Review and prospect of transgenic rice research

Chinese Science Bulletin

© 2009 SCIENCE IN CHINA PRESS

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Citation: Chen H, Lin Y J, Zhang Q F. Review and prospect of transgenic rice research. Chinese Sci Bull, 2009, 54: 4049―4068, doi: 10.1007/s11434-009-0645-x

Review and prospect of transgenic rice research

CHEN Hao, LIN YongJun & ZHANG QiFa† National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China

Rice is one of the most important crops as the staple food for more than half of the world’s population. Rice improvement has achieved remarkable success in the past half-century, with the yield doubled in most parts of the world and even tripled in certain regions, which has contributed greatly to food se-curity globally. Rapid population growth and economic development pose a constantly increased food requirement. However, rice yield has been hovering in the past decade, which is mainly caused by the absence of novel breeding technologies, reduction of genetic diversity of rice cultivars, and serious yield loss due to increasingly severe occurrences of insects, diseases, and abiotic stresses. To address these challenges, Chinese scientists proposed a novel rice breeding goal of developing Green Super Rice to improve rice varieties and realize the sustainable development of agriculture, by focusing on the following 5 classes of traits: insect and disease resistance, drought-tolerance, nutrient-use effi-ciency, quality and yield potential. As a modern breeding approach, transgenic strategy will play an important role in realizing the goal of Green Super Rice. Presently, many transgenic studies of rice have been conducted, and most of target traits are consistent with the goal of Green Super Rice. In this paper, we firstly review technical advances of rice transformation, and then outline the main progress in transgenic rice research with respect to the most important traits: insect and disease-resistance, drought-tolerance, nutrient-use efficiency, quality, yield potential and herbicide-tolerance. The pros-pects of developing transgenic rice are also discussed.

Oryza sativa, transgenic rice, Green Super Rice

Rice is one of the most important crops as the staple food for more than half of the global population. Rice breeding has achieved remarkable success in the past half-century, due to two breakthroughs: increasing harvest index and yield potential by reducing plant height making use of the semidwarf varieties since the 1960s, and second yield leap through developing and applying of rice hybrids since the 1970s. However, rice production in the new century is still confronting enormous challenges. For in-stance, consistent yield pressure due to global population increase is presented, associated with the reduction of arable land worldwide, while rice yield has reached the ceiling since the 1990s, mainly caused by decrease of genetic diversity of rice cultivars, increasingly severe oc-currence of insects and diseases in rice production, water shortage and increasingly frequent occurrence of drought. Meanwhile, overuse of chemical pesticides and fertilizers

also leads to environmental pollution and ecological dis-ruption. Moreover, higher quality requirements were posed with the development of social economy and peo-ple’s living conditions.

To address these challenges, Zhang[1] proposed the goal to develop Green Super Rice (GSR) aiming at re-ducing the use of pesticides and fertilizers, water-saving and drought-tolerance, improving quality and yield in rice production by improving the following five classes of traits: insect and disease-resistance, drought-tolerance, nutrient-use efficiency, quality and yield potential. He suggested taking a strategy of combining conventional breeding program, marker-assisted selection (MAS), and transgenic approach to make the best use of rice germ- Received September 18, 2009; accepted September 26, 2009 doi: 10.1007/s11434-009-0645-x †Corresponding author (email: [email protected])

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plasm resource to realize the goal of GRS. The transgenic approach provides new opportunities for rice breeding with the capacity to break the reproductive isolation be-tween species and realize the free communication of ge-netic materials. Reviewing the history of the development of transgenic rice in the past two decades, most target traits are consistent with the goal of GSR. In this article, we firstly give a brief account of the technical advances of rice transformation, and then outline the main progress in transgenic rice research with respect to the five classes of traits, and finally discuss the prospects for the development of transgenic rice.

1 Rice transformation

Rice transformation achieved important success in the late 1980s. Three independent groups reported on re-generated transgenic rice plants using rice protoplast as the recipient via electroporation-mediated or PEG-me-diated methods in 1988[2–4]. Rice transformation via par-ticle bombardment succeeded in 1991[5], which later became one of the most common methods of rice trans-formation. Chan et al.[6] acquired transgenic rice plants by Agrobacterium-mediated method in 1993. Hiei et al.[7] established the highly efficient Agrobacterium-mediated transformation system for japonica rice using the mature seed-derived callus as the explant, which subsequently became the most common rice transformation method. The transformation system of japonica varieties was further improved to shorten the transformation proce-dure[8]. Although Hiei et al.’s protocol established in 1994 made the transformation very amenable for japon-ica rice[7], that of indica rice was still obstinate. Some modifications were made to improve the transformation efficiency of indica rice[9,10]. Recently, Hiei and Komari[11] published a protocol of Agrobacterium-mediated trans-formation adaptable to both japonica and indica varieties. According to Hiei and Komari [11], transformation of in-dica rice can be done within 2.5 months using the imma-ture embryo with extremely high transformation effi-ciency (a single immature embryo may produce 5―13 independent transformants). However, the disadvantage of the protocol is that collection of immature embryos is laborious and limited by the season.

With the development of rice transformation, simple introduction of foreign genes into the genomes of target organisms can not meet scientists’ requirements any-more. Some special transformation technologies have

been developed according to different research purposes. The following is a brief introduction of some special transformation technologies.

1.1 Multigene transformation

Transformation of multiple genes is mainly applied to two purposes. Firstly, it facilitates the procedure of map-based gene cloning. A key step of map-based gene cloning is to validate the candidate genes. Transformation of multiple genes with a single construct is very important to this step, because the more candidate genes that can be transformed once, the less labor of transformation.

Secondly, multigene transformation may play an im-portant role in rice transgenic breeding. The introduced foreign genes in commercialized transgenic crops are generally single genes to control qualitative traits such as insect-resistance, disease-resistance, or herbicide-resistance. However, many crop traits are actually controlled by multiple genes. To improve these traits, the multiple genes must be introduced into the crop simultaneously. Moreover, transformation of multiple genes is also needed in case of promptly pyramiding multiple qualitative traits or introducing novel metabolic pathways consist-ing of multiple genes. Golden rice is a famous example, in which a novel β-carotenoid biosynthesis pathway is established in rice endosperm by introducing two for-eign genes into transgenic rice[12]. There are two com-monly available strategies of multigene transformation. One is to construct foreign genes in different vectors firstly, and then the multigene pyramiding is performed by ways of co-transformation, repetitive transformation, or separate transformations in combination with hy-bridization. The production of golden rice took this strategy. Another one is to construct foreign genes in a single vector, and multiple genes are then introduced into the recipient by a transformation event[13]. Obvi-ously, the latter strategy is more amenable and economic compared with the former one. However, transformation with large DNA fragments is the main difficulty of mul-tigene transformation. The cloning capacity of common Ti binary vectors such as pCAMBIA series is limited, because their replicons derive from plasmid. The cloning capacity of a common Ti binary vector is usually less than 20 kb, which can approximately carry 2－3 foreign genes and appears inadequate for multigene transforma-tion. Some special Ti vectors have been developed to en-hance cloning capacity of large DNA fragments. There are two main Ti vectors for transformation of large DNA

Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4051

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fragments: BIBAC (Binary BAC) derived from bacterial artificial chromosome[14] and TAC (Transformation-competent Artificial Chromosome) derived from P1 artificial chromosome[15], both of which can accept a foreign DNA fragment more than 100 kb. BIBAC and TAC have been successfully applied in rice transformation[16,17]. With the development of BIBAC and TAC vectors, multigene transformation would have a huge potential for rice transgenic breeding.

