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 special
with the capacity to break the reproductive isolation be- transformation technologies.
tween species and realize the free communication of ge- 1.1 Multigene transformation
netic materials. Reviewing the history of the development
Transformation of multiple genes is mainly applied to two
of transgenic rice in the past two decades, most target
purposes. Firstly, it facilitates the procedure of map-based
traits are consistent with the goal of GSR. In this article,
gene cloning. A key step of map-based gene cloning is to
we firstly give a brief account of the technical advances of
validate the candidate genes. Transformation of multiple
rice 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 transformed
traits, 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 introduced
1 Rice transformation
foreign genes in commercialized transgenic crops are
Rice transformation achieved important success in the generally single genes to control qualitative traits such
late 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 controlled
the recipient via electroporation-mediated or PEG-me- by multiple genes. To improve these traits, the multiple
diated 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 needed
became one of the most common methods of rice trans- in case of promptly pyramiding multiple qualitative
formation. 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 is
transformation 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 vectors
further improved to shorten the transformation proce- firstly, and then the multigene pyramiding is performed
dure[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 this
modifications were made to improve the transformation strategy. Another one is to construct foreign genes in a
efficiency of indica rice[9,10]. Recently, Hiei and Komari[11] single vector, and multiple genes are then introduced
published 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 economic
According to Hiei and Komari [11], transformation of in- compared with the former one. However, transformation
dica 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 common
ciency (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 cloning
of the protocol is that collection of immature embryos is capacity of a common Ti binary vector is usually less
laborious 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. There
more. Some special transformation technologies have are two main Ti vectors for transformation of large DNA
4050 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
3. REVIEW
fragments: BIBAC (Binary BAC) derived from bacterial 1.3 Chloroplast transformation
artificial chromosome[14] and TAC (Transformation-com-
Chloroplast transformation is usually implemented by
petent Artificial Chromosome) derived from P1 artificial
delivering plasmid vectors containing transgenes into
chromosome[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 been
successfully 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 of
formation would have a huge potential for rice transgenic homologous sequences flanking transgenes. There are
breeding. 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 extremely
Constitutive CaMV 35S and maize Ubiquitin promoters high due to high transgene copies. There are generally 10
are the two most common promoters used in rice trans-
-100 chloroplast genome copies per chloroplast and 10
genic research. There are certain problems to express
-100 chloroplasts per cell, resulting in theoretically as
transgenes in all plant tissues and organs at all growth
stages using a strong constitutive promoter, for instance, many as up to 10000 transgene copies per cell that is
increasing 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 is
due 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 (up
abiotic 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 genome
growth 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 field
inducible promoters. experiment or commercial production of transgenic
Transgenic Bt rice is the most promising transgenic plants acquired via chloroplast transformation is safer
rice for commercialization. However, the public’s con-
and more environment-friendly. Furthermore, there are
cern 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 arranged
rium-mediated transformation. In acquired transgenic
in operons, that means a promoter is able to control the
plants, Bt protein is expressed predominantly in green
parts of the plant such as the leaf and stem that are expression of multigenes as a polycistron, which may
mainly targets attacked by insect pests, while barely in facilitate multigene transformation; gene silencing of
the 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 been
is less than 1/1000 of that when driven by the maize applied as widely as nuclear transformation due to many
Ubiquitin promoter compared with the results of Tang practically technical difficulties. So far, chloroplast trans-
GENE ENGINEERING
et al.[18,19]. It is supposed that Bt rice with green part- formation has been achieved only for more than 10 plant
specific 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. 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]. There
Insect 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 resistance
Spraying 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 of
costs, but also pollutes the environment and threatens transgenic rice harboring a cry1Ab/Ac fusion gene. Their
human health. Enhancing insect-resistance of rice itself results showed that transgenic cry1Ab/Ac Minghui 63
by breeding approaches is a more economic and envi- (an elite rice restorer line) and its hybrid Bt Shanyou 63
ronment-friendly strategy. However, developing insect- exhibited high insect-resistance in field conditions
resistant cultivars by conventional breeding approaches without spraying any chemical insecticides during the
is time-consuming. Moreover, no effective resistance whole growth period, indicating huge use value of Bt
germplasm 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 might
locrocis medinalis), which are main rice pests. The most evolve resistance against Bt crop, which would impair
promising method currently is to develop transgenic in- its durability. Although no insect species with resistance
sect-resistant varieties by introducing foreign insect- re- to Bt crops have been identified under natural conditions
