeric oryzacystatin Oc gene. Later, the corncystatin Cc gene was introduced into rice by Irie et al. [19]. The transgenic rice plants were resistant to the insect pest Sitophilus zeamais. Xu et al. [83] engineered rice with a cowpea trypsin inhibitor Cpti gene driven by the Actin 1 promoter for high level of expression. Re- sults of a small scale field test suggested increased resistance of transgenic rice to the striped stem borer and pink stem borer. Duan et al. [81] intro- duced potato proteinase inhibitor II PinII gene driven by PinII promoter for wound-inducible ex- pression of transgene in rice. The stability of this gene up to four generations was confirmed by molecular analyses. The transgenic plants were found to have increased resistance to pink stem borer. These results suggest that proteinase in- hibitor genes could be used as a general strategy for the control of insect pests. Zhen et al. [84] reported that a modified cowpea trypsin inhibitor gene with ER targeting signal KDEL resulted in two to four times higher average proteinase in- hibitor activity than that of plants transformed with CpTi gene. Bioassays proved the efficacy of this modified gene as revealed by 60 – 72 mortal- ity rate of Chilo suppressalis larvae. Another class of proteinase inhibitors, the Kunitz family repre- sented by soybean Kunitz trypsin inhibitor, has also been engineered in rice [85]. A vector was constructed by fusing full-length cDNA with 35 S promoter. Protoplasts isolated from japonica vari- ety ‘Nagdongbyeo’ were transformed using PEG- mediated gene delivery. The integration, expression and inheritance of the transgene were demonstrated up to R2. The transgene product accumulated between 0.05 and 2.5 of total solu- ble leaf protein. Insect bioassays with the progeny of the above plant lines revealed that transgenic plants are more resistant to Nilapar6ata lugens Sta¨l than control rice plants. However, one third of the plants obtained in this study were sterile. The snowdrop Galanthus ni6alis lectin GNA gene has also been used for obtaining resistance against insect pests in rice. Transformation of rice with the gene driven by the phloem-specific su- crose synthase promoter of rice RSSGNA through electroporation as well as with maize ubiquitin promoter driving GNA gene UBIGNA by biolistics has been achieved [86,87]. Western blot analysis revealed the presence of 12-kDa band in both types of transgenic rice plants correspond- ing to the standard GNA polypeptide. Semi-quan- titative estimates of expression levels of GNA from Western blots revealed 0.01 – 0.25 of total soluble protein for RSSGNA plants and up to 2 of total soluble protein for UBIGNA plants. In- sect bioassay showed that by expressing lectin GNA, transgenic rice plants can be partially pro- tected against brown planthopper. Immunolocal- ization studies revealed the phloem tissue-specific expression of rice sucrose synthase 1 promoter driven GNA [88]. 3 . 3 . Resistance against 6iruses Hayakawa et al. [89] engineered the coat protein Cp gene of rice stripe virus into two japonica rice varieties by electroporation of protoplasts result- ing in significant levels of resistance against the virus in the transgenic plants. Huntley and Hall [90] obtained transgenic rice with four different constructs containing regions of RNA-2, RNA-3 and capsid protein genes derived from the brome mosaic virus BMV. When challenged with virion RNA, protoplasts obtained from transgenic plants or cell lines showed up to 95 reduction in the accumulation of viral RNA. To achieve resistance against rice dwarf virus, Zheng et al. [91] intro- duced the outer coat protein gene S 8 of this virus into rice. Rice tungro disease may cause an estimated US 343 million annual loss in crop in South East Asia alone. Two viruses, rice tungro spherical virus RTSV and rice tungro bacilliform virus RTBV, are known to be causative agents. RTBV is a double stranded virus and RTSV is a positive- sense, single stranded RNA virus. Of these, RTBV causes severe disease symptoms. These viruses are transmitted by green leafhopper GLH Nepho- tettix 6irescens. To obtain resistance against RTBV, it is necessary to understand the molecular biology of the viral cycle. Therefore, the RTBV genome has been characterized and mutant viral proteins have been created which can act as com- petitive inhibitors of viral functions [92]. Further, transgenic indica rice plants have been produced expressing RTBV proteins which serve as precur- sors to viral coat protein and reverse transcriptase [92]. Element which provides specificity to the RTBV promoter has also been analyzed [93,94]. Sivamani et al. [95] reported detailed analysis of transgenic plants with three different coat protein genes, Cp 1 , Cp 2 , and Cp 3 of RTSV. Northern blot analysis revealed the presence of detectable levels of mRNA in all except three lines. However, no signal was obtained in Western blot analysis prob- ably because of the low amount of transgene product. Inheritance of the Cp gene was analyzed up to R2 progeny. When challenged with RTSV, moderate level of resistance was observed in differ- ent R1 and R2 lines accompanied by delay in virus