1.2 Tissue-specific/inducible expression

Constitutive CaMV 35S and maize Ubiquitin promoters are the two most common promoters used in rice trans-genic research. There are certain problems to express transgenes in all plant tissues and organs at all growth stages using a strong constitutive promoter, for instance, increasing the metabolic burden of transgenic plants, and causing the public’s concerns about the food safety due to accumulation of the protein products of trans-genes in the edible parts of transgenic plants. Moreover, constitutive expression of some good genes, such as abiotic stress-resistance related transcription factor genes in transgenic plants would lead to abnormal plant growth and development. Thus, tissue-specific/inducible expression is crucial for transgenic breeding, which is usually implemented by making use of tissue-specific/ inducible promoters.

Transgenic Bt rice is the most promising transgenic rice for commercialization. However, the public’s con-cern about the food safety of Bt protein is a major bar-rier to its release. Ye et al.[18] introduced a synthetic cry1C* driven by the rice rbcs (a small subunit of ribu-lose-1,5-bisphosphate carboxylase/oxygenase) promoter into a japonica variety Zhonghua 11 by Agrobacte-rium-mediated transformation. In acquired transgenic plants, Bt protein is expressed predominantly in green parts of the plant such as the leaf and stem that are mainly targets attacked by insect pests, while barely in the edible endosperm. The expression level of Cry1C* in the leaf of transgenic plants when driven by rice rbcs promoter is almost three times of that when driven by the maize Ubiquitin promoter; contrarily Cry1C* con-tent in endosperm when driven by the rice rbcs promoter is less than 1/1000 of that when driven by the maize Ubiquitin promoter compared with the results of Tang et al.[18,19]. It is supposed that Bt rice with green part- specific expression is more acceptable to the consumers and therefore more promising to commercialization.

1.3 Chloroplast transformation

Chloroplast transformation is usually implemented by delivering plasmid vectors containing transgenes into chloroplasts with a direct method, such as particle bom-bardment. The transgenes are integrated into the chloro-plast genome through homologous recombination of homologous sequences flanking transgenes. There are two main advantages of chloroplast transformation com-pared with the common nuclear transformation. Firstly, expression efficiency of foreign proteins is extremely high due to high transgene copies. There are generally 10－100 chloroplast genome copies per chloroplast and 10－100 chloroplasts per cell, resulting in theoretically as many as up to 10000 transgene copies per cell that is much more than that by nuclear transformation. Therefore, the expression efficiency of chloroplast transformation is supposed to be much higher than that of nuclear trans-formation. Transgenic plants of chloroplast transforma-tion can have a high accumulation of foreign proteins (up to 47% of total soluble protein)[20]. Secondly, the inheri-tance of transgenes integrated in chloroplast genome shows a maternal pattern, which can prevent the trans-gene flow from transgenic plants to non- transgenic va-rieties or wild relatives by pollination. Thus, the field experiment or commercial production of transgenic plants acquired via chloroplast transformation is safer and more environment-friendly. Furthermore, there are some other advantages, for instance, transgene is inte-grated through homologous recombination at a precise, predetermined location resulting in elimination of “posi-tion effect” and uniform expression level among differ-ent transformants; chloroplast genes are often arranged in operons, that means a promoter is able to control the expression of multigenes as a polycistron, which may facilitate multigene transformation; gene silencing of chloroplast transformation has never been reported so far, while which is often observed in nuclear transforma-tion[20].

Although chloroplast transformation is a very promis-ing technology with many advantages, it has not been applied as widely as nuclear transformation due to many practically technical difficulties. So far, chloroplast transformation has been achieved only for more than 10 plant species, and there are few reports about chloroplast trans-formation in rice[21–24].


2 Transgenic insect-resistant rice

Insect destroy is one of the major causations of yield loss, which leads to about 10% yield loss annually. Spraying chemical insecticides is the major way to pre-vent insect destroy in rice production. However, overuse of chemical insecticides not only increases production costs, but also pollutes the environment and threatens human health. Enhancing insect-resistance of rice itself by breeding approaches is a more economic and envi-ronment-friendly strategy. However, developing insect- resistant cultivars by conventional breeding approaches is time-consuming. Moreover, no effective resistance germplasm resources have been identified in rice against striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas), and leaffolder (Cnapha-locrocis medinalis), which are main rice pests. The most promising method currently is to develop transgenic in-sect-resistant varieties by introducing foreign insect- re-sistant genes into rice. Many useful insect-resistant genes have been identified and isolated from plants, animals, and even microorganisms. Transgenic insect-resistant rice lines have been obtained by introducing these in-sect-resistant genes. Some of them have been tested under field conditions and showed broad potential application for production.

2.1 Transgenic Bt rice

Bt toxin genes derived from Bacillus thuringiesis (Bt) is one of the most broadly-used insecticidal genes world-wide. Bt forms various crystals upon sporulation, which are a class of proteins with specific insecticidal activities, referred to as Bt toxins or insecticidal crystal proteins. Transgenic Bt crops acquire insect-resistance due to the accumulation of Bt toxin in the plant. Bt genes have been successfully transferred and expressed in different crops including rice. Among them, Bt cotton, corn, and potato have been commercially growing and bringing huge economic benefits[25].

Various Bt toxins with specific insecticidal activities against species of the orders lepidoptera, coleoptera, diptera, and invertebrata (acarids, nematodes, and pro-tozoa) have been identified and isolated from different Bt strains. Totally more than 400 Bt genes have been cloned so far (http://www.lifesci.sussex.ac.uk/home/Neil_ Crickmore/Bt/toxins2.html). However, in spite of so many Bt genes, only a small proportion of them have been used in transgenic plants. The most common Bt genes

used in transgenic rice are cry1A including cry1Ab[26–35], cry1Ac[30,36–39], and cry1Ab/Ac fusion gene[40,41]. There are limited studies involving other Bt genes[19,42–47]. Most of these transgenic Bt rice showed high resistance against striped stem borer, yellow stem borer, and leaf-folder.

