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REVIEWChinese Science Bulletin© 2009        SCIENCE IN CHINA PRESSReview and prospect of transgenic rice researchCHEN Hao,...
plasm resource to realize the goal of GRS. The transgenic           been developed according to different research purpose...
REVIEWfragments: BIBAC (Binary BAC) derived from bacterial                1.3 Chloroplast transformationartificial chromos...
2 Transgenic insect-resistant rice                                  used in transgenic rice are cry1A including cry1Ab[26–...
REVIEWrice pests. Transgenic Cry2A* rice and Cry1C* rice may                    the laboratory showed that most combinatio...
GNA gene is that GNA has certain insecticidal activity                 All transgenic insect-resistant rice described abov...
REVIEWmany physiological races with high variability, a Blast-            QTLs is very valuable to develop the resistant v...
The activated transcription factors can subsequently                             group is referred to as functional or str...
REVIEWfactor gene SNAC1 with great potential application,                 SOS2, Actin1:ZAT10, and CBF3, LOS5, ZAT10, andwh...
rus ore can barely sustain to the end of this century[136].        Zhonghua 11, and found that all GS-overexpressed (in-Th...
REVIEWdifference in main agronomic traits and yield compared              tassium because they were not concomitantly incr...
ever, it should be noted that too high protein content             transgenic rice, however overexpressing C3 plant-would ...
REVIEWherbicides to decrease their susceptivity or increase the           fore, overexpressing P450 monooxygenases in plan...
production, because BPH has multiple biotypes and is                           transgenic rice as one of the most importan...
REVIEW    monocotyledonous cereal crop, rice (Oryza sativa) and transmis-              pressing modified Cry1Ac endotoxin ...
Review and prospect of transgenic rice research
Review and prospect of transgenic rice research
Review and prospect of transgenic rice research
Review and prospect of transgenic rice research
Review and prospect of transgenic rice research
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Review and prospect of transgenic rice research

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2009. Zhang Qifa. Review and prospect of transgenic rice research

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  1. 1. REVIEWChinese Science Bulletin© 2009 SCIENCE IN CHINA PRESSReview and prospect of transgenic rice researchCHEN Hao, LIN YongJun & ZHANG QiFa†National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, ChinaRice 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 inmost 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 foodrequirement. However, rice yield has been hovering in the past decade, which is mainly caused by theabsence of novel breeding technologies, reduction of genetic diversity of rice cultivars, and seriousyield loss due to increasingly severe occurrences of insects, diseases, and abiotic stresses. To addressthese challenges, Chinese scientists proposed a novel rice breeding goal of developing Green SuperRice to improve rice varieties and realize the sustainable development of agriculture, by focusing onthe 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 animportant role in realizing the goal of Green Super Rice. Presently, many transgenic studies of rice havebeen 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 intransgenic 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 RiceRice is one of the most important crops as the staple food also leads to environmental pollution and ecological dis-for more than half of the global population. Rice breeding ruption. Moreover, higher quality requirements werehas achieved remarkable success in the past half-century, posed with the development of social economy and peo-due to two breakthroughs: increasing harvest index and ple’s living conditions.yield potential by reducing plant height making use of the To address these challenges, Zhang[1] proposed thesemidwarf varieties since the 1960s, and second yield goal to develop Green Super Rice (GSR) aiming at re- GENE ENGINEERINGleap through developing and applying of rice hybrids ducing the use of pesticides and fertilizers, water-savingsince the 1970s. However, rice production in the new and drought-tolerance, improving quality and yield incentury is still confronting enormous challenges. For in- rice production by improving the following five classesstance, consistent yield pressure due to global population of traits: insect and disease-resistance, drought-tolerance,increase is presented, associated with the reduction of nutrient-use efficiency, quality and yield potential. Hearable land worldwide, while rice yield has reached the suggested taking a strategy of combining conventionalceiling since the 1990s, mainly caused by decrease of breeding program, marker-assisted selection (MAS), andgenetic diversity of rice cultivars, increasingly severe oc- transgenic approach to make the best use of rice germ-currence of insects and diseases in rice production, water Received September 18, 2009; accepted September 26, 2009shortage and increasingly frequent occurrence of drought. doi: 10.1007/s11434-009-0645-xMeanwhile, overuse of chemical pesticides and fertilizers † Corresponding author (email: qifazh@mail.hzau.edu.cn)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
  2. 2. plasm resource to realize the goal of GRS. The transgenic been developed according to different research purposes.approach provides new opportunities for rice breeding The following is a brief introduction of some specialwith the capacity to break the reproductive isolation be- transformation technologies.tween species and realize the free communication of ge- 1.1 Multigene transformationnetic materials. Reviewing the history of the development Transformation of multiple genes is mainly applied to twoof transgenic rice in the past two decades, most target purposes. Firstly, it facilitates the procedure of map-basedtraits are consistent with the goal of GSR. In this article, gene cloning. A key step of map-based gene cloning is towe firstly give a brief account of the technical advances of validate the candidate genes. Transformation of multiplerice transformation, and then outline the main progress in genes with a single construct is very important to this step,transgenic rice research with respect to the five classes of because the more candidate genes that can be transformedtraits, and finally discuss the prospects for the development once, the less labor of transformation.of transgenic rice. Secondly, multigene transformation may play an im- portant role in rice transgenic breeding. The introduced1 Rice transformation foreign genes in commercialized transgenic crops areRice transformation achieved important success in the generally single genes to control qualitative traits suchlate 1980s. Three independent groups reported on re- as insect-resistance, disease-resistance, or herbicide-resis-generated transgenic rice plants using rice protoplast as tance. However, many crop traits are actually controlledthe recipient via electroporation-mediated or PEG-me- by multiple genes. To improve these traits, the multiplediated methods in 1988[2–4]. Rice transformation via par- genes must be introduced into the crop simultaneously.ticle bombardment succeeded in 1991[5], which later Moreover, transformation of multiple genes is also neededbecame one of the most common methods of rice trans- in case of promptly pyramiding multiple qualitativeformation. Chan et al.