infection. One noteworthy feature of the study was that no cumulative effect on the resistance was observed when these genes were expressed to- gether. This is contrary to the popular belief that pyramiding of these genes would help. Pinto et al. [96] constructed transformation vec- tors by fusing either full length rice yellow mottle virus RYMV cDNA encoding RNA-dependent RNA polymerase or C-terminal deletions of this to the 35 S promoter. Transgenic plants were re- generated following gene delivery by biolistics. Some of the transgenic plants obtained were highly resistant against different isolates of RYMV up to R3 progeny. Northern analysis of these resistant plants revealed the presence of very low levels of RYMV transcript. In comparison, the plants having partial resistance had high level of RYMV transcript. A post-transcriptional gene silencing of the RYMV transgene in the resistant lines has been implicated as the basis of RYMV resistance mechanism. This is the first report of homology-dependent resistance in rice. Mun˜oz et al. [97] reported transformation of Costa Rican indica rice with coat protein gene of rice hoja blanca virus RHBV under the control of the rice Actin promoter together with MAR sequences. The bar gene was used for the selection of transformed tissues. Molecular analysis showed the presence of RHBV coat protein gene in low copy number. 3 . 4 . Herbicide resistance Genes for herbicide resistance have also been introduced in rice. The bar gene is advantageous as it serves the dual purpose of selectable marker gene as well as conferring resistance to the herbi- cide, phosphinothricin PPT. Christou et al. [21] and Datta et al. [98] were able to engineer several rice cultivars to express the bar gene. The trans- genics were resistant to high doses of the commer- cial formulations of PPT. A field study, conducted for 3 years, with 1.12 or 2.24 kgha glufosinate showed significant improvement in the perfor- mance of transgenics [40]. 3 . 5 . Resistance against fungal pathogens Transgenic rice plants, expressing bar gene, were not infected by Rhizoctonia solani [99] and showed decreased symptoms of rice blast disease, follow- ing bialaphos treatment [100], probably because of the sensitivity of pathogens to the herbicide. The rice chitinase gene Chi 11 under the control of the constitutive 35 S promoter was used to transform rice protoplasts to obtain rice plants resistant to the sheath blight pathogen R. solani [18]. Simi- larly, a basic chitinase gene from rice could be employed to engineer resistance to pathogens [101]. Nishizawa et al. [65] introduced two rice chitinase genes Cht- 2 and Cht- 3 into japonica rice under the control of an enhanced 35 S promoter. In transgenic plants, the Cht- 2 product was targeted extracellularly, whereas the Cht- 3 product accumulated intracellularly. Transgenic plants ex- pressing both the genes constitutively were found to show enhanced resistance to two races 007.0 and 333 of the rice blast pathogen, Magnaporthe grisea. With a similar objective, Stark-Lorenzen et al. [102] employed a stilbene synthase gene from grapevine, under the control of its own promoter, to raise transgenic rice plants. The accumulation of stilbene synthase mRNA in response to inocula- tion with Pyricularia oryzae as well as wounding, elicitor treatment and UV irradiation was ob- served in R2 plants. Plants expressing this gene showed resistance against P. oryzae. Kim et al. [103] reported transgenic rice plants expressing the maize ribosome inactivating protein gene, Rip b- 32 . Southern hybridization revealed that 30 of the regenerated plants had a single transgene in- sert. Transmission of the Rip b- 32 as well as selectable marker bar gene was observed up to R2 progeny. Gene silencing was observed in some of the transgenic plants having multiple copies of transgene. Expression levels of the protein were 0.5 – 1 of total soluble leaf protein. However, when R2 transgenic plants were challenged with R. solani and M. grisea, there was no significant reduction in disease severity as compared to con- trol suggesting that normal processing of b-32 protein may be required for in planta antifungal activity. To achieve resistance against sheath blight disease, transformation with thaumatin-like protein PR-5 gene Tlp under the control of 35 S promoter has also been attempted in Chinsurah BoroII, IR72 and IR51500 [39]. Transformation was achieved by PEG-mediated transformation of protoplasts as well as gene delivery to immature embryos by biolistics. Southern hybridization data revealed transmission of the transgenes up to R2 progeny and fertility was 80. Western analysis showed the presence of 23-kDa TLP-D34 protein. Insect bioassay revealed that several transgenic plant lines were having limited infection compared to the control. 3 . 