sistant genes into rice. Many useful insect-resistant genes so far, some insects have evolved resistances against Bt
have 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 green
lines 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 have
field conditions and showed broad potential application the potential to evolve the resistance against Bt crops in
for 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 durability
one 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 resistance
referred to as Bt toxins or insecticidal crystal proteins. against two Bt toxins simultaneously is much lower than
Transgenic Bt crops acquire insect-resistance due to the that against one Bt toxin. Therefore, two-toxin Bt rice
accumulation of Bt toxin in the plant. Bt genes have can greatly delay the development of insect-resistance
been successfully transferred and expressed in different and is more durable. However, the two Bt toxins in
crops including rice. Among them, Bt cotton, corn, and combination must bind to different receptor sites on insect
potato have been commercially growing and bringing gut cells to avoid the occurrence of “cross-resistance”. As
huge 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. It
against 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 and
used in transgenic plants. The most common Bt genes Cry1C* rice were highly resistant against lepidopteran
4052 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
5. REVIEW
rice pests. Transgenic Cry2A* rice and Cry1C* rice may the laboratory showed that most combinations of two Bt
provide new gene resources for the development of two- toxins had synergistic effects and exhibited significantly
toxin 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 genes
suitable to combine because insects unlikely develop a that have been applied in transgenic rice so far, which
cross-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 in
10 two-toxin Bt rice lines 1Ab/1C, 1C/1Ab, 1Ab/2A, Iran 2005. Bt rice has been well-developed in China and
2A/1Ab, 1Ac/1C, 1C/1Ac, 1Ac/2A, 2A/1Ac, 1C/2A and can be commercialized promptly as soon as the policy
2A/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-derived
cry1Ac (1Ac), cry1C* (1C), and cry2A* (2A), in five
genes
combination patterns (1Ab+1C, 1Ab+2A, 1Ac+1C,
1Ac+2A, 1C+2A) (Yang Zhou and Lin Yongjun, un- Plant-derived insect-resistant genes commonly include
published data). The transgenic line 1Ab/1C means the plant lectin genes and protease inhibitor genes. Plant
maternal 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) gene
may reason by analogy. The results of bioassay in has been widely applied. The principal advantage to use
GENE ENGINEERING
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.
Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4053
6. GNA gene is that GNA has certain insecticidal activity All transgenic insect-resistant rice described above
against sap-sucking (homoptera) insects such as rice acquired their resistance through directly expressing
planthoppers, 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 key
GNA rice lines via particle bombardment. Their data genes for pest development or biochemical metabolism
showed that the homogenous transgenic lines can control via RNA interference (RNAi) using gene fragment from
brown 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 strategy
development 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 be
effects on planthoppers, leafhoppers, and aphids[38,43,52–59]. noted that if using RNAi strategy, the targeting sites
However, toxicity of GNA to sap-sucking rice pests is not must be pest gene-specific to ensure that the transgenic
comparable 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 rice
other study involving an Allium sativum agglutinin from Bacterial blight (BB), fungal diseases blast, and sheath
leaf (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. Oryzae
hanced resistance to BPH and green leafhopper (Nepho- (Xoo) is the most devastating rice bacterial disease
tettix 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 caused
dence of rice tungro diseases, caused by co-infection of
by Magnaporthe grisea (M. grisea) may arise in all rice
green leafhopper-vectored rice tungro bacilliform virus
organs at any growth stage. Sheath blight caused by
and 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 against
in transgenic rice include: potato protease inhibitor gene
Xoo have been identified in rice so far. Among them, six
pinII[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]. BB
gene BTI-Cme[67]. These transgenic rice plants exhibited
is effectively controlled in rice production due to the
certain 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 Minghui
have 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 Minghui
cation of plant-derived insect-resistant genes is still lim- 63 and WanB (a rice maintainer line) by introducing
ited because of their relatively inadequate insecticidal Xa21 into the corresponding wild-type recipients, and
activities. 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 been
transgenic insect-resistant rice against striped stem borer identified in rice so far[76], among which 10 resistant
and 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 has
4054 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
7. REVIEW
many physiological races with high variability, a Blast- QTLs is very valuable to develop the resistant varieties
resistant 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 some
no 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 OsWRKY13
like 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 the
proteins encoded by the host plants but induced exclu- expression of a resistance-related QTL OsDR10 in rice
sively in pathological or related situations, and many of via RNAi, and the transgenic plants showed enhanced
them showed antifungal activity in vitro[78]. Some stud- resistances to multiple Xoo strains compared with the
ies 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 not
grisea[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 not
rice enhanced resistance against M. grisea; Datta et al.[85] comparable with that of qualitative resistance conferred
found that overexpressing thaumatin-like protein in by major resistance genes, but they are still worthy of
transgenic rice enhanced resistance against R. solani. research and utilization because of their broad-spectrum
Besides using single PR genes, pyramiding different PR and durability.