Tu et al.[40] have been performed field experiment of transgenic rice harboring a cry1Ab/Ac fusion gene. Their results showed that transgenic cry1Ab/Ac Minghui 63 (an elite rice restorer line) and its hybrid Bt Shanyou 63 exhibited high insect-resistance in field conditions without spraying any chemical insecticides during the whole growth period, indicating huge use value of Bt rice in production.

As applying other insecticides or resistant varieties, one of the major risks of Bt crops is that insects might evolve resistance against Bt crop, which would impair its durability. Although no insect species with resistance to Bt crops have been identified under natural conditions so far, some insects have evolved resistances against Bt spray reagents in the field. Moreover, many Bt toxins- resistant insect strains have been selected in the green house or laboratory, and some of them were able to sur-vive on Bt crops[48], indicating the risk that insects have the potential to evolve the resistance against Bt crops in field conditions.

Utilization of two-toxin Bt rice is an important strat-egy to delay insect-resistance and prolong the durability of Bt rice[49]. Two-toxin Bt rice is a transgenic rice ex-pressing two different Bt toxins in combination. In prin-ciple, the frequency that insects evolve a resistance against two Bt toxins simultaneously is much lower than that against one Bt toxin. Therefore, two-toxin Bt rice can greatly delay the development of insect-resistance and is more durable. However, the two Bt toxins in combination must bind to different receptor sites on insect gut cells to avoid the occurrence of “cross-resistance”. As described previously, common Bt genes used in rice are cry1A such as cry1Ab, cry1Ac, and fused cry1Ab/Ac. It is not suitable to combine two cry1A genes because in-sects are prone to develop a cross-resistance to over-come them because they shared very high protein se-quence homology each other. Therefore, Chen et al.[47] and Tang et al.[19] developed transgenic rice with syn-thetic cry2A* and cry1C*, respectively. Field experi-ments showed that both transgenic Cry2A* rice and Cry1C* rice were highly resistant against lepidopteran


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rice pests. Transgenic Cry2A* rice and Cry1C* rice may provide new gene resources for the development of two- toxin Bt rice.

Studies showed that cry1A, cry1C and cry2A are suitable to combine because insects unlikely develop a cross-resistance to them due to their low protein se-quence homology each other[50,51]. Yang et al. developed 10 two-toxin Bt rice lines 1Ab/1C, 1C/1Ab, 1Ab/2A, 2A/1Ab, 1Ac/1C, 1C/1Ac, 1Ac/2A, 2A/1Ac, 1C/2A and 2A/1C by reciprocal hybridizations of 4 transgenic Minghui 63 lines with different Bt genes cry1Ab (1Ab), cry1Ac (1Ac), cry1C* (1C), and cry2A* (2A), in five combination patterns (1Ab+1C, 1Ab+2A, 1Ac+1C, 1Ac+2A, 1C+2A) (Yang Zhou and Lin Yongjun, un-published data). The transgenic line 1Ab/1C means the maternal line of the hybrid is 1Ab, and the paternal line is 1C; while 1C/1Ab means contrary parents. The rest may reason by analogy. The results of bioassay in

the laboratory showed that most combinations of two Bt toxins had synergistic effects and exhibited significantly higher insect-resistance than single Bt gene.

Bt genes are the most successful insect-resistant genes that have been applied in transgenic rice so far, which can effectively control lepidopteran rice pests (Figure 1(a) and (b)). Bt rice has been temporarily commercialized in Iran 2005. Bt rice has been well-developed in China and can be commercialized promptly as soon as the policy permits.

2.2 Transgenic rice with plant or animal-derived genes

Plant-derived insect-resistant genes commonly include plant lectin genes and protease inhibitor genes. Plant lectin genes have a relatively high insecticidal activity, among which Galanthus nivalis agglutinin (GNA) gene has been widely applied. The principal advantage to use

Figure 1 Transgenic insect-resistant rice ((a) and (b)) and transgenic drought-tolerant rice ((c)and (d)). (a) WT, wild-type Minghui63 control; 1Ac+1C, two-toxin Bt Minghui63 (1Ac+1C). (b) WT, wild-type Minghui63 control; 1Ac+2A, two-toxin Bt Minghui63 (1Ac+2A). (c) WT, wild-type Nipponbare control; SNAC1, SNAC1-overexpressing transgenic Nipponbare. (d) WT, wild-type Zhonghua 11 control; S58S, OsSKIPa-over expressing transgenic Zhonghua 11.


GNA gene is that GNA has certain insecticidal activity against sap-sucking (homoptera) insects such as rice planthoppers, which is not able to be controlled by Bt toxins. Sun et al.[52] obtained homogenous transgenic GNA rice lines via particle bombardment. Their data showed that the homogenous transgenic lines can control brown planthopper (Nilaparvata lugens, BPH) by sig-nificantly decreasing survival rate and fecundity, retarding development and declining feeding. More studies have proved that transgenic GNA rice had some insecticidal effects on planthoppers, leafhoppers, and aphids[38,43,52–59]. However, toxicity of GNA to sap-sucking rice pests is not comparable to that of Bt toxin to lepidopteran rice pests. The effects of GNA are to significantly restrain the in-sect’s growth, development, and fecundity. There is an-other study involving an Allium sativum agglutinin from leaf (ASAL) gene. Saha et al.[60] obtained transgenic rice overexpressing ASAL gene, which also exhibited en-hanced resistance to BPH and green leafhopper (Nepho-tettix cinciteps). Moreover, expressing ASAL in trans-genic rice plants significantly reduced the infection inci-dence of rice tungro diseases, caused by co-infection of green leafhopper-vectored rice tungro bacilliform virus and rice tungro spherical virus[60].

In addition to plant lectin genes, protease inhibitor genes are another group of plant-derived insect-resistant genes. The protease inhibitor genes that have been tested in transgenic rice include: potato protease inhibitor gene pinII[61,62], cowpea trypsin inhibitor gene CpTI[63], soy-bean kunitz trypsin inhibitor gene SKTI[64], corn cystatin gene[65], rice cystatin gene[66] and barley trypsin inhibitor gene BTI-Cme[67]. These transgenic rice plants exhibited certain resistance to BPH, striped stem borer, leaffolder, nematode, etc.

Utilization of plant-derived insect-resistant genes has some special advantages, for instance, they generally have a broad-spectrum insect resistance, and especially GNA has some resistance against homoptera rice pests that Bt toxins are unable to control. However, the appli-cation of plant-derived insect-resistant genes is still lim-ited because of their relatively inadequate insecticidal activities.

There are very few studies to use animal-derived in-sect-resistant genes. Huang et al.[68] reported to acquire transgenic insect-resistant rice against striped stem borer and leaffolder by introducing an insecticidal gene SpI from spider into rice varieties Xiushui 11 and Chunjiang 11.