[6] acquired transgenic rice plants traits or introducing novel metabolic pathways consist-by Agrobacterium-mediated method in 1993. Hiei et al.[7] ing of multiple genes. Golden rice is a famous example,established the highly efficient Agrobacterium-mediated in which a novel β-carotenoid biosynthesis pathway istransformation system for japonica rice using the mature established in rice endosperm by introducing two for-seed-derived callus as the explant, which subsequently eign genes into transgenic rice[12]. There are two com-became the most common rice transformation method. monly available strategies of multigene transformation.The transformation system of japonica varieties was One is to construct foreign genes in different vectorsfurther improved to shorten the transformation proce- firstly, and then the multigene pyramiding is performeddure[8]. Although Hiei et al.’s protocol established in by ways of co-transformation, repetitive transformation,1994 made the transformation very amenable for japon- or separate transformations in combination with hy-ica rice[7], that of indica rice was still obstinate. Some bridization. The production of golden rice took thismodifications were made to improve the transformation strategy. Another one is to construct foreign genes in aefficiency of indica rice[9,10]. Recently, Hiei and Komari[11] single vector, and multiple genes are then introducedpublished a protocol of Agrobacterium-mediated trans- into the recipient by a transformation event[13]. Obvi-formation adaptable to both japonica and indica varieties. ously, the latter strategy is more amenable and economicAccording to Hiei and Komari [11], transformation of in- compared with the former one. However, transformationdica rice can be done within 2.5 months using the imma- with large DNA fragments is the main difficulty of mul-ture embryo with extremely high transformation effi- tigene transformation. The cloning capacity of commonciency (a single immature embryo may produce 5―13 Ti binary vectors such as pCAMBIA series is limited,independent transformants). However, the disadvantage because their replicons derive from plasmid. The cloningof the protocol is that collection of immature embryos is capacity of a common Ti binary vector is usually lesslaborious and limited by the season. than 20 kb, which can approximately carry 2-3 foreign With the development of rice transformation, simple genes and appears inadequate for multigene transforma-introduction of foreign genes into the genomes of target tion. Some special Ti vectors have been developed to en-organisms can not meet scientists’ requirements any- hance cloning capacity of large DNA fragments. Theremore. Some special transformation technologies have are two main Ti vectors for transformation of large DNA4050 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  3. 3. REVIEWfragments: BIBAC (Binary BAC) derived from bacterial 1.3 Chloroplast transformationartificial chromosome[14] and TAC (Transformation-com- Chloroplast transformation is usually implemented bypetent Artificial Chromosome) derived from P1 artificial delivering plasmid vectors containing transgenes intochromosome[15], both of which can accept a foreign DNA chloroplasts with a direct method, such as particle bom-fragment more than 100 kb. BIBAC and TAC have beensuccessfully applied in rice transformation[16,17]. With the bardment. The transgenes are integrated into the chloro-development of BIBAC and TAC vectors, multigene trans- plast genome through homologous recombination offormation would have a huge potential for rice transgenic homologous sequences flanking transgenes. There arebreeding. two main advantages of chloroplast transformation com- pared with the common nuclear transformation. Firstly,1.2 Tissue-specific/inducible expression expression efficiency of foreign proteins is extremelyConstitutive CaMV 35S and maize Ubiquitin promoters high due to high transgene copies. There are generally 10are the two most common promoters used in rice trans- -100 chloroplast genome copies per chloroplast and 10genic research. There are certain problems to express -100 chloroplasts per cell, resulting in theoretically astransgenes in all plant tissues and organs at all growthstages using a strong constitutive promoter, for instance, many as up to 10000 transgene copies per cell that isincreasing the metabolic burden of transgenic plants, much more than that by nuclear transformation. Therefore,and causing the public’s concerns about the food safety the expression efficiency of chloroplast transformation isdue to accumulation of the protein products of trans- supposed to be much higher than that of nuclear trans-genes in the edible parts of transgenic plants. Moreover, formation. Transgenic plants of chloroplast transforma-constitutive expression of some good genes, such as tion can have a high accumulation of foreign proteins (upabiotic stress-resistance related transcription factor to 47% of total soluble protein)[20]. Secondly, the inheri-genes in transgenic plants would lead to abnormal plant tance of transgenes integrated in chloroplast genomegrowth and development. Thus, tissue-specific/inducible shows a maternal pattern, which can prevent the trans-expression is crucial for transgenic breeding, which is gene flow from transgenic plants to non- transgenic va-usually implemented by making use of tissue-specific/ rieties or wild relatives by pollination. Thus, the fieldinducible promoters. experiment or commercial production of transgenic Transgenic Bt rice is the most promising transgenic plants acquired via chloroplast transformation is saferrice for commercialization. However, the public’s con- and more environment-friendly. Furthermore, there arecern about the food safety of Bt protein is a major bar- some other advantages, for instance, transgene is inte-rier to its release. Ye et al.[18] introduced a synthetic grated through homologous recombination at a precise,cry1C* driven by the rice rbcs (a small subunit of ribu- predetermined location resulting in elimination of “posi-lose-1,5-bisphosphate carboxylase/oxygenase) promoter tion effect” and uniform expression level among differ-into a japonica variety Zhonghua 11 by Agrobacte- ent transformants; chloroplast genes are often arrangedrium-mediated transformation. In acquired transgenic in operons, that means a promoter is able to control theplants, Bt protein is expressed predominantly in greenparts of the plant such as the leaf and stem that are expression of multigenes as a polycistron, which maymainly targets attacked by insect pests, while barely in facilitate multigene transformation; gene silencing ofthe edible endosperm. The expression level of Cry1C* chloroplast transformation has never been reported so far,in the leaf of transgenic plants when driven by rice rbcs while which is often observed in nuclear transforma-promoter is almost three times of that when driven by tion[20].the maize Ubiquitin promoter; contrarily Cry1C* con- Although chloroplast transformation is a very promis-tent in endosperm when driven by the rice rbcs promoter ing technology with many advantages, it has not beenis less than 1/1000 of that when driven by the maize applied as widely as nuclear transformation due to manyUbiquitin promoter compared with the results of Tang practically technical difficulties. So far, chloroplast trans- GENE ENGINEERINGet al.[18,19]. It is supposed that Bt rice with green part- formation has been achieved only for more than 10 plantspecific expression is more acceptable to the consumers species, and there are few reports about chloroplast trans-and therefore more promising to commercialization. formation in rice[21–24]. Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4051