genes is also common. For instance, combinations of
chintinase with β-1,3-glucanase can enhance the resis- 4 Transgenic drought-tolerant rice
tance of transgenic rice to blast[86–88]; combination of
Drought is one of the major factors causing yield loss in
chitinase with a modified maize ribosome-inactivating
rice production for a long time and is getting worse as
protein[89] or a thaumatin-like protein[90] can enhance
the climate changes worldwide. Rice production need
resistance 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 our
fungal diseases by overexpressing antifungal proteins or
country. While China is water deficient, and the average
peptides from plants or microorganisms in rice, and also
capita water capacity is only a quarter of that of the
achieved 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 the
and 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 is
sistance. Shao et al.[95] reported that overexpression of a that plants are not “movable”. Correspondingly, plants
protein elicitor harpin from Xoo in transgenic rice con- evolve a complex biological mechanism to resist various
ferred high non-specific resistance to multiple M. grisea environmental stresses. When under an environmental
races. 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, and
are 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 phosphates
pared with the major resistance genes, their advantages that can transfer further in the plant cell. Then, protein
are broad-spectrum and more durable. Nevertheless, no phosphorylation cascades of Ca2+-dependent protein
GENE ENGINEERING
major resistance genes have been found in rice for some kinases (CDPKs), mitogen-activated protein kinases
rice diseases such as rice sheath blight, false smut, and (MAPKs), etc. triggered by the second messenger
bacterial 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. 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 as
proteins to help the plant re-establish osmotic homeostasis, superoxide dismutase (SOD). This group was generally
scavenge 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-like
it 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 of
engineering has been broadly applied to developing drought- these genes generally can influence the expression level
tolerant 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 more
Table 1 summarizes some representative experiments effective than those functional or structural genes with
about 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 transcription
Table 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. REVIEW
factor gene SNAC1 with great potential application, SOS2, Actin1:ZAT10, and CBF3, LOS5, ZAT10, and
which is a member of NAC (NAM, ATAF, and CUC) NHX1 by both promoters) showed significantly higher
plant-specific gene family. SNAC1 is specifically ex- relative spikelet fertility than the wild-type control in the
pressed in leaf guard cells under drought stress condi- PVC pipes under drought stress. In the field drought
tions. 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 significantly
without showing any phenotypic changes or yield pen- higher yield per plant than the wild-type control, and
alty. 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, and
maintained turgor pressure under lower relative water Actin1:NHX1) had higher spikelet fertility than the
content[128]. The transgenic rice also showed signifi- wild-type control. In conclusion, LOS5 and ZAT10
cantly improved drought and salt-tolerance at the vege- showed relatively better effects than the other five genes
tative stage (80% higher survival rate compared with the in improving drought resistance of transgenic rice under
control) (Figure 1(c)). DNA microarray analysis re- field conditions. The results of this study were based on
vealed that over 150 stress-related genes were up-regu- field experiments and might be a useful reference for
lated 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 have
related 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 practical
SOD and monodehydroascorbate (MDA) in plants under drought-tolerant rice variety. In view of the complex
drought stress. Moreover, the transcript levels of many mechanism of drought-tolerance, it is crucial to pyramid
stress-related genes are significantly higher than the various drought-tolerant genes by taking an integrated
wild-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 were
obtained under greenhouse conditions, and very few 5 Transgenic nutrient-use efficient rice
studies 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 crop
resistant 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 decades
homolog (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, worldwide due to the green evolution, associated with a
and 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 on
under field conditions. Their results showed that trans- a high investment is not sustainable. The increase of
genic families of eight constructs (HVA22P:CBF3, HVA22P: food production is not so significant anymore even if the
NPK1, 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 ENGINEERING
significantly higher relative yield than the wild-type con- of environmental issues, such as eutrophication of water
trol in both field and PVC pipes conditions with drought body, groundwater pollution, soil acidification, etc. As
stress. 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. 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 showed
rity 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 N
efficient rice varieties in combination with scientific content in the whole plant. However, both grain yield
fertilization and cultivation management to substantially and total amino acids in seeds of GS-overexpressed rice
decrease the use of fertilizer is very important to ensure plants decreased compared with the wild-type control
food security and realize the sustainable development of under field conditions with N deficit stress.