All transgenic insect-resistant rice described above acquired their resistance through directly expressing foreign insecticidal protein. Recently, a novel insect- resistant strategy, suppressing the expression of key genes for pest development or biochemical metabolism via RNA interference (RNAi) using gene fragment from the target pest itself, has succeeded in developing trans-genic insect resistant corn[69] and cotton[70]. This strategy might become a new research trend to develop trans-genic insect-resistant plants. However, it should be noted that if using RNAi strategy, the targeting sites must be pest gene-specific to ensure that the transgenic plant is harmless to other species especially to humans.

3 Transgenic disease-resistant rice

Bacterial blight (BB), fungal diseases blast, and sheath blight are three main diseases in rice production. Bacte-rial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) is the most devastating rice bacterial disease worldwide[71], which may cause 20%－30% yield loss, or even 100% in case of severe occurrence. Blast caused by Magnaporthe grisea (M. grisea) may arise in all rice organs at any growth stage. Sheath blight caused by Rhizoctonia solani (R. solani) may lead to whitehead, reductions of fertility and grain weight, and 10%－30% yield loss, even more than 50% if serious.

More than 30 BB resistance (R) genes or loci against Xoo have been identified in rice so far. Among them, six R genes (Xa1, Xa3/ Xa26, xa5, xa13, Xa21, and Xa27) have been cloned and many (Xa4, Xa7, Xa10, Xa22(t), Xa23, xa24, Xa25(t), and Xa31(t)) fine-mapped[72,73]. BB is effectively controlled in rice production due to the application of R genes and resistant varieties. For trans-genic breeding, introducing R genes into the desired rice varieties is a direct and convenient way. Zhang et al.[74] introduced a broad-spectrum R gene Xa21 into Minghui 63, and the acquired transgenic Minghui 63 showed sig-nificantly enhanced resistance to Xoo. Wu et al.[75] ob-tained marker-free BB-resistant transgenic Minghui 63 and WanB (a rice maintainer line) by introducing Xa21 into the corresponding wild-type recipients, and their hybrids also exhibited significantly enhanced BB-resistance.

More than 60 major Blast-resistant genes have been identified in rice so far[76], among which 10 resistant genes (Pib, Pi-d2, Pikm, Pi-ta, Pizt, Pi2, Pi5, Pi9, Pi36, and Pi37) have been cloned[77]. Because M. grisea has


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many physiological races with high variability, a Blast- resistant cultivar might lose the resistance 3－5 years after it is adopted in production widely. As for R. solani, no major resistant genes have been identified in rice.

Overexpressing pathogenesis-related proteins (PRs), including chitinase, β-1,3-glucanases, and thaumatin- like proteins, and other plant- or microorganism-derived antifungal proteins, is a common strategy to develop transgenic fungus-resistant rice. PRs are a battery of proteins encoded by the host plants but induced exclu-sively in pathological or related situations, and many of them showed antifungal activity in vitro[78]. Some stud-ies have confirmed that overexpressing chitinases in transgenic rice enhanced the resistance against both M. grisea[79–82] and R. solani[83]. Nishizawa et al.[84] re-ported that overexpressing β-1,3-glucanase in transgenic rice enhanced resistance against M. grisea; Datta et al.[85] found that overexpressing thaumatin-like protein in transgenic rice enhanced resistance against R. solani. Besides using single PR genes, pyramiding different PR genes is also common. For instance, combinations of chintinase with β-1,3-glucanase can enhance the resis-tance of transgenic rice to blast[86–88]; combination of chitinase with a modified maize ribosome-inactivating protein[89] or a thaumatin-like protein[90] can enhance resistance to sheath blight. Moreover, some studies at-tempted to enhance the resistance of transgenic rice to fungal diseases by overexpressing antifungal proteins or peptides from plants or microorganisms in rice, and also achieved some effects[91–94].

Expressing pathogen-derived protein elicitors in trans-genic rice to induce the plant general defense response and system-acquired resistance (SAR) is another strategy for developing transgenic rice with enhanced disease re-sistance. Shao et al.[95] reported that overexpression of a protein elicitor harpin from Xoo in transgenic rice con-ferred high non-specific resistance to multiple M. grisea races.

Besides qualitative major resistance genes, recent studies of quantitative resistance genes (resistant QTLs) are worth noting. Although the resistance of single quantitative resistance gene is relatively limited com-pared with the major resistance genes, their advantages are broad-spectrum and more durable. Nevertheless, no major resistance genes have been found in rice for some rice diseases such as rice sheath blight, false smut, and bacterial leaf streak, and thus the research of resistant

QTLs is very valuable to develop the resistant varieties against those diseases.

A few studies have shown that overexpressing some resistant QTLs in rice may obtain satisfying results too, although most natural resistant QTLs have minor effects. Qiu et al.[96] overexpressed a resistant QTL OsWRKY13 driven by the maize Ubiquitin promoter in a BB suscep-tive rice variety, and the transgenic plants exhibited en-hanced resistance to Xoo. Xiao et al.[97] suppressed the expression of a resistance-related QTL OsDR10 in rice via RNAi, and the transgenic plants showed enhanced resistances to multiple Xoo strains compared with the non-transgenic control. It should be noted that the resis-tance reaction regulated by the resistant QTLs is not species or race-specific but broad-spectrum basic resis-tance. The resistance level of the resistant QTLs is not comparable with that of qualitative resistance conferred by major resistance genes, but they are still worthy of research and utilization because of their broad-spectrum and durability.

4 Transgenic drought-tolerant rice

Drought is one of the major factors causing yield loss in rice production for a long time and is getting worse as the climate changes worldwide. Rice production need consume a huge amount of water, accounting for ap-proximate 70% water consumption of agriculture in our country. While China is water deficient, and the average capita water capacity is only a quarter of that of the world. Therefore, developing drought-tolerant rice va-rieties and reducing water consumption in rice produc-tion is crucial to increasing rice yield and ensuring the food security of China.

One distinguishing feature of plants from animals is that plants are not “movable”. Correspondingly, plants evolve a complex biological mechanism to resist various environmental stresses. When under an environmental stress such as drought, the initial signals are perceived by the sensors (including ion proteins, histidine kinases, and G-protein coupled receptors) of the plant cell, and transduced to second messenger molecules such as Ca2+, reactive oxygen species (ROS), and inositol phosphates that can transfer further in the plant cell. Then, protein phosphorylation cascades of Ca2+-dependent protein kinases (CDPKs), mitogen-activated protein kinases (MAPKs), etc. triggered by the second messenger molecules activate the downstream transcription factors.


The activated transcription factors can subsequently regulate the expression of a many of downstream func-tional or structural genes such as late embryogenesis abundant (LEA) proteins, various catalytic enzymes that synthesize osmoprotectants, antifreeze proteins, channel proteins to help the plant re-establish osmotic homeostasis, scavenge harmful compounds, protect and repair dam-aged proteins and membrane systems caused by the stresses[98–100].