6 . Resistance against bacterial diseases The rice Xa 21 gene which confers resistance to blight pathogen, Xanthomonas oryzae was cloned by Song et al. [104]. Transgenic rice plants har- bouring the cloned gene displayed high levels of resistance. The gene has been found to be effective against several isolates [105]. An elite indica rice cultivar IR72 has also been transformed with the Xa 21 gene [32] and transgenic plants from R1 generation were found to be resistant to bacterial blight. However, in some of the lines gene silenc- ing was observed. Zhang et al. [106] reported production of transgenic elite indica rice IR64, IR72 and Minghui 63, a restorer line for Chinese indica hybrid rice with Xa 21 gene. Bioassays re- vealed that a number of lines up to R2 generation were resistant against the bacteria. R3 generation of one line of IR72 was also found to be resistant. However, since the plants analyzed include ho- mozygous as well as heterozygous plants, efficacy of these transgenic plants would be clear only after the field trials, which are being performed. 3 . 7 . Engineering of rice for abiotic stress tolerance To engineer rice for tolerance to abiotic stresses, Xu et al. [107] introduced a late embryogenesis abundant LEA protein gene H6a 1 from barley using biolistics method. Use of rice Actin 1 pro- moter led to high level constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic plants and the second generation showed increased tolerance to water deficit and salinity. Another gene, Cor 47 , has also been shown to confer resistance against several abiotic stresses [108,109]. Yokoi et al. [50] employed Ara- bidopsis cDNA for glycerol-3-phosphate acyltrans- ferase Gpat under the control of maize Ubi 1 gene promoter to obtain transgenic plants with high levels of unsaturated fatty acids and chilling toler- ance of photosynthesis. The codA gene for the enzyme choline oxidase from Arthrobacter globiformis, which can catalyze conversion of choline to glycine betaine in a single step, fused to a rice RbcS transit peptide chl- COD for targeting codA gene product to the chloroplast was employed for transformation of japonica rice [63]. In the other construct cyt- COD, no targeting signal was used and it was expected to enhance glycine betaine in the cytosol. Additionally, for better processing of RNA, rice Sod gene intron was incorporated in both the constructs. It was reported that cyt-COD and chl-COD plants accumulated glycine betaine at a level of 1 and 5 mmol per gram fresh weight, respectively. Both types of transgenic plants were tolerant to low temperature as well as salinity stress as revealed by PSII activity. Since the glycine betaine concentration achieved was not significant for osmotic adjustment, the authors suggest that a possible mechanism for glycine be- taine action could be the stabilization of the struc- ture of large protein complexes. This gene has also been transferred to an elite indica rice variety, ‘Pusa Basmati 1’, and a preliminary study with R0 plants showed effectiveness of this gene for en- hancing salinity tolerance Mohanty and co-work- ers, unpublished. The effectiveness of superoxide dismutase Sod gene in providing salt tolerance has also been examined [110]. A yeast mitochondrial Mn-Sod gene, activity of whose product is not inhibited by H 2 O 2 , fused to the chloroplast targeting signal of glutamine synthase gene and 35 S promoter was used for transformation by electroporation into protoplasts of the japonica variety ‘Sasanishiki’. Studies with R2 homozygous lines revealed in- creased total SOD activity and enhanced tolerance to salt stress in these transgenic plants. It is obvi- ous that much more work is needed to engineer such complex traits as abiotic stress tolerance [111 – 114]. 3 . 8 . Engineering of rice for nutritional quality Improvement of the nutritional quality of rice is a very important consideration keeping in view its importance as food for a large number of people worldwide. Shimada et al. [115] produced trans- genic rice plants with antisense construct of rice Waxy gene coding for granule-bound starch syn- thase under the control of 35 S promoter. A signifi- cant reduction in amylose content of grain starch was observed in the seeds of these plants. In another study, Zheng et al. [116] obtained trans- genic rice with the gene for seed storage protein b-phaseolin of the common bean Phaseolus 6ul- garis L.. With the ultimate aim of increasing the lysine content of rice grain, the gene was driven by 5.1- or 1.8-kb promoter fragment of rice seed storage protein glutelin Gt 1 gene. This resulted in the expression of three glycoforms of 51, 48 and 45, in the endosperm. The antisense strategy was also applied to suppress the expression of the maturing rice seed 14 – 16-kDa allergen gene [117]. The seeds of transgenic plants showed much lower amounts of this protein and its transcripts, as compared to the parental wild type rice, for at least three generations. Most interestingly, to confer the capability of producing precursor b-carotene of vitamin A in rice endosperm, Burkhardt et al. [29] engineered rice with the cDNA coding for phytoene synthase from daffodil, the first of the four specific enzymes involved in b-carotene provitamin A biosynthesis in plants. In the endosperm of these transgenic plants, phytoene synthase accumulation was ob- served indicating that engineering of provitamin A biosynthesis pathway is possible in non-photosyn- thetic, carotenoid lacking tissue. Recently, the same group reported