modern agriculture. Ammonium transporters are crucial for the plant root
5.1 Nitrogen-use efficiency to take up NH4+ from the soil. Ten ammonium trans-
porter genes have been identified in rice, among which
N is an essential nutrient that plants require in the most
OsAMT1;1, OsAMT1;2 and OsAMT1;3 belong to ATM1
quantity 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, and
compounds 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 rice
key 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 is
tively absorbed from the soil by different ammonium supposed that biomass decrease of the transgenic plants
transporters 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 transgenic
nantly in seeds, roots, nodules, flowers, and phloem, plants has also been attempted to change the level of
which 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. Shrawat
location. GS2 is the predominant isoenzyme in leaves et al.[144] reported that tissue-specifically expressing a
that may function in assimilation of ammonia reduced barley alanine aminotransferase (AlaAT) cDNA in rice
from nitrate in chloroplasts and/or in the reassimilation roots significantly increased the biomass and grain yield
of 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 transgenic
GS2 that exists predominantly in leaf cells, and three rice plants also increased, indicating enhanced N uptake
ones encoding cytosolic GS1 that exists predominantly in efficiency. Zhou et al.[145] overexpressed separately all of
the 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 gene
dependent 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 grain
compared with the non-transgenic control, indicating amino acid and protein contents compared with the
that NADH-GOGAT is indeed a key step for N utiliza- non-transgenic control. No significant changes were
tion 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 significant
4058 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
11. REVIEW
difference in main agronomic traits and yield compared tassium because they were not concomitantly increased
with the wild-type control. with an enhanced P acquisition.
5.2 Phosphorus-use efficiency
6 Transgenic high quality rice
Phosphorus (P) is one of the essential macroelements
too. Although the absolute P amount in the soil is com- Rice quality is recently getting more and more attention
paratively abundant, the available P is deficient (usually with the improvement of people’s living conditions. The
less 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 quality
developing 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 Wx
scription factor OsPTF1 from Kasalath, a P-use efficient for amylase content[154], have been cloned, and some
indica landrace. Overexpressing OsPTF1 in a low-P quality related genes have been fine-mapped, which
sensitive 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 to
higher than those of the wild-type plants in P-deficient improving the nutritional quality of rice at present. Other
culture solution. In pot and field experiments with low-P than providing energy, rice is also an important source of
levels, 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 showed
wild-type control. Moreover, total root length, root sur- that the PC varied between 9.3% and 17.7%, and the
face 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 that
deficient 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 lysine
mis 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 in
porters, and finally transported to the shoots of the plant
transgenic rice. For instance, Gao et al.[156] introduced a
via 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 rice
dicating that they are involved in the acquisition of P
through the roots under low external P concentrations. plants increased up to 16.04%. Tang et al.[157] introduced
Seo et al.[149] identified a phosphate transporter gene a winged bean-derived lysine-rich protein gene into rice
OsPT1 that is expressed primarily in roots and leaves via the Agrobacterium-mediated method, and obtained
regardless of external phosphate concentrations. Trans- maker-free transgenic rice with significantly improved
genic 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 an
twice as much phosphate in the shoots compared with indica variety LongTeFuB. The average content protein
the 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 essential
ter root development. However, transgenic rice overex- amino acids also increased in varying degree. Li et al.[159]
GENE ENGINEERING
pressing OsPT1 was 30% shorter than the wild-type introduced sb401 into Nipponbare, and the content of
control, which was supposed to be caused by the com- protein, lysine, and other essential amino acids in seeds
parative 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. 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 rice
hance rice micronutrients such as β-carotene, iron and plants exhibited some photosynthetic characteristics of
zinc. Golden rice, which is transgenic rice with enhanced C4 plants. O2 inhibition in photosynthesis of transgenic
β-carotene, was an outstanding paradigm. Golden rice is plants reduced about 20% compared with the wild-type
generated by introducing two foreign genes into trans- control. Later, more C4 cycle-related genes have been
genic 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 diverse
thy issues such as blindness, susceptibility for diseases, effects, it is still far from the purpose of increasing the
and increased child mortality caused by vitamin yield greatly, and even overexpressing some C4-related
A-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 of
rice grain two- to threefold by tissue-specifically over- multiple C4-related genes synchronously have also been
expressing 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 of
strategies and obtained similar results[162–165]. Taking this transgenic rice, Taniguchi et al.[171] overexpressed four
transgenic 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 sorghum
in 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 increased
trated on seeking C4 rice in the past decade. As known,
22%―24% through co-expressing C4-specific PEPC and
higher 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 challenging
cording to the initial photosynthates of CO2 in the car-
subjects for transgenic rice research. C4 rice research is
bon 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 still
higher photosynthesis efficiency, which have competi-
valuable as an attempt to change the current status that
tive 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 rice
soybean are C3 plants. For a long time, botanists and
breeders dreamed to change C3 crops into C4 crops, and Herbicide-tolerance has been continuously the number
recently the advances of genetic engineering provide new one trait of GM crops, with the largest growing area
opportunities. 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 of
4060 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
13. REVIEW
herbicides to decrease their susceptivity or increase the fore, overexpressing P450 monooxygenases in plants is
expression 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 or
income; 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 organic
first 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 transgenic
the resistance to the herbicide phosphinothricin (PPT), rice plants possibly varies due to the alteration of P450
which can non-selectively kill various plants (trade species and activities. However, what effects on human
names: 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 cause
monia 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 described
studies of transgenic rice with bar gene have been re- above, there are other types of transgenic rice against
ported[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 acquired
86B with bar gene. Risk assessment of intermediate trial by modifying the genes of target proteins.