Due to the complex mechanism of drought-tolerance, it is difficult to develop drought-tolerant varieties only relying on conventional approaches. Nowadays, genetic engineering has been broadly applied to developing drought- tolerant rice, and a common strategy is to overexpress drought-responsive or related genes in transgenic rice. Table 1 summarizes some representative experiments about transgenic drought-resistant rice. The applied transgenes can be roughly classified into two groups according to their functions and action patterns. One

group is referred to as functional or structural genes, including LEA proteins, water channel proteins, cata-lytic enzymes that synthesize osmoprotectants (com-patible solutes) including proline, trehalose, glycinebe-taine, polyamines, etc., and detoxifying genes such as superoxide dismutase (SOD). This group was generally used in the initial transgenic studies because the mecha-nism is comparatively simple. Another group is regula-tory genes, which function in the upstream of the drought response network, including CDPKs, calcineurin B-like protein-interacting protein kinases (CIPKs), MAPKs, transcription factors, etc. Modifying the expression of these genes generally can influence the expression level of a battery of downstream drought-related genes. Ap-plication of the regulatory genes is thought to be more effective than those functional or structural genes with simple functions, considering the complexity of drought- tolerant mechanism.

Hu et al.[128] reported a drought-tolerance transcription Table 1 Summarization of recent transgenic rice trials of drought-tolerance

Gene Gene type/function Source Effect Reference

P5CS improve proline synthesis mothbean (Vigna aconitifolia L.) proline increase, drought and salt-tolerance [101,102]

TPSP improve trehalose synthesis E. coli trehalose increase, drought, salt, and cold-tolerance [103,104]CodA improve glycine betaine synthesis Arthrobacter globiformis glycine betaine increase, drought-tolerance [105] adc improve polyamine synthesis Oat, Datura stramonium putrescine increase, drought-tolerance [106,107]HAV 1 LEA protein barley drought and salt-tolerance [108―110]PMA80 PMA1959 LEA protein wheat drought and salt-tolerance [111] OsLEA3-1 LEA protein rice drought-tolerance [112] sHSP17.7 heat shock protein rice drought-tolerance [113] MnSOD detoxification pea drought-tolerance [114] Sod1 detoxification Avicennia marina drought and salt-tolerance [115] RWC3 water channel protein rice drought-tolerance [116] OsCDPK7 CDPK rice drought, salt, and cold-tolerance [117] OsMAPK5 MAPK rice drought, salt, and cold-tolerance [118] OsCIPK 12 CIPK rice drought-tolerance [119] CBF3 transcription factor Arabidopsis drought, salt, and cold-tolerance [120] ABF3 transcription factor Arabidopsis drought tolerance [120] OsDREB1A,1B; DREB1A, 1B, and 1C transcription factor rice, Arabidopsis drought, salt, and cold-tolerance, growth retardation [121]

OsDREB1F transcription factor rice drought, salt, and cold-tolerance [122] ZFP25 transcription factor rice drought and salt-tolerance [123] OsDREBs transcription factor rice drought-tolerance [124] OsWRKY11 transcription factor rice drought and heat-tolerance [125] OsbZIP23 transcription factor rice drought and salt-tolerance [126] SNAC1 transcription factor rice drought and salt-tolerance [127] OsSKIPa SKI-interacting protein homolog rice drought and salt-tolerance [128] OsiSAP8 stress associated protein rice drought, salt, and cold-tolerance [129] OCPI1 proteinase inhibitor rice drought-tolerance [130] ZFP177 A20/AN1-type zinc finger rice drought-tolerance [131] OsMT1a type 1 metallothionein rice drought-tolerance [132] OsCOIN cold-induced zinc finger rice drought, salt, and cold-tolerance [133]


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factor gene SNAC1 with great potential application, which is a member of NAC (NAM, ATAF, and CUC) plant-specific gene family. SNAC1 is specifically ex-pressed in leaf guard cells under drought stress condi-tions. Overexpressing SNAC1 significantly enhanced drought resistance in transgenic rice (22%－34% higher seed setting rate than the control) at the reproductive stage in the field under severe drought stress conditions without showing any phenotypic changes or yield pen-alty. Compared with the control, transgenic rice plants were more sensitive to abscisic acid (ABA) and lost wa-ter more slowly by closing more stomatal pores, and maintained turgor pressure under lower relative water content[128]. The transgenic rice also showed signifi-cantly improved drought and salt-tolerance at the vege-tative stage (80% higher survival rate compared with the control) (Figure 1(c)). DNA microarray analysis re-vealed that over 150 stress-related genes were up-regu-lated in the SNAC1-overexpressing rice plants.

Hou et al.[129] recently published a drought-tolerance related gene OsSKIPa. Drought-tolerance of OsSKIPa- overexpressing rice plants increased 2―4 fold compared with the control at the adult stage (Figure 1(d)). The OsS-KIPa-overexpressing rice showed significantly increased ROS-scavenging ability by analyzing the relative levels of SOD and monodehydroascorbate (MDA) in plants under drought stress. Moreover, the transcript levels of many stress-related genes are significantly higher than the wild-type control after drought stress treatment.

Although many studies about transgenic drought- resistant rice have been reported (Table 1), the data were obtained under greenhouse conditions, and very few studies under field conditions have been reported. Xiao et al.[134] introduced seven well-documented stress- resistant genes under the control of constitutive Actin1 promoter and stress-inducible promoter of a rice HVA22 homolog (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, and NHX1) into Zhonghua 11, and then the drought- resistance of regenerated transgenic rice lines was tested under field conditions. Their results showed that trans-genic families of eight constructs (HVA22P:CBF3, HVA22P: NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, Actin1:NHX1, and HVA22P:NHX1) had significantly higher relative yield than the wild-type con-trol in both field and PVC pipes conditions with drought stress. Transgenic families of 10 constructs (HVA22P:

SOS2, Actin1:ZAT10, and CBF3, LOS5, ZAT10, and NHX1 by both promoters) showed significantly higher relative spikelet fertility than the wild-type control in the PVC pipes under drought stress. In the field drought resistance testing of T2 and T3 families, transgenic fami-lies of seven constructs (HVA22P:CBF3, Actin1:NPK1, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1: ZAT10, and HVA22P:ZAT10) showed significantly higher yield per plant than the wild-type control, and families of nine constructs (Actin1:CBF3, HVA22P: CBF3, HVA22P:SOS2, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, and Actin1:NHX1) had higher spikelet fertility than the wild-type control. In conclusion, LOS5 and ZAT10 showed relatively better effects than the other five genes in improving drought resistance of transgenic rice under field conditions. The results of this study were based on field experiments and might be a useful reference for developing practical transgenic drought-resistant rice.