  4. 4. 2 Transgenic insect-resistant rice used in transgenic rice are cry1A including cry1Ab[26–35], cry1Ac[30,36–39], and cry1Ab/Ac fusion gene[40,41]. ThereInsect destroy is one of the major causations of yield are limited studies involving other Bt genes[19,42–47].loss, which leads to about 10% yield loss annually. Most of these transgenic Bt rice showed high resistanceSpraying chemical insecticides is the major way to pre- against striped stem borer, yellow stem borer, and leaf-vent insect destroy in rice production. However, overuse folder.of chemical insecticides not only increases production Tu et al.[40] have been performed field experiment ofcosts, but also pollutes the environment and threatens transgenic rice harboring a cry1Ab/Ac fusion gene. Theirhuman health. Enhancing insect-resistance of rice itself results showed that transgenic cry1Ab/Ac Minghui 63by breeding approaches is a more economic and envi- (an elite rice restorer line) and its hybrid Bt Shanyou 63ronment-friendly strategy. However, developing insect- exhibited high insect-resistance in field conditionsresistant cultivars by conventional breeding approaches without spraying any chemical insecticides during theis time-consuming. Moreover, no effective resistance whole growth period, indicating huge use value of Btgermplasm resources have been identified in rice against rice in production.striped stem borer (Chilo suppressalis), yellow stem As applying other insecticides or resistant varieties,borer (Tryporyza incertulas), and leaffolder (Cnapha- one of the major risks of Bt crops is that insects mightlocrocis medinalis), which are main rice pests. The most evolve resistance against Bt crop, which would impairpromising method currently is to develop transgenic in- its durability. Although no insect species with resistancesect-resistant varieties by introducing foreign insect- re- to Bt crops have been identified under natural conditionssistant genes into rice. Many useful insect-resistant genes so far, some insects have evolved resistances against Bthave been identified and isolated from plants, animals, spray reagents in the field. Moreover, many Bt toxins-and even microorganisms. Transgenic insect-resistant rice resistant insect strains have been selected in the greenlines have been obtained by introducing these in- house or laboratory, and some of them were able to sur-sect-resistant genes. Some of them have been tested under vive on Bt crops[48], indicating the risk that insects havefield conditions and showed broad potential application the potential to evolve the resistance against Bt crops infor production. field conditions.2.1 Transgenic Bt rice Utilization of two-toxin Bt rice is an important strat-Bt toxin genes derived from Bacillus thuringiesis (Bt) is egy to delay insect-resistance and prolong the durabilityone of the most broadly-used insecticidal genes world- of Bt rice[49]. Two-toxin Bt rice is a transgenic rice ex-wide. Bt forms various crystals upon sporulation, which pressing two different Bt toxins in combination. In prin-are a class of proteins with specific insecticidal activities, ciple, the frequency that insects evolve a resistancereferred to as Bt toxins or insecticidal crystal proteins. against two Bt toxins simultaneously is much lower thanTransgenic Bt crops acquire insect-resistance due to the that against one Bt toxin. Therefore, two-toxin Bt riceaccumulation of Bt toxin in the plant. Bt genes have can greatly delay the development of insect-resistancebeen successfully transferred and expressed in different and is more durable. However, the two Bt toxins incrops including rice. Among them, Bt cotton, corn, and combination must bind to different receptor sites on insectpotato have been commercially growing and bringing gut cells to avoid the occurrence of “cross-resistance”. Ashuge economic benefits[25]. described previously, common Bt genes used in rice are Various Bt toxins with specific insecticidal activities cry1A such as cry1Ab, cry1Ac, and fused cry1Ab/Ac. Itagainst species of the orders lepidoptera, coleoptera, is not suitable to combine two cry1A genes because in-diptera, and invertebrata (acarids, nematodes, and pro- sects are prone to develop a cross-resistance to over-tozoa) have been identified and isolated from different come them because they shared very high protein se-Bt strains. Totally more than 400 Bt genes have been quence homology each other. Therefore, Chen et al.[47]cloned so far (http://www.lifesci.sussex.ac.uk/home/Neil_ and Tang et al.[19] developed transgenic rice with syn-Crickmore/Bt/toxins2.html). However, in spite of so many thetic cry2A* and cry1C*, respectively. Field experi-Bt genes, only a small proportion of them have been ments showed that both transgenic Cry2A* rice andused in transgenic plants. The most common Bt genes Cry1C* rice were highly resistant against lepidopteran4052 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  5. 5. REVIEWrice pests. Transgenic Cry2A* rice and Cry1C* rice may the laboratory showed that most combinations of two Btprovide new gene resources for the development of two- toxins had synergistic effects and exhibited significantlytoxin Bt rice. higher insect-resistance than single Bt gene. Studies showed that cry1A, cry1C and cry2A are Bt genes are the most successful insect-resistant genessuitable to combine because insects unlikely develop a that have been applied in transgenic rice so far, whichcross-resistance to them due to their low protein se- can effectively control lepidopteran rice pests (Figure 1(a)quence homology each other[50,51]. Yang et al. developed and (b)). Bt rice has been temporarily commercialized in10 two-toxin Bt rice lines 1Ab/1C, 1C/1Ab, 1Ab/2A, Iran 2005. Bt rice has been well-developed in China and2A/1Ab, 1Ac/1C, 1C/1Ac, 1Ac/2A, 2A/1Ac, 1C/2A and can be commercialized promptly as soon as the policy2A/1C by reciprocal hybridizations of 4 transgenic permits.Minghui 63 lines with different Bt genes cry1Ab (1Ab), 2.2 Transgenic rice with plant or animal-derivedcry1Ac (1Ac), cry1C* (1C), and cry2A* (2A), in five genescombination patterns (1Ab+1C, 1Ab+2A, 1Ac+1C,1Ac+2A, 1C+2A) (Yang Zhou and Lin Yongjun, un- Plant-derived insect-resistant genes commonly includepublished data). The transgenic line 1Ab/1C means the plant lectin genes and protease inhibitor genes. Plantmaternal line of the hybrid is 1Ab, and the paternal line lectin genes have a relatively high insecticidal activity,is 1C; while 1C/1Ab means contrary parents. The rest among which Galanthus nivalis agglutinin (GNA) genemay reason by analogy. The results of bioassay in has been widely applied. The principal advantage to use GENE ENGINEERINGFigure 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-typeNipponbare control; SNAC1, SNAC1-overexpressing transgenic Nipponbare. (d) WT, wild-type Zhonghua 11 control; S58S, OsSKIPa-overexpressing transgenic Zhonghua 11. Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4053