and environmental release for transgenic Minghui 86B
with bar gene and its hybrids have been done, and the 9 Prospects
production 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 including
which is a key enzyme involved in the synthesis of aro-
Arabidopsis and rice, many agronomically important
matic amino acids in bacteria and plants. Glyphosate
genes have been discovered and isolated, which enriches
kills plants by inhibiting EPSP and the synthesis of aro- strongly the available gene resources for transgenic rice
matic amino acids. The common strategy of glyphosate- research. However, there are still many needs for further
resistant genetic engineering is to decrease the suscepti- improvement of transgenic rice research from various
bility 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 further
erated 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 still
mutant 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 ENGINEERING
the 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], a
in all organisms, which play an important role in de- BPH-resistant rice variety is probably overcome by BPH
toxifying 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. 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 isolate
genes or QTLs of N-use efficiency have been identified more gene resources with practical use. Meanwhile, the
in rice too. The current status of lacking gene resources research of underlying biological mechanisms of related
has become the bottleneck to develop novel rice varie- traits should be conducted too, because the related trait
ties. Thirdly, a transgenic approach still has many limi- improvement would be more effective if the underlying
tations on the improvement of complicated traits. For biological mechanism has been well-documented. On
instance, to develop transgenic drought-tolerant rice or the other hand, scientists must intensify popularization
C4 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’s
to 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 procedures
soybean, corn, and cotton have been grown commer- can transgenic approaches exert their advantages fully to
cially for over 10 years. People have too much doubt on develop more and better rice varieties.
1 Zhang Q. Strategies for developing green super rice. Proc Natl 11 Hiei Y, Komari T. Agrobacterium-mediated transformation of rice
Acad Sci USA, 2007, 104: 16402―16409 using immature embryos or calli induced from mature seed. Nat
2 Toriyama K, Arimotoa Y, Uchimiyaa H, et al. Transgenic rice plants Protoc, 2008, 3: 824―834
after direct gene transfer into protoplasts. Bio/Technology, 1988, 6: 12 Ye X, Al-Babili S, Kloti A, et al. Engineering the pro-Vitamin A
1072―1074 (beta-carotene) biosynthetic pathway into (carotenoid-free) rice
3 Zhang H M, Yang H, Rech E L. Transgenic rice plants produced by endosperm. Science, 2000, 287: 303―305
electroporation-mediated plasmid uptake into protoplasts. Plant 13 Daniell H, Dhingra A. Multigene engineering: dawn of an exciting
Cell Rep, 1988, 7: 379―384 new era in biotechnology. Curr Opin Biotechnol, 2002, 13: 136―
4 Zhang W, Wu R. Efficient regeneration of transgenic plants from 141
rice protoplasts and correctly regulated expression of the foreign 14 Hamilton C M, Frary A, Lewis C, et al. Stable transfer of intact
gene in the plants. Theor Appl Genet, 1988, 76: 835―840 high molecular weight DNA into plant chromosomes. Proc Natl
5 Christou P, Ford T, Kofron M. Production of transgenic Rice (Oryza Acad Sci USA, 1996, 93: 9975―9979
Sativa L.) plants from agronomically important indica and japonica 15 Liu Y G, Shirano Y, Fukaki H, et al. Complementation of plant mu-
varieties via electric discharge particle acceleration of exogenous tant with large genomic DNA fragments by a transformation-
DNA into immature zygotic embryos. Bio/Technology, 1991, 9: competent artificial chromosome vector accelerates positional
957―962 cloning. Proc Natl Acad Sci USA, 1999, 96: 6535―6540
6 Chan M T, Chang H H, Ho S L, et al. Agrobacterium-mediated 16 Zhou Y, Jiang D, Wu H, et al. Development of transformation system
production of transgenic rice plants expressing a chimeric al- of rice based on transformation-competent artificial chromosome
pha-amylase promoter/beta-glucuronidase gene. Plant Mol Biol, (TAC) vector. Acta Genet Sin, 2005, 32: 514―518
1993, 22: 491―506 17 He R, W Y, Du P, et al. Development of transformation system of
7 Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice rice based on binary bacterial artificial chromosome (BIBAC)
(Oryza sativa L.) mediated by Agrobacterium and sequence analy- vector. Acta Genet Sin, 2006, 33: 269―276
sis of the boundaries of the T-DNA. Plant J, 1994, 6: 271―282 18 Ye R, Huang H, Zhou Y, et al. Development of insect-resistant
8 Toki S, Hara N, Ono K, et al. Early infection of scutellum tissue transgenic rice with Cry1C*-free endosperm. Pest Manag Sci, 2009,
with Agrobacterium allows high-speed transformation of rice. Plant 65: 1015―1020
J, 2006, 47: 969―976 19 Tang W, Chen H, Xu C G, et al. Development of insect-resistant
9 Lin Y J, Zhang Q. Optimizing the tissue culture conditions for high transgenic indica rice with a synthetic cry1C* gene. Mol Breed,
efficiency transformation of indica rice. Plant Cell Rep, 2005, 23: 2006, 18: 1―10
540―547 20 Daniell H, Muhammad S, Allison K L. Milestones in chloroplast
10 Hiei Y, Komari T. Improved protocols for transformation of indica genetic engineering: an environmentally friendly era in biotechnology.
rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Or- Trends Plant Sci, 2002, 7: 84―91
gan Cult, 2006, 85: 271―283 21 Lee S M, Kang K, Chung H et al. Plastid transformation in the
4062 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com
15. REVIEW
monocotyledonous cereal crop, rice (Oryza sativa) and transmis- pressing modified Cry1Ac endotoxin of Bacillus thuringiensis
sion of transgenes to their progeny. Mol Cells, 2006, 21: 401―410 show enhanced resistance to yellow stem borer (Scirpophaga in-
22 Su N, Sun M, Yang B, et al.The insect resistance of OC and Bt certulas). Transgenic Res, 2002, 11: 411―423
transplastomic plant and the phenotype of their progenies. 38 Loc N T, Tinjuangjun P, Gatehouse A M R, et al. Linear transgene
Hereditas, 2002, 24: 288―292 constructs lacking vector backbone sequences generate transgenic
23 Li Y, Sun B, Su N, et al. Establishment of a gene expression system rice plants which accumulate higher levels of proteins conferring
in rice chloroplast and obtainment of PPT-resistant rice plants. Sci insect resistance. Mol Breed, 2002, 9: 231―244
Agric Sin, 2007, 40: 1849―1855 39 Zeng Q C, Wu Q, Zhou K D, et al. Obtaining stem borer-resistant
24 Qian X, Yang X, Guo D, et al. Advances in the research of plant homozygous transgenic lines of Minghui 81 harboring novel
chloroplast genetic transformation. Mol Plant Breed, 2008, 6: cry1Ac gene via particle bombardment. Acta Genet Sin, 2002, 29:
959―966 519―524
25 James C. Global status of commercialized biotech/GM crops: 2008. 40 Tu J, Zhang G, Datta K, et al. Field performance of transgenic elite
ISAAA Brief No. 39. Ithaca, N.Y.: ISAAA, 2008 commercial hybrid rice expressing bacillus thuringiensis
26 Fujimoto H, Itoh K, Yamamoto M, et al. Insect resistant rice delta-endotoxin. Nat Biotechnol, 2000, 18: 1101―1104
generated by introduction of a modified delta-endotoxin gene of 41 Ramesh S, Nagadhara D, Pasalu I C, et al. Development of stem
Bacillus thuringiensis. Bio/Technology, 1993, 11: 1151―1155 borer resistant transgenic parental lines involved in the production
27 Wünn J, Kloti A, Burkhardt P K, et al. Transgenic indica rice of hybrid rice. J Biotechnol, 2004, 111: 131―141
breeding line IR58 expressing a synthetic cry1A(b) gene from 42 Maqbool S B, Husnain T, Riazuddin S et al. Effective control of
Bacillus thuringiensis provides effective insect pest control. Bio/ yellow stem borer and rice leaf folder in transgenic rice indica
Technology, 1996, 14: 171―176 varieties Basmati 370 and M7 using the novel δ-endotoxin cryIIA
28 Ghareyazie B, Alinia F, Menguito C A, et al. Enhanced resistance to Bacillus thuringiensis gene. Mol Breed, 1998, 4: 1―7
two stem borers in an aromatic rice containing a synthetic cryIA(b) 43 Maqbool S B, Riazuddin S, Loc N T, et al. Expression of multiple
gene. Mol Breed, 1997, 3: 401―414 insecticidal genes confers broad resistance against a range of
29 Wu C, Fan Y, Zhang C, et al. Transgenic fertile japonica rice plants different rice pests. Mol Breed, 2001, 7: 85―93
expressing a modified cry1A(b) gene resistant to yellow stem borer. 44 Breitler J C, Marfa V, Royer M, et al. Expression of a Bacillus
Plant Cell Rep, 1997, 17: 129―132 thuringiensis cry1B synthetic gene protects Mediterranean rice
30 Cheng X, Sardana R, Kaplan H, et al. Agrobacterium-transformed against the striped stem borer. Plant Cell Rep, 2000, 19: 1195―
rice plants expressing synthetic cryIA(b) and cryIA(c) genes are 1202
highly toxic to striped stem borer and yellow stem borer. Proc Natl 45 Breitler J C, Cordero M J, Royer M, et al. The –689/+197 region of