An ideal drought-tolerant rice variety should have high yield and good quality when water is adequate, while higher yield than the best rice cultivars under wa-ter-deficit or drought conditions. Although certain ad-vances have been made in transgenic breeding of drought- tolerant rice, it is still far from developing a practical drought-tolerant rice variety. In view of the complex mechanism of drought-tolerance, it is crucial to pyramid various drought-tolerant genes by taking an integrated strategy of transgenic approaches, MAS and conven-tional breeding programs.

5 Transgenic nutrient-use efficient rice

Chemical fertilizer is the basis of modern agriculture, which ever contributes greatly to improving food crop production and ensuring food security. Food crop pro-duction has been doubled in the past four decades worldwide due to the green evolution, associated with a seven-fold increase in the use of nitrogen (N) fertiliz-ers[135]. However, this high-production pattern relying on a high investment is not sustainable. The increase of food production is not so significant anymore even if the use of fertilizer still keeps growing in the past decade. Nevertheless, overuse of fertilizer is leading to a series of environmental issues, such as eutrophication of water body, groundwater pollution, soil acidification, etc. As an unrenewable resource, the global supply of phospho-


rus ore can barely sustain to the end of this century[136]. These challenges threaten not only the ecological security but also sustainable development of agriculture. Therefore, research and development of nutrient-use efficient rice varieties in combination with scientific fertilization and cultivation management to substantially decrease the use of fertilizer is very important to ensure food security and realize the sustainable development of modern agriculture.

5.1 Nitrogen-use efficiency

N is an essential nutrient that plants require in the most quantity and is also a major limiting factor in crop pro-duction. NO3

− and NH4+ are two major inorganic N

compounds presenting in agricultural soils. NO3− is

converted to NH4+ by two reductases: nitrate reductase

and nitrite reductase in the plant after it is absorbed from the soil. NH4

+ is converted to glutamine (Gln) and glu-tamate (Glu) by the GS/GOGAT cycle consisting of two key enzymes glutamine synthetase (GS) and glutamate synthetase (GOGAT). Glu can be further transferred to many other amino acids by different aminotransferases. Rice prefers NH4

+ as the major N source, which is ac-tively absorbed from the soil by different ammonium transporters in rice roots, and subsequently assimilated by GS and NADH-GOGAT in roots[137].

GS is tissue/cell-type specific. GS1 exists predomi-nantly in seeds, roots, nodules, flowers, and phloem, which is inducible by water-flood, pathogens, and se-nescence, and may function in N assimilation and trans-location. GS2 is the predominant isoenzyme in leaves that may function in assimilation of ammonia reduced from nitrate in chloroplasts and/or in the reassimilation of photorespiratory ammonia[138]. There are four GS genes in rice: one encoding the chloroplastic/plastidic GS2 that exists predominantly in leaf cells, and three ones encoding cytosolic GS1 that exists predominantly in the root (GS1;2), stem (GS1;1) and spikelet (GS1;3)[138,139].

Yamaya et al.[140] found that expression of a NADH- dependent glutamate synthase (NADH-GOGAT) gene from a japonica variety Sasanishiki in an indica cultivar Kasalath increased significantly grain weight (up to 80%) compared with the non-transgenic control, indicating that NADH-GOGAT is indeed a key step for N utiliza-tion and grain-filling in rice.

Cai et al.[141] overexpressed GS1;1, GS1;2 from Ming hui 63 and an Escherichia coli (E. coli)-derived GS gene glnA under the control of CaMV 35S promoter in

Zhonghua 11, and found that all GS-overexpressed (in-cluding GS1;1, G1;2 and glnA) transgenic plants showed higher total GS activities and soluble protein concentra-tions in leaves and higher total amino acids and total N content in the whole plant. However, both grain yield and total amino acids in seeds of GS-overexpressed rice plants decreased compared with the wild-type control under field conditions with N deficit stress.

Ammonium transporters are crucial for the plant root to take up NH4

+ from the soil. Ten ammonium transporter genes have been identified in rice, among which OsAMT1;1, OsAMT1;2 and OsAMT1;3 belong to ATM1 subfamily, and the other seven (OsAMT2;1, OsAMT2; 2, OsAMT2;3, OsAMT3;1, OsAMT3;1, OsAMT3;3, and OsAMT4) belong to ATM2 subfamily. Kumar et al.[142] found that the flow of 15NH4

+ in transgenic plants over-expressing OsAMT1;1 changed, and the biomass of transgenic plants decreased compared with the control. Ho-que et al.[143] found that the biomass of transgenic rice overexpressing OsAMT1;1 significantly decreased at vegetative growth stage compared with the wild-type control. Moreover, the transgenic plants showed increased am-monium uptake and ammonium content in roots. It is supposed that biomass decrease of the transgenic plants at the early growth stages might be caused by phytotox-icity due to the accumulation of ammonium in the root.

Overexpressing some aminotransferases in transgenic plants has also been attempted to change the level of amino acid synthesis and N metabolism, which is ex-pected to improve N-use efficiency in rice. Shrawat et al.[144] reported that tissue-specifically expressing a barley alanine aminotransferase (AlaAT) cDNA in rice roots significantly increased the biomass and grain yield compared with the control. Moreover, some key me-tabolites such as Gln and total N content in transgenic rice plants also increased, indicating enhanced N uptake efficiency. Zhou et al.[145] overexpressed separately all of three rice aspartate aminotransferase (AAT) genes (OsAAT1-3) from rice and an E. coli-derived AAT gene (EcAAT) in transgenic rice. The transgenic plants over-expressing OsAAT1, OsAAT2 and EcATT showed sig-nificantly increased leaf AAT activity and higher grain amino acid and protein contents compared with the non-transgenic control. No significant changes were found in leaf AAT activity, grain amino acid content, or protein content in OsAAT3 overexpressed rice plants. Moreover, transgenic rice plants overexpressing OsAAT1, OsAAT2, OsAAT3, and EcAAT did not show significant


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difference in main agronomic traits and yield compared with the wild-type control.

5.2 Phosphorus-use efficiency

Phosphorus (P) is one of the essential macroelements too. Although the absolute P amount in the soil is com-paratively abundant, the available P is deficient (usually less than 10 μmol/L or even less) due to its low solubil-ity and high adsorptive capacity[136,146]. As a result, im-proving the capacity of rice plants to activate and utilize the fixed P in the soil is a major research objective of developing P-use efficiency varieties.