  6. 6. GNA gene is that GNA has certain insecticidal activity All transgenic insect-resistant rice described aboveagainst sap-sucking (homoptera) insects such as rice acquired their resistance through directly expressingplanthoppers, which is not able to be controlled by Bt foreign insecticidal protein. Recently, a novel insect-toxins. Sun et al.[52] obtained homogenous transgenic resistant strategy, suppressing the expression of keyGNA rice lines via particle bombardment. Their data genes for pest development or biochemical metabolismshowed that the homogenous transgenic lines can control via RNA interference (RNAi) using gene fragment frombrown planthopper (Nilaparvata lugens, BPH) by sig- the target pest itself, has succeeded in developing trans-nificantly decreasing survival rate and fecundity, retarding genic insect resistant corn[69] and cotton[70]. This strategydevelopment and declining feeding. More studies have might become a new research trend to develop trans-proved that transgenic GNA rice had some insecticidal genic insect-resistant plants. However, it should beeffects on planthoppers, leafhoppers, and aphids[38,43,52–59]. noted that if using RNAi strategy, the targeting sitesHowever, toxicity of GNA to sap-sucking rice pests is not must be pest gene-specific to ensure that the transgeniccomparable to that of Bt toxin to lepidopteran rice pests. plant is harmless to other species especially to humans.The effects of GNA are to significantly restrain the in-sect’s growth, development, and fecundity. There is an- 3 Transgenic disease-resistant riceother study involving an Allium sativum agglutinin from Bacterial blight (BB), fungal diseases blast, and sheathleaf (ASAL) gene. Saha et al.[60] obtained transgenic rice blight are three main diseases in rice production. Bacte-overexpressing ASAL gene, which also exhibited en- rial blight caused by Xanthomonas oryzae pv. Oryzaehanced resistance to BPH and green leafhopper (Nepho- (Xoo) is the most devastating rice bacterial diseasetettix cinciteps). Moreover, expressing ASAL in trans- worldwide[71], which may cause 20%-30% yield loss,genic rice plants significantly reduced the infection inci- or even 100% in case of severe occurrence. Blast causeddence of rice tungro diseases, caused by co-infection of by Magnaporthe grisea (M. grisea) may arise in all ricegreen leafhopper-vectored rice tungro bacilliform virus organs at any growth stage. Sheath blight caused byand rice tungro spherical virus[60]. Rhizoctonia solani (R. solani) may lead to whitehead, In addition to plant lectin genes, protease inhibitor reductions of fertility and grain weight, and 10%-30%genes are another group of plant-derived insect-resistant yield loss, even more than 50% if serious.genes. The protease inhibitor genes that have been tested More than 30 BB resistance (R) genes or loci againstin transgenic rice include: potato protease inhibitor gene Xoo have been identified in rice so far. Among them, sixpinII[61,62], cowpea trypsin inhibitor gene CpTI[63], soy- R genes (Xa1, Xa3/ Xa26, xa5, xa13, Xa21, and Xa27)bean kunitz trypsin inhibitor gene SKTI[64], corn cystatin have been cloned and many (Xa4, Xa7, Xa10, Xa22(t),gene[65], rice cystatin gene[66] and barley trypsin inhibitor Xa23, xa24, Xa25(t), and Xa31(t)) fine-mapped[72,73]. BBgene BTI-Cme[67]. These transgenic rice plants exhibited is effectively controlled in rice production due to thecertain resistance to BPH, striped stem borer, leaffolder, application of R genes and resistant varieties. For trans-nematode, etc. genic breeding, introducing R genes into the desired rice Utilization of plant-derived insect-resistant genes has varieties is a direct and convenient way. Zhang et al.[74]some special advantages, for instance, they generally introduced a broad-spectrum R gene Xa21 into Minghuihave a broad-spectrum insect resistance, and especially 63, and the acquired transgenic Minghui 63 showed sig-GNA has some resistance against homoptera rice pests nificantly enhanced resistance to Xoo. Wu et al.[75] ob-that Bt toxins are unable to control. However, the appli- tained marker-free BB-resistant transgenic Minghuication of plant-derived insect-resistant genes is still lim- 63 and WanB (a rice maintainer line) by introducingited because of their relatively inadequate insecticidal Xa21 into the corresponding wild-type recipients, andactivities. their hybrids also exhibited significantly enhanced There are very few studies to use animal-derived in- BB-resistance.sect-resistant genes. Huang et al.[68] reported to acquire More than 60 major Blast-resistant genes have beentransgenic insect-resistant rice against striped stem borer identified in rice so far[76], among which 10 resistantand leaffolder by introducing an insecticidal gene SpI from genes (Pib, Pi-d2, Pikm, Pi-ta, Pizt, Pi2, Pi5, Pi9, Pi36,spider into rice varieties Xiushui 11 and Chunjiang 11. and Pi37) have been cloned[77]. Because M. grisea has4054 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  7. 7. REVIEWmany physiological races with high variability, a Blast- QTLs is very valuable to develop the resistant varietiesresistant cultivar might lose the resistance 3-5 years against those diseases.after it is adopted in production widely. As for R. solani, A few studies have shown that overexpressing someno major resistant genes have been identified in rice. resistant QTLs in rice may obtain satisfying results too, Overexpressing pathogenesis-related proteins (PRs), although most natural resistant QTLs have minor effects.including chitinase, β-1,3-glucanases, and thaumatin- Qiu et al.[96] overexpressed a resistant QTL OsWRKY13like proteins, and other plant- or microorganism-derived driven by the maize Ubiquitin promoter in a BB suscep-antifungal proteins, is a common strategy to develop tive rice variety, and the transgenic plants exhibited en-transgenic fungus-resistant rice. PRs are a battery of hanced resistance to Xoo. Xiao et al.[97] suppressed theproteins encoded by the host plants but induced exclu- expression of a resistance-related QTL OsDR10 in ricesively in pathological or related situations, and many of via RNAi, and the transgenic plants showed enhancedthem showed antifungal activity in vitro[78]. Some stud- resistances to multiple Xoo strains compared with theies have confirmed that overexpressing chitinases in non-transgenic control. It should be noted that the resis-transgenic rice enhanced the resistance against both M. tance reaction regulated by the resistant QTLs is notgrisea[79–82] and R. solani[83]. Nishizawa et al.[84] re- species or race-specific but broad-spectrum basic resis-ported that overexpressing β-1,3-glucanase in transgenic tance. The resistance level of the resistant QTLs is notrice enhanced resistance against M. grisea; Datta et al.[85] comparable with that of qualitative resistance conferredfound that overexpressing thaumatin-like protein in by major resistance genes, but they are still worthy oftransgenic rice enhanced resistance against R. solani. research and utilization because of their broad-spectrumBesides using single PR genes, pyramiding different PR and durability.genes is also common. For instance, combinations ofchintinase with β-1,3-glucanase can enhance the resis- 4 Transgenic drought-tolerant ricetance of transgenic rice to blast[86–88]; combination of Drought is one of the major factors causing yield loss inchitinase with a modified maize ribosome-inactivating rice production for a long time and is getting worse asprotein[89] or a thaumatin-like protein[90] can enhance the climate changes worldwide. Rice production needresistance to sheath blight. Moreover, some studies at- consume a huge amount of water, accounting for ap-tempted to enhance the resistance of transgenic rice to proximate 70% water consumption of agriculture in ourfungal diseases by overexpressing antifungal proteins or country. While China is water deficient, and the averagepeptides from plants or microorganisms in rice, and also capita water capacity is only a quarter of that of theachieved some effects[91–94]. world. Therefore, developing drought-tolerant rice va- Expressing pathogen-derived protein elicitors in trans- rieties and reducing water consumption in rice produc-genic rice to induce the plant general defense response tion is crucial to increasing rice yield and ensuring theand system-acquired resistance (SAR) is another strategy food security of China.for developing transgenic rice with enhanced disease re- One distinguishing feature of plants from animals issistance. Shao et al.