Acad Sci USA, 1998, 95: 2767―2772 the maize protease inhibitor gene directs high level,
31 Datta K, Vasquez A, Tu J, et al. Constitutive and tissue specific wound-inducible expression of the cry1B gene which protects
differential expression of the cry1A(b) gene in transgenic rice plants transgenic rice plants from stemborer attack. Mol Breed, 2001, 7:
conferring resistance to rice insect pest. Theor Appl Genet, 1998, 259―274
97: 20―30 46 Gahakwa D, Maqbool S B, Fu X, et al. Transgenic rice as a system
32 Su Q, Ye G, Cui H, et al. Development of transgenic Bacillus to study the stability of transgene expression: multiple heterologous
thuriengiensis rice resistant to rice stem borers and leaf folders. J transgenes show similar behaviour in diverse genetic backgrounds.
Zhejiang Agric Univ, 1998, 24: 579―580 Theor Appl Genet, 2000, 101: 388―399
33 Alam M F, Datta K, Abrigo E, et al. Transgenic insect resistant 47 Chen H, Tang W, Xu C G, et al. Transgenic indica rice plants
maintainer line (IR68899B) for improvement of hybrid rice. Plant harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit
Cell Rep, 1999, 18: 572―575 enhanced resistance against lepidopteran rice pests. Theor Appl
34 Ye G Y, Shu Q Y, Yao H W, et al. Field evaluation of resistance of Genet, 2005, 111: 1330―1337
transgenic rice containing a synthetic cry1Ab gene from Bacillus 48 Bates S, Zhao J, Roush R, et al. Insect resistance management in
thuringiensis Berliner to two stem borers. J Econ Entomol, 2001, GM crops: Past, present and future. Nat Biotechnol, 2005, 23: 57―
94: 271―276 62
35 Wu G, Cui H, Ye G, et al. Inheritance and expression of the cry1Ab 49 High S M, Cohen M B, Shu Q Y, et al. Achieving successful
gene in Bt (Bacillus thuringiensis) transgenic rice. Theor Appl deployment of Bt rice. Trends Plant Sci, 2004, 9: 286―292
Genet, 2002, 104:727―734 50 Karim S, Dean D H. Toxicity and receptor binding properties of
36 Nayak P, Basu D, Das S, et al. 1997. Transgenic elite indica rice Bacillus thuringiensis δ-endotoxins to the midgut brush border
plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis membrane vesicles of the rice leaf folders, Cnaphalocrocis medinalis
GENE ENGINEERING
are resistant against yellow stem borer (Scirpophaga incertulas). and Marasmia patnalis. Curr Microbiol, 2000, 41: 276―283
Proc Natl Acad Sci USA, 1997, 94: 2111―2116 51 Alcantara E P, Aguda R M, Curtiss A, et al. Bacillus thuringiensis
37 Khanna H K, Raina S K. Elite Indica transgenic rice plants ex- δ-endotoxin binding to brush border membrane vesicles of rice
Chen H et al. Chinese Science Bulletin | November 2009 | vol. 54 | no. 22 4063
16. stem borers. Arch Insect Biochem Physiol, 2004, 55: 169―177 confers resistance to the rice weevil Sitophilus oryzae. Transgenic
52 Sun X F, Tang K X, Wan B L, et al. Transgenic rice pure lines Res, 2003, 12: 23―31
expressing GNA resistant to brown planthopper. Chinese Sci Bull, 68 Huang J Q, Wei Z M, An H L, et al. Agrobacterium tumefaciens-
2001, 46: 1698―1703 mediated transformation of rice with the spider insecticidal gene
53 Rao K V, Rathore K S, Hodges T K, et al. Expression of snowdrop conferring resistance to leaffolder and striped stem borer. Cell Res,
lectin (GNA) in transgenic rice plants confers resistance to rice 2001, 11: 149―55
brown planthopper. Plant J, 1998, 15: 469―477 69 James A B, Thierry B, William C, et al. Control of coleopteran
54 Tang K, Tinjuangjun P, Xu Y, et al. Particle-bombardment-mediated insect pests through RNA interference. Nat Biotech, 2007, 10:
co-transformation of elite Chinese rice cultivars with genes 1038―1359
conferring resistance to bacterial blight and sap-sucking insect pests. 70 Mao Y B, Cai W J, Wang J W, et al. Silencing a cotton bollworm
Planta, 1999, 208: 552―563 P450 monooxygenase gene by plant-mediated RNAi impairs larval
55 Foissac X, Loc N T, Christou P, et al. Resistance to green tolerance of gossypol. Nat Biotech, 2007, 25: 1307―1313
leafhopper (Nephotettix virescens) and brown planthopper 71 Guo C. Rice Bacterial Blight. Crop Disease and Insect Pest of
(Nilaparvata lugens) in transgenic rice expressing snowdrop lectin China. Beijing: China Agricultural Press, 1995. 14―24
(Galanthus nivalis agglutinin; GNA). J Insect Physiol, 2000, 46: 72 Wu X, Li X, Xu C, et al. Fine genetic mapping of xa24, a recessive
573―583 gene for resistance against Xanthomonas oryzae pv. oryzae in rice.
56 Sun X, Wu A, Tang K. Transgenic rice lines with enhanced resistance Theor Appl Genet, 2008, 118: 185―191
to the small brown plant hopper. Crop Prot, 2002, 21: 511―514 73 Wang C T, Wen G S, Lin X H, et al. Identification and fine mapping
57 Nagadhara D, Ramesh S, Pasalu I C, et al. Transgenic indica rice of the new bacterial blight resistance gene, Xa31(t), in rice. Eur J
plants resistant to sap-sucking insects. Plant Biotechnol J, 2003, 1: Plant Pathol, 2009, 123: 235―240
231―240 74 Zhang S P, Song W Y, Chen L L, et al. Transgenic elite indica rice
58 Nagadhara D, Ramesh S, Pasalu I C, et al. Transgenic rice plants varieties, resistance to Xanthomonas oryzae pv. Oryzae. Mol Breed,
expressing the snowdrop lectin gene (gna) exhibit high-level 1998, 4: 551―558
resistance to the whitebacked planthopper (Sogatella furcifera). 75 Wu J, Yang J, Xu C, et al. Study on resistance gene to bacterial
Theor Appl Genet, 2004, 109: 1399―1405 blight Xa21 transgenic rice and their hybrid combinations. Acta
59 Tinjuangjun P, Loc N T, Gatehouse A M R, et al. Enhanced insect Agron Sin, 2001, 27: 29―34
resistance in Thai rice varieties generated by particle bombardment. 76 E Z, Zhang L, Jiao G, et al. Highlights in identification and
Mol Breed, 2000, 6: 391―399 application of resistance genes to rice blast. Chin J Rice Sci, 2008,
60 Saha P, Majumder P, Dutta I, et al. Transgenic rice expressing 22: 533―540
Allium sativum leaf lectin with enhanced resistance against 77 Lee S K, Song M Y, Seo Y S, et al. Rice Pi5-mediated resistance to
sap-sucking insect pests. Planta, 2006, 223: 1329―1343 Magnaporthe oryzae requires the presence of two
61 Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an coiled-coil-nucleotide-binding-leucine-rich repeat genes. Genetics,
introduced potato proteinase inhibitor Ⅱ gene are insect resistant. 2009, 181: 1627―1638
Bio/Technology, 1996, 14: 494―498 78 Van Loon L C, Van Sterin E A. The families of pathogenesis-related
62 Ding Y, Zeng L, Cheng Z. et al. Studies on transforming high proteins, their activities, and comparative analysis of PR-1 type
efficiency insect-resistant gene PinⅡ into rice. Southwest Chin J proteins. Physiol Mol Plant Pathol, 1999, 55: 85―97
Agric Sci, 2003, 16: 27―32 79 Lin W, Anuratha C S, Datta K, et al. Genetic engineering of rice for
63 Xu D, Xue Q, McElroy D. Constitutive expression of a cowpea resistance to sheath blight. Bio/Technology, 1995, 13: 686―691
trypsin inhibitor gene, CpTi, in transgenic rice plants confers 80 Datta K, Baisakh N, Thet K M, et al. Pyramiding transgenes for
resistance to two major rice insect pests. Mol Breeding, 1996