Yi et al.[147] identified a P-deficiency responsive tran-scription factor OsPTF1 from Kasalath, a P-use efficient indica landrace. Overexpressing OsPTF1 in a low-P sensitive rice variety Nipponbare significantly enhanced P-use efficiency. Tillering ability, root and shoot bio-mass, and P content of the transgenic plants were >30% higher than those of the wild-type plants in P-deficient culture solution. In pot and field experiments with low-P levels, tiller number, panicle weight, and P content in-creased >20% in transgenic plants, compared with the wild-type control. Moreover, total root length, root sur-face area, and P uptake rate of transgenic rice plants were also significantly higher than the control in P- deficient conditions.

For phosphate uptake of plants, phosphate firstly en-ters the rice apoplast made up of the cell wall of epider-mis and cortex cells from the soil, and then is transferred through membrane into the symplast by phosphate transporters, and finally transported to the shoots of the plant via xylem and distributed to various organs[148]. Most of high-affinity P transporter genes are expressed pre-dominantly in roots and are induced by P depletion, in-dicating that they are involved in the acquisition of P through the roots under low external P concentrations. Seo et al.[149] identified a phosphate transporter gene OsPT1 that is expressed primarily in roots and leaves regardless of external phosphate concentrations. Trans-genic rice plants overexpressing OsPT1 under the con-trol of the CaMV 35S promoter accumulated almost twice as much phosphate in the shoots compared with the wild-type control under both normal and P-null ferti-lizations. The transgenic plants had more tillers and bet-ter root development. However, transgenic rice overex-pressing OsPT1 was 30% shorter than the wild-type control, which was supposed to be caused by the com-parative deficiency of other nutrients such as N and po-

tassium because they were not concomitantly increased with an enhanced P acquisition.

6 Transgenic high quality rice

Rice quality is recently getting more and more attention with the improvement of people’s living conditions. The physical and chemical indexes of good quality rice gen-erally include processing quality, appearance quality, cooking and eating quality, nutritional quality[150]. Actu-ally, several important genes controlling rice quality traits such as GS3 for grain length[151], GW2 for grain width[152], Alk for gelatinization temperature[153], and Wx for amylase content[154], have been cloned, and some quality related genes have been fine-mapped, which greatly facilitate the improvement of rice quality by us-ing MAS or transgenic strategies.

Transgenic approaches have been applied mainly to improving the nutritional quality of rice at present. Other than providing energy, rice is also an important source of proteins. Zhou et al.[155] analyzed the crude protein con-tents (PC) in 351 rice varieties, and the results showed that the PC varied between 9.3% and 17.7%, and the average value is 12.4%. The average PC of indica varie-ties is 13.2% that is approximately 1% higher than that of japonica varieties. The nutritional quality of rice would be enhanced by increasing protein content espe-cially the amount of essential amino acids such as lysine in rice endosperm using transgenic approaches. A com-mon strategy is to express lysine-rich foreign proteins in transgenic rice. For instance, Gao et al.[156] introduced a lysine-rich protein gene (lys) from winged bean (Pso-phocarpus tetragonolobus) into rice by particle bom-bardment, and lysine content in seeds of transgenic rice plants increased up to 16.04%. Tang et al.[157] introduced a winged bean-derived lysine-rich protein gene into rice via the Agrobacterium-mediated method, and obtained maker-free transgenic rice with significantly improved lysine content in seeds. Wang et al.[158] introduced a ly-sine-rich protein gene sb401 from potato pollen into an indica variety LongTeFuB. The average content protein and lysine in seeds of transgenic rice increased 18.7% and 10% respectively, and the content of other essential amino acids also increased in varying degree. Li et al.[159] introduced sb401 into Nipponbare, and the content of protein, lysine, and other essential amino acids in seeds of transgenic plants increased in varying degree. How-


ever, it should be noted that too high protein content would affect the taste, and impair the eating quality of rice.

Moreover, some studies have been conducted to en-hance rice micronutrients such as β-carotene, iron and zinc. Golden rice, which is transgenic rice with enhanced β-carotene, was an outstanding paradigm. Golden rice is generated by introducing two foreign genes into trans-genic rice phytoene synthase gene (psy) from daffodil (Narcissus pseudonarcissus), and bacterial phytoene desaturase (crtI) from Erwinia uredovora to establish a novel carotenoid biosynthesis pathway in rice endosperm[12,160]. β-carotene is the precursor of vitamin A, and taking golden rice is thus supposed to address the heal- thy issues such as blindness, susceptibility for diseases, and increased child mortality caused by vitamin A-deficiency which prevails in the population living in the poor areas.

Besides golden rice, high iron content rice has also been developed. Goto et al.[161] increased iron content in rice grain two- to threefold by tissue-specifically over-expressing an iron storage protein gene ferritin in rice endosperm. Several other groups attempted similar strategies and obtained similar results[162–165]. Taking this transgenic rice is expected to alleviate the symptoms such as anemia caused by iron-deficiency which prevails in the population, especially children and women, living in the poor areas.

7 Transgenic high yield rice

Much effort to develop high yield rice has been concen-trated on seeking C4 rice in the past decade. As known, higher plants can be divided into three groups: C3, C4 and crassulacean acid metabolism (CAM) plants ac-cording to the initial photosynthates of CO2 in the car-bon assimilation pathway during photosynthesis. C4 plants which evolved from C3 plants are the type with higher photosynthesis efficiency, which have competi-tive advantages in photosynthesis efficiency and stresses tolerance over C3 plants. Unfortunately, many agronomi-cally important crops such as rice, wheat, barley, and soybean are C3 plants. For a long time, botanists and breeders dreamed to change C3 crops into C4 crops, and recently the advances of genetic engineering provide new opportunities.

The common strategy to develop C4 rice is to over-express C4 plant-derived genes involved in C4 cycle in

transgenic rice, however overexpressing C3 plant- derived orthologs has also been attempted. Ku et al.[166] firstly introduced a maize phosphoenolpyruvate carbo-cylase (PEPC) gene into rice, and the transgenic rice plants exhibited some photosynthetic characteristics of C4 plants. O2 inhibition in photosynthesis of transgenic plants reduced about 20% compared with the wild-type control. Later, more C4 cycle-related genes have been introduced into rice including PEPC[166–171], pyruvate, orthophosphate dikinase (PPDK) gene[171,172], phosphoenolpyruvate carboxykinase (PEPCK) gene[169,173], NADP- malic enzyme gene (ME) gene[171,174,175], and NADP- malate dehydrogenase (MDH) gene[176]. Although over-expressing these C4-related genes in rice showed diverse effects, it is still far from the purpose of increasing the yield greatly, and even overexpressing some C4-related gene led to severe negative effects. For instance, overex-pression of maize C4-specific ME resulted in serious stunting, leaf chlorophyll bleaching, and enhanced pho- toinhibition of photosynthesis[171,174,175]. Combinations of multiple C4-related genes synchronously have also been attempted, which was expected to achieve better effects. To establish a C4-like pathway in mesophyll cells of transgenic rice, Taniguchi et al.[171] overexpressed four C4-related genes with different origins in combination: the maize C4-specific PEPC and PPDK, the sorghum MDH, and the rice C3-specific ME. However, the trans-genic rice plants only exhibited slightly improved photo-synthesis accompanied with slight but reproducible stunt-ing phenotype compared with the wild-type control. How- ever, some reports were optimistic anyway. Jiao et al.[167] reported that grain yield of transgenic rice increased 22%―24% through co-expressing C4-specific PEPC and PPDK in rice.

C4 rice is undoubtedly one of the most challenging subjects for transgenic rice research. C4 rice research is very arduous due to huge distances of antimony and ge-netics between C3 and C4 plants. However, it is still valuable as an attempt to change the current status that rice yield has been hovering for a long period.

8 Transgenic herbicide-tolerant rice Herbicide-tolerance has been continuously the number one trait of GM crops, with the largest growing area since GM crops were first commercially grown in 1996. There are two main strategies to develop herbicide- tolerant rice: (ⅰ) modifying the target protein genes of


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herbicides to decrease their susceptivity or increase the expression level; (ⅱ) introducing novel enzyme systems via genetic engineering to enhance the metabolic capacity of herbicides. There are three main purposes to pro-duce herbicide-tolerant rice: to use chemical herbicides in the field that can decrease the cost and increase the income; to remove the false hybrid seeds and increase the seed purity for rice hybrid production; to generate transgenic rice plants as selection markers.

The bar gene from Streptomyces hygroscopicus is the first and most common herbicide-resistant gene used in transgenic rice. The bar gene can confer transgenic rice the resistance to the herbicide phosphinothricin (PPT), which can non-selectively kill various plants (trade names: Liberty, Finale, Basta, etc.). PPT kills plants by inhibiting plant GS and causes the accumulation of am-monia in plant cells. Bar gene encodes a PPT acetyl-transferase (PAT) that can deactivate PPT. To date, many studies of transgenic rice with bar gene have been re-ported[176–178]. Novel hybrids IIyou 86B and Teyou 86B were developed by South China Botanical Garden, Chi-nese Academy of Sciences using transgenic Minghui 86B with bar gene. Risk assessment of intermediate trial and environmental release for transgenic Minghui 86B with bar gene and its hybrids have been done, and the production trial would be conducted in 2005[179].

Glyphosate is the active ingredient of the herbicide Roundup of Monsanto Company, which has been broadly applied worldwide due to its high efficiency, low toxicity, and broad-spectrum. The targeting enzyme of glyphosate is 5-enolpyrulyshikimate-3-phosphate synthase (EPSP), which is a key enzyme involved in the synthesis of aro-matic amino acids in bacteria and plants. Glyphosate kills plants by inhibiting EPSP and the synthesis of aro-matic amino acids. The common strategy of glyphosate- resistant genetic engineering is to decrease the suscepti-bility to glyphosate by modifying EPSP. Hu et al.[180] introduced a bacterium-derived citrate synthase gene (CS) into an elite indica restorer Minghui 86 using a synthetic EPSP gene as the selection marker. The regen-erated transgenic plants showed significantly enhanced resistance to Roundup. Su et al.[181] obtained an EPSP mutant gene by error-prone PCR and introducing this EPSP mutant gene into rice could significantly enhance the glyphosate-tolerance of transgenic plants.

The cytochrome P450 monooxygenases exist broadly in all organisms, which play an important role in de-toxifying hydrophobic xenobiotic chemicals[182]. There-

fore, overexpressing P450 monooxygenases in plants is able to enhance the herbicide resistance, and the resis-tance is generally broad-spectrum against multiple her-bicides with different modes of action. Japanese re-searchers have done much work about it. They intro-duced P450 monooxygenase genes from mammals or even humans into transgenic rice to obtain herbicide- tolerant rice[183–188]. Moreover, the transgenic rice over-expressing P450 monooxygenases can be used to phy-toremediate pesticides or other environmental organic pollutants[186,187,189,190]. It should be noted that the com-position of the secondary metabolites in these transgenic rice plants possibly varies due to the alteration of P450 species and activities. However, what effects on human health and the environment the variation of the secon-dary metabolites in transgenic rice plant would cause still needs further evaluations.

In addition to the herbicide-tolerant rice described above, there are other types of transgenic rice against different herbicides targeting protoporphyringen oxi-dases[191–193] and acetolactate synthase[194]. The herbi-cide-tolerance of these transgenic rice plants is acquired by modifying the genes of target proteins.

9 Prospects

Tremendous progress in the development of transgenic research in rice has been shown in the past two decades. Not only transformation system has been established, but also a many of transgenic rice materials with poten-tial application acquired. With the deployment of func-tional genomics research in model plants including Arabidopsis and rice, many agronomically important genes have been discovered and isolated, which enriches strongly the available gene resources for transgenic rice research. However, there are still many needs for further improvement of transgenic rice research from various aspects. Firstly, from a technology perspective, trans-formation with large DNA fragments and chloroplast transformation have shown huge potential application, but which have not been used widely and need further technical modifications. Secondly, the comparative scar-city of the gene resource for transgenic research is still a limitation. For instance, no highly effective insect- resistance genes are available for transgenic rice to con-trol rice planthoppers currently. Although 19 resistant genes against BPH have been identified in rice[1], a BPH-resistant rice variety is probably overcome by BPH within few years after it has been adopted widely in


production, because BPH has multiple biotypes and is prone to evolve a resistance. How to develop durable planthopper-resistant varieties is one of the most urgent issues for transgenic rice research at present. Moreover, no major resistant genes to rice sheath blight and major genes or QTLs of N-use efficiency have been identified in rice too. The current status of lacking gene resources has become the bottleneck to develop novel rice varie-ties. Thirdly, a transgenic approach still has many limi-tations on the improvement of complicated traits. For instance, to develop transgenic drought-tolerant rice or C4 rice, although certain effects have been observed by introducing some foreign genes, there is still a long way to go. Finally, we must be aware that the commercializa-tion of transgenic rice is still difficult, even if transgenic soybean, corn, and cotton have been grown commer-cially for over 10 years. People have too much doubt on

transgenic rice as one of the most important food crops. The best achievements would not have any values if transgenic rice can not be used in production.

To address these challenges, functional genomics re-search should be further deepened to identify and isolate more gene resources with practical use. Meanwhile, the research of underlying biological mechanisms of related traits should be conducted too, because the related trait improvement would be more effective if the underlying biological mechanism has been well-documented. On the other hand, scientists must intensify popularization of science and education to improve the public know- ledge of transgenic technology and dispel the people’s prejudice and doubt about it. Finally, only when inte-grated with MAS and conventional breeding procedures can transgenic approaches exert their advantages fully to develop more and better rice varieties.

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Review and prospect of transgenic rice research