[95] reported that overexpression of a that plants are not “movable”. Correspondingly, plantsprotein elicitor harpin from Xoo in transgenic rice con- evolve a complex biological mechanism to resist variousferred high non-specific resistance to multiple M. grisea environmental stresses. When under an environmentalraces. stress such as drought, the initial signals are perceived Besides qualitative major resistance genes, recent by the sensors (including ion proteins, histidine kinases,studies of quantitative resistance genes (resistant QTLs) and G-protein coupled receptors) of the plant cell, andare worth noting. Although the resistance of single transduced to second messenger molecules such as Ca2+,quantitative resistance gene is relatively limited com- reactive oxygen species (ROS), and inositol phosphatespared with the major resistance genes, their advantages that can transfer further in the plant cell. Then, proteinare broad-spectrum and more durable. Nevertheless, no phosphorylation cascades of Ca2+-dependent protein GENE ENGINEERINGmajor resistance genes have been found in rice for some kinases (CDPKs), mitogen-activated protein kinasesrice diseases such as rice sheath blight, false smut, and (MAPKs), etc. triggered by the second messengerbacterial leaf streak, and thus the research of resistant molecules activate the downstream transcription factors. Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4055
  8. 8. The activated transcription factors can subsequently group is referred to as functional or structural genes,regulate the expression of a many of downstream func- including LEA proteins, water channel proteins, cata-tional or structural genes such as late embryogenesis lytic enzymes that synthesize osmoprotectants (com-abundant (LEA) proteins, various catalytic enzymes that patible solutes) including proline, trehalose, glycinebe-synthesize osmoprotectants, antifreeze proteins, channel taine, polyamines, etc., and detoxifying genes such asproteins to help the plant re-establish osmotic homeostasis, superoxide dismutase (SOD). This group was generallyscavenge harmful compounds, protect and repair dam- used in the initial transgenic studies because the mecha-aged proteins and membrane systems caused by the nism is comparatively simple. Another group is regula-stresses[98–100]. tory genes, which function in the upstream of the drought Due to the complex mechanism of drought-tolerance, response network, including CDPKs, calcineurin B-likeit is difficult to develop drought-tolerant varieties only protein-interacting protein kinases (CIPKs), MAPKs,relying on conventional approaches. Nowadays, genetic transcription factors, etc. Modifying the expression ofengineering has been broadly applied to developing drought- these genes generally can influence the expression leveltolerant rice, and a common strategy is to overexpress of a battery of downstream drought-related genes. Ap-drought-responsive or related genes in transgenic rice. plication of the regulatory genes is thought to be moreTable 1 summarizes some representative experiments effective than those functional or structural genes withabout transgenic drought-resistant rice. The applied simple functions, considering the complexity of drought-transgenes can be roughly classified into two groups tolerant mechanism.according to their functions and action patterns. One Hu et al.[128] reported a drought-tolerance transcriptionTable 1 Summarization of recent transgenic rice trials of drought-tolerance Gene Gene type/function Source Effect Reference mothbean P5CS improve proline synthesis proline increase, drought and salt-tolerance [101,102] (Vigna aconitifolia L.) 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, transcription factor rice, Arabidopsis drought, salt, and cold-tolerance, growth retardation [121] 1B, and 1C 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]4056 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  9. 9. REVIEWfactor gene SNAC1 with great potential application, SOS2, Actin1:ZAT10, and CBF3, LOS5, ZAT10, andwhich is a member of NAC (NAM, ATAF, and CUC) NHX1 by both promoters) showed significantly higherplant-specific gene family. SNAC1 is specifically ex- relative spikelet fertility than the wild-type control in thepressed in leaf guard cells under drought stress condi- PVC pipes under drought stress. In the field droughttions. Overexpressing SNAC1 significantly enhanced resistance testing of T2 and T3 families, transgenic fami-drought resistance in transgenic rice (22%-34% higher lies of seven constructs (HVA22P:CBF3, Actin1:NPK1,seed setting rate than the control) at the reproductive HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:stage in the field under severe drought stress conditions ZAT10, and HVA22P:ZAT10) showed significantlywithout showing any phenotypic changes or yield pen- higher yield per plant than the wild-type control, andalty. Compared with the control, transgenic rice plants families of nine constructs (Actin1:CBF3, HVA22P:were more sensitive to abscisic acid (ABA) and lost wa- CBF3, HVA22P:SOS2, HVA22P:NPK1, Actin1:LOS5,ter more slowly by closing more stomatal pores, and HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, andmaintained turgor pressure under lower relative water Actin1:NHX1) had higher spikelet fertility than thecontent[128]. The transgenic rice also showed signifi- wild-type control. In conclusion, LOS5 and ZAT10cantly improved drought and salt-tolerance at the vege- showed relatively better effects than the other five genestative stage (80% higher survival rate compared with the in improving drought resistance of transgenic rice undercontrol) (Figure 1(c)). DNA microarray analysis re- field conditions. The results of this study were based onvealed that over 150 stress-related genes were up-regu- field experiments and might be a useful reference forlated in the SNAC1-overexpressing rice plants. developing practical transgenic drought-resistant rice. Hou et al.[129] recently published a drought-tolerance An ideal drought-tolerant rice variety should haverelated gene OsSKIPa. Drought-tolerance of OsSKIPa- high yield and good quality when water is adequate,overexpressing rice plants increased 2―4 fold compared while higher yield than the best rice cultivars under wa-with the control at the adult stage (Figure 1(d)). The OsS- ter-deficit or drought conditions. Although certain ad-KIPa-overexpressing rice showed significantly increased vances have been made in transgenic breeding of drought-ROS-scavenging ability by analyzing the relative levels of tolerant rice, it is still far from developing a practicalSOD and monodehydroascorbate (MDA) in plants under drought-tolerant rice variety. In view of the complexdrought stress. Moreover, the transcript levels of many mechanism of drought-tolerance, it is crucial to pyramidstress-related genes are significantly higher than the various drought-tolerant genes by taking an integratedwild-type control after drought stress treatment. strategy of transgenic approaches, MAS and conven- Although many studies about transgenic drought- tional breeding programs.resistant rice have been reported (Table 1), the data wereobtained under greenhouse conditions, and very few 5 Transgenic nutrient-use efficient ricestudies under field conditions have been reported. Xiao Chemical fertilizer is the basis of modern agriculture,et al.