Agrobacterium-mediated transformation of rice with all the genes necessary for the accumulation of provitamin A in trans- genic rice seeds [66]. The genes transferred were daffodil phytoene synthase, Erwinia phytoene de- saturase and Narcissus lycopene b-cyclase. Glutelin promoter was used for endosperm-spe- cific expression and transit peptide sequences were used for targeting the products in endosperm plas- tids. The seeds of R0 transgenic rice showed re- markable accumulation of b-carotene providing yellow colour to seeds. It is expected that the homozygous derivative would be capable of providing enough b-carotene to fulfil the nutri- tional requirements. In yet another significant development, Goto et al. [64] reported Agrobacterium-mediated transfor- mation of the japonica variety ‘Kitaake’ with soy- bean ferritin gene. They used rice Glu-B 1 promoter for endosperm-specific expression of the transgene. Southern hybridization analysis re- vealed the presence of independent transformation events in different lines. Transgene expression was confirmed by RT-PCR. A polyclonal antibody raised against this protein detected a band of 28-kDa size by Western analysis. This protein accumulated up to 0.01 – 0.3 of the level of total soluble protein in seeds and the iron content of transgenic rice seeds increased up to threefold over control. The group led by Potrykus also reported their efforts to counter iron deficiency, which is the most prevalent micro-nutrient defi- ciency, by a three pronged approach [118,119]. They engineered rice with a ferritin gene from P. 6 ulgaris to increase iron content. As the phytate present in the gut inhibits absorption of iron, they engineered rice with a heat stable phytase gene from Aspergillus fumigatus. The sulphur-rich metallothionin-like protein exhibits resorption en- hancing effects, hence they also engineered rice with one such gene from rice. Preliminary studies revealed that transgenic plants have twofold higher iron content. Different transgenic plants harbouring these genes are being crossed to com- bine these useful traits [119]. These work may pave the way for reducing iron deficiency, which affects 30 of the total world population. 3 . 9 . Other engineered genes Several other examples of the introduction of genes into rice with variable objectives are avail- able. Interestingly, engineering of a proteinase in- hibitor gene for oryzacystatin Oc, IDD86, in rice also resulted in resistance against nematodes [34]. Although the level of expression was low up to 0.2 of total soluble protein, it resulted in 55 reduction in egg production by the nematode Meloidogyne incognita. Capell et al. [120] reported production of transgenic rice with oat arginine decarboxylase Adc cDNA keeping in view the role played by polyamines in several plant pro- cesses including stress. This led to morphological abnormalities in tissues and plants producing high level Adc transcripts accompanied with high level of putrescine. Tissues expressing very high level of Adc mRNA failed to differentiate. The effect was more prominent when tissues were exposed to light. Kisaka et al. [121] reported transformation of japonica rice by electroporation of protoplast with Rgp 1 gene, a small GTP binding protein from rice. Only one fertile transgenic plant could be regenerated. Southern hybridization analysis re- vealed the presence of single copy of transgene. R0 transgenic plants were short in comparison to control and produced three times more tillers but mostly without fertile seeds. Ku et al. [122] trans- formed japonica rice cultivars ‘Kitaake’ and ‘Nip- ponbare’ with an intact maize gene encoding C4- specific phosphoenolpyruvate carboxylase Pepc. Of the regenerated plants 10 showed abnor- mal morphology such as growth retardation, al- binism, narrow leaves, infertility, etc. The transgene was inherited up to R3 progeny in a Mendelian fashion and active enzyme was present in these generations. Western blot analysis re- vealed the presence of protein of expected size 106 kDa. The level of expression was very high as the transgene product accumulated up to 12 of total soluble protein. This is in fact the highest level of transgene expression reported in monocots to date. The probable reasons for such high expres- sion could be the use of an intact monocot gene together with its introns in another monocot driven by its own promoter as well as the strength of Pepc promoter. They also concluded that high level of expression is positively correlated with transgene copy number, which is contrary to con- temporary data that high copy number results in gene silencing. These transgenic plants resulted in reduced sensitivity of photosynthesis to O 2 inhibi- tion, which could be positively correlated with PEPC activity in these plants. Sung-In et al. [123] reported transformation of rice with an antimicro- bial protein Asa-AMP gene from Allium sati6um. They used MAR sequences from chicken lysozyme for position-independent expression of the transge- nes. Molecular analysis revealed the positive effect of MAR in obtaining low copy number transgen- ics and reduced variability of transgene expression.

4. Rice and functional genomics