[134] introduced seven well-documented stress- which ever contributes greatly to improving food cropresistant genes under the control of constitutive Actin1 production and ensuring food security. Food crop pro-promoter and stress-inducible promoter of a rice HVA22 duction has been doubled in the past four decadeshomolog (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, worldwide due to the green evolution, associated with aand NHX1) into Zhonghua 11, and then the drought- seven-fold increase in the use of nitrogen (N) fertiliz-resistance of regenerated transgenic rice lines was tested ers[135]. However, this high-production pattern relying onunder field conditions. Their results showed that trans- a high investment is not sustainable. The increase ofgenic families of eight constructs (HVA22P:CBF3, HVA22P: food production is not so significant anymore even if theNPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, use of fertilizer still keeps growing in the past decade.HVA22P:ZAT10, Actin1:NHX1, and HVA22P:NHX1) had Nevertheless, overuse of fertilizer is leading to a series GENE ENGINEERINGsignificantly higher relative yield than the wild-type con- of environmental issues, such as eutrophication of watertrol in both field and PVC pipes conditions with drought body, groundwater pollution, soil acidification, etc. Asstress. Transgenic families of 10 constructs (HVA22P: an unrenewable resource, the global supply of phospho- Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4057
  10. 10. rus ore can barely sustain to the end of this century[136]. Zhonghua 11, and found that all GS-overexpressed (in-These challenges threaten not only the ecological secu- cluding GS1;1, G1;2 and glnA) transgenic plants showedrity but also sustainable development of agriculture. higher total GS activities and soluble protein concentra-Therefore, research and development of nutrient-use tions in leaves and higher total amino acids and total Nefficient rice varieties in combination with scientific content in the whole plant. However, both grain yieldfertilization and cultivation management to substantially and total amino acids in seeds of GS-overexpressed ricedecrease the use of fertilizer is very important to ensure plants decreased compared with the wild-type controlfood security and realize the sustainable development of under field conditions with N deficit stress.modern agriculture. Ammonium transporters are crucial for the plant root5.1 Nitrogen-use efficiency to take up NH4+ from the soil. Ten ammonium trans- porter genes have been identified in rice, among whichN is an essential nutrient that plants require in the most OsAMT1;1, OsAMT1;2 and OsAMT1;3 belong to ATM1quantity and is also a major limiting factor in crop pro- subfamily, and the other seven (OsAMT2;1, OsAMT2; 2,duction. NO3− and NH4+ are two major inorganic N OsAMT2;3, OsAMT3;1, OsAMT3;1, OsAMT3;3, andcompounds presenting in agricultural soils. NO3− is OsAMT4) belong to ATM2 subfamily. Kumar et al.[142]converted to NH4+ by two reductases: nitrate reductase found that the flow of 15NH4+ in transgenic plants over-and nitrite reductase in the plant after it is absorbed from expressing OsAMT1;1 changed, and the biomass of trans-the soil. NH4+ is converted to glutamine (Gln) and glu- genic plants decreased compared with the control. Ho-tamate (Glu) by the GS/GOGAT cycle consisting of two que et al.[143] found that the biomass of transgenic ricekey enzymes glutamine synthetase (GS) and glutamate overexpressing OsAMT1;1 significantly decreased at vege-synthetase (GOGAT). Glu can be further transferred to tative growth stage compared with the wild-type control.many other amino acids by different aminotransferases. Moreover, the transgenic plants showed increased am-Rice prefers NH4+ as the major N source, which is ac- monium uptake and ammonium content in roots. It istively absorbed from the soil by different ammonium supposed that biomass decrease of the transgenic plantstransporters in rice roots, and subsequently assimilated at the early growth stages might be caused by phytotox-by GS and NADH-GOGAT in roots[137]. icity due to the accumulation of ammonium in the root. GS is tissue/cell-type specific. GS1 exists predomi- Overexpressing some aminotransferases in transgenicnantly in seeds, roots, nodules, flowers, and phloem, plants has also been attempted to change the level ofwhich is inducible by water-flood, pathogens, and se- amino acid synthesis and N metabolism, which is ex-nescence, and may function in N assimilation and trans- pected to improve N-use efficiency in rice. Shrawatlocation. GS2 is the predominant isoenzyme in leaves et al.[144] reported that tissue-specifically expressing athat may function in assimilation of ammonia reduced barley alanine aminotransferase (AlaAT) cDNA in ricefrom nitrate in chloroplasts and/or in the reassimilation roots significantly increased the biomass and grain yieldof photorespiratory ammonia[138]. There are four GS compared with the control. Moreover, some key me-genes in rice: one encoding the chloroplastic/plastidic tabolites such as Gln and total N content in transgenicGS2 that exists predominantly in leaf cells, and three rice plants also increased, indicating enhanced N uptakeones encoding cytosolic GS1 that exists predominantly in efficiency. Zhou et al.[145] overexpressed separately all ofthe root (GS1;2), stem (GS1;1) and spikelet (GS1;3)[138,139]. three rice aspartate aminotransferase (AAT) genes Yamaya et al.[140] found that expression of a NADH- (OsAAT1-3) from rice and an E. coli-derived AAT genedependent glutamate synthase (NADH-GOGAT) gene (EcAAT) in transgenic rice. The transgenic plants over-from a japonica variety Sasanishiki in an indica cultivar expressing OsAAT1, OsAAT2 and EcATT showed sig-Kasalath increased significantly grain weight (up to 80%) nificantly increased leaf AAT activity and higher graincompared with the non-transgenic control, indicating amino acid and protein contents compared with thethat NADH-GOGAT is indeed a key step for N utiliza- non-transgenic control. No significant changes weretion and grain-filling in rice. found in leaf AAT activity, grain amino acid content, or Cai et al.[141] overexpressed GS1;1, GS1;2 from Ming protein content in OsAAT3 overexpressed rice plants.hui 63 and an Escherichia coli (E. coli)-derived GS gene Moreover, transgenic rice plants overexpressing OsAAT1,glnA under the control of CaMV 35S promoter in OsAAT2, OsAAT3, and EcAAT did not show significant4058 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  11. 11. REVIEWdifference in main agronomic traits and yield compared tassium because they were not concomitantly increasedwith the wild-type control. with an enhanced P acquisition.5.2 Phosphorus-use efficiency 6 Transgenic high quality ricePhosphorus (P) is one of the essential macroelementstoo. Although the absolute P amount in the soil is com- Rice quality is recently getting more and more attentionparatively abundant, the available P is deficient (usually with the improvement of people’s living conditions. Theless than 10 μmol/L or even less) due to its low solubil- physical and chemical indexes of good quality rice gen-ity and high adsorptive capacity[136,146]. As a result, im- erally include processing quality, appearance quality,proving the capacity of rice plants to activate and utilize cooking and eating quality, nutritional quality[150]. Actu-the fixed P in the soil is a major research objective of ally, several important genes controlling rice qualitydeveloping P-use efficiency varieties. traits such as GS3 for grain length[151], GW2 for grain Yi et al.[147] identified a P-deficiency responsive tran- width[152], Alk for gelatinization temperature[153], and Wxscription factor OsPTF1 from Kasalath, a P-use efficient for amylase content[154], have been cloned, and someindica landrace. Overexpressing OsPTF1 in a low-P quality related genes have been fine-mapped, whichsensitive rice variety Nipponbare significantly enhanced greatly facilitate the improvement of rice quality by us-P-use efficiency. Tillering ability, root and shoot bio- ing MAS or transgenic strategies.mass, and P content of the transgenic plants were >30% Transgenic approaches have been applied mainly tohigher than those of the wild-type plants in P-deficient improving the nutritional quality of rice at present. Otherculture solution. In pot and field experiments with low-P than providing energy, rice is also an important source oflevels, tiller number, panicle weight, and P content in- proteins. Zhou et al.[155] analyzed the crude protein con-creased >20% in transgenic plants, compared with the tents (PC) in 351 rice varieties, and the results showedwild-type control. Moreover, total root length, root sur- that the PC varied between 9.3% and 17.7%, and theface area, and P uptake rate of transgenic rice plants average value is 12.4%. The average PC of indica varie-were also significantly higher than the control in P- ties is 13.2% that is approximately 1% higher than thatdeficient conditions. of japonica varieties. The nutritional quality of rice For phosphate uptake of plants, phosphate firstly en- would be enhanced by increasing protein content espe-ters the rice apoplast made up of the cell wall of epider- cially the amount of essential amino acids such as lysinemis and cortex cells from the soil, and then is transferred in rice endosperm using transgenic approaches. A com-through membrane into the symplast by phosphate trans- mon strategy is to express lysine-rich foreign proteins inporters, and finally transported to the shoots of the plant transgenic rice. For instance, Gao et al.[156] introduced avia xylem and distributed to various organs[148]. Most of lysine-rich protein gene (lys) from winged bean (Pso-high-affinity P transporter genes are expressed pre- phocarpus tetragonolobus) into rice by particle bom-dominantly in roots and are induced by P depletion, in- bardment, and lysine content in seeds of transgenic ricedicating that they are involved in the acquisition of Pthrough the roots under low external P concentrations. plants increased up to 16.04%. Tang et al.[157] introducedSeo et al.[149] identified a phosphate transporter gene a winged bean-derived lysine-rich protein gene into riceOsPT1 that is expressed primarily in roots and leaves via the Agrobacterium-mediated method, and obtainedregardless of external phosphate concentrations. Trans- maker-free transgenic rice with significantly improvedgenic rice plants overexpressing OsPT1 under the con- lysine content in seeds. Wang et al.[158] introduced a ly-trol of the CaMV 35S promoter accumulated almost sine-rich protein gene sb401 from potato pollen into antwice as much phosphate in the shoots compared with indica variety LongTeFuB. The average content proteinthe wild-type control under both normal and P-null ferti- and lysine in seeds of transgenic rice increased 18.7%lizations. The transgenic plants had more tillers and bet- and 10% respectively, and the content of other essentialter root development. However, transgenic rice overex- amino acids also increased in varying degree. Li et al.[159] GENE ENGINEERINGpressing OsPT1 was 30% shorter than the wild-type introduced sb401 into Nipponbare, and the content ofcontrol, which was supposed to be caused by the com- protein, lysine, and other essential amino acids in seedsparative deficiency of other nutrients such as N and po- of transgenic plants increased in varying degree. How- Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4059
  12. 12. ever, it should be noted that too high protein content transgenic rice, however overexpressing C3 plant-would affect the taste, and impair the eating quality of derived orthologs has also been attempted. Ku et al.[166]rice. firstly introduced a maize phosphoenolpyruvate carbo- Moreover, some studies have been conducted to en- cylase (PEPC) gene into rice, and the transgenic ricehance rice micronutrients such as β-carotene, iron and plants exhibited some photosynthetic characteristics ofzinc. Golden rice, which is transgenic rice with enhanced C4 plants. O2 inhibition in photosynthesis of transgenicβ-carotene, was an outstanding paradigm. Golden rice is plants reduced about 20% compared with the wild-typegenerated by introducing two foreign genes into trans- control. Later, more C4 cycle-related genes have beengenic rice phytoene synthase gene (psy) from daffodil introduced into rice including PEPC[166–171], pyruvate,(Narcissus pseudonarcissus), and bacterial phytoene orthophosphate dikinase (PPDK) gene[171,172], phosphoe-desaturase (crtI) from Erwinia uredovora to establish a nolpyruvate carboxykinase (PEPCK) gene[169,173], NADP-novel carotenoid biosynthesis pathway in rice endos- malic enzyme gene (ME) gene[171,174,175], and NADP-perm[12,160]. β-carotene is the precursor of vitamin A, and malate dehydrogenase (MDH) gene[176]. Although over-taking golden rice is thus supposed to address the heal- expressing these C4-related genes in rice showed diversethy issues such as blindness, susceptibility for diseases, effects, it is still far from the purpose of increasing theand increased child mortality caused by vitamin yield greatly, and even overexpressing some C4-relatedA-deficiency which prevails in the population living in gene led to severe negative effects. For instance, overex-the poor areas. pression of maize C4-specific ME resulted in serious Besides golden rice, high iron content rice has also stunting, leaf chlorophyll bleaching, and enhanced pho-been developed. Goto et al.[161] increased iron content in toinhibition of photosynthesis[171,174,175]. Combinations ofrice grain two- to threefold by tissue-specifically over- multiple C4-related genes synchronously have also beenexpressing an iron storage protein gene ferritin in rice attempted, which was expected to achieve better effects.endosperm. Several other groups attempted similar To establish a C4-like pathway in mesophyll cells ofstrategies and obtained similar results[162–165]. Taking this transgenic rice, Taniguchi et al.[171] overexpressed fourtransgenic rice is expected to alleviate the symptoms C4-related genes with different origins in combination:such as anemia caused by iron-deficiency which prevails the maize C4-specific PEPC and PPDK, the sorghumin the population, especially children and women, living MDH, and the rice C3-specific ME. However, the trans-in the poor areas. genic rice plants only exhibited slightly improved photo- synthesis accompanied with slight but reproducible stunt-7 Transgenic high yield rice ing phenotype compared with the wild-type control. How- ever, some reports were optimistic anyway. Jiao et al.[167]Much effort to develop high yield rice has been concen- reported that grain yield of transgenic rice increasedtrated on seeking C4 rice in the past decade. As known, 22%―24% through co-expressing C4-specific PEPC andhigher plants can be divided into three groups: C3, C4 PPDK in rice.and crassulacean acid metabolism (CAM) plants ac- C4 rice is undoubtedly one of the most challengingcording to the initial photosynthates of CO2 in the car- subjects for transgenic rice research. C4 rice research isbon assimilation pathway during photosynthesis. C4 very arduous due to huge distances of antimony and ge-plants which evolved from C3 plants are the type with netics between C3 and C4 plants. However, it is stillhigher photosynthesis efficiency, which have competi- valuable as an attempt to change the current status thattive advantages in photosynthesis efficiency and stresses rice yield has been hovering for a long period.tolerance over C3 plants. Unfortunately, many agronomi-cally important crops such as rice, wheat, barley, and 8 Transgenic herbicide-tolerant ricesoybean are C3 plants. For a long time, botanists andbreeders dreamed to change C3 crops into C4 crops, and Herbicide-tolerance has been continuously the numberrecently the advances of genetic engineering provide new one trait of GM crops, with the largest growing areaopportunities. since GM crops were first commercially grown in 1996. The common strategy to develop C4 rice is to over- There are two main strategies to develop herbicide-express C4 plant-derived genes involved in C4 cycle in tolerant rice: (ⅰ) modifying the target protein genes of4060 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
  13. 13. REVIEWherbicides to decrease their susceptivity or increase the fore, overexpressing P450 monooxygenases in plants isexpression level; (ⅱ) introducing novel enzyme systems able to enhance the herbicide resistance, and the resis-via genetic engineering to enhance the metabolic capac- tance is generally broad-spectrum against multiple her-ity of herbicides. There are three main purposes to pro- bicides with different modes of action. Japanese re-duce herbicide-tolerant rice: to use chemical herbicides searchers have done much work about it. They intro-in the field that can decrease the cost and increase the duced P450 monooxygenase genes from mammals orincome; to remove the false hybrid seeds and increase even humans into transgenic rice to obtain herbicide-the seed purity for rice hybrid production; to generate tolerant rice[183–188]. Moreover, the transgenic rice over-transgenic rice plants as selection markers. expressing P450 monooxygenases can be used to phy- The bar gene from Streptomyces hygroscopicus is the toremediate pesticides or other environmental organicfirst and most common herbicide-resistant gene used in pollutants[186,187,189,190]. It should be noted that the com-transgenic rice. The bar gene can confer transgenic rice position of the secondary metabolites in these transgenicthe resistance to the herbicide phosphinothricin (PPT), rice plants possibly varies due to the alteration of P450which can non-selectively kill various plants (trade species and activities. However, what effects on humannames: Liberty, Finale, Basta, etc.). PPT kills plants by health and the environment the variation of the secon-inhibiting plant GS and causes the accumulation of am- dary metabolites in transgenic rice plant would causemonia in plant cells. Bar gene encodes a PPT acetyl- still needs further evaluations.transferase (PAT) that can deactivate PPT. To date, many In addition to the herbicide-tolerant rice describedstudies of transgenic rice with bar gene have been re- above, there are other types of transgenic rice againstported[176–178]. Novel hybrids IIyou 86B and Teyou 86B different herbicides targeting protoporphyringen oxi-were developed by South China Botanical Garden, Chi- dases[191–193] and acetolactate synthase[194]. The herbi-nese Academy of Sciences using transgenic Minghui cide-tolerance of these transgenic rice plants is acquired86B with bar gene. Risk assessment of intermediate trial by modifying the genes of target proteins.and environmental release for transgenic Minghui 86Bwith bar gene and its hybrids have been done, and the 9 Prospectsproduction trial would be conducted in 2005[179]. Tremendous progress in the development of transgenic Glyphosate is the active ingredient of the herbicide research in rice has been shown in the past two decades.Roundup of Monsanto Company, which has been broadly Not only transformation system has been established,applied worldwide due to its high efficiency, low toxicity, but also a many of transgenic rice materials with poten-and broad-spectrum. The targeting enzyme of glyphosate tial application acquired. With the deployment of func-is 5-enolpyrulyshikimate-3-phosphate synthase (EPSP), tional genomics research in model plants includingwhich is a key enzyme involved in the synthesis of aro- Arabidopsis and rice, many agronomically importantmatic amino acids in bacteria and plants. Glyphosate genes have been discovered and isolated, which enricheskills plants by inhibiting EPSP and the synthesis of aro- strongly the available gene resources for transgenic ricematic amino acids. The common strategy of glyphosate- research. However, there are still many needs for furtherresistant genetic engineering is to decrease the suscepti- improvement of transgenic rice research from variousbility to glyphosate by modifying EPSP. Hu et al.[180] aspects. Firstly, from a technology perspective, trans-introduced a bacterium-derived citrate synthase gene formation with large DNA fragments and chloroplast(CS) into an elite indica restorer Minghui 86 using a transformation have shown huge potential application,synthetic EPSP gene as the selection marker. The regen- but which have not been used widely and need furthererated transgenic plants showed significantly enhanced technical modifications. Secondly, the comparative scar-resistance to Roundup. Su et al.[181] obtained an EPSP city of the gene resource for transgenic research is stillmutant gene by error-prone PCR and introducing this a limitation. For instance, no highly effective insect-EPSP mutant gene into rice could significantly enhance resistance genes are available for transgenic rice to con- GENE ENGINEERINGthe glyphosate-tolerance of transgenic plants. trol rice planthoppers currently. Although 19 resistant The cytochrome P450 monooxygenases exist broadly genes against BPH have been identified in rice[1], ain all organisms, which play an important role in de- BPH-resistant rice variety is probably overcome by BPHtoxifying hydrophobic xenobiotic chemicals[182]. There- within few years after it has been adopted widely in Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4061
  14. 14. production, because BPH has multiple biotypes and is transgenic rice as one of the most important food crops.prone to evolve a resistance. How to develop durable The best achievements would not have any values if trans-planthopper-resistant varieties is one of the most urgent genic rice can not be used in production.issues for transgenic rice research at present. Moreover, To address these challenges, functional genomics re-no major resistant genes to rice sheath blight and major search should be further deepened to identify and isolategenes or QTLs of N-use efficiency have been identified more gene resources with practical use. Meanwhile, thein rice too. The current status of lacking gene resources research of underlying biological mechanisms of relatedhas become the bottleneck to develop novel rice varie- traits should be conducted too, because the related traitties. Thirdly, a transgenic approach still has many limi- improvement would be more effective if the underlyingtations on the improvement of complicated traits. For biological mechanism has been well-documented. Oninstance, to develop transgenic drought-tolerant rice or the other hand, scientists must intensify popularizationC4 rice, although certain effects have been observed by of science and education to improve the public know-introducing some foreign genes, there is still a long way ledge of transgenic technology and dispel the people’sto go. Finally, we must be aware that the commercializa- prejudice and doubt about it. Finally, only when inte-tion of transgenic rice is still difficult, even if transgenic grated with MAS and conventional breeding proceduressoybean, corn, and cotton have been grown commer- can transgenic approaches exert their advantages fully tocially for over 10 years. People have too much doubt on develop more and better rice varieties. 1 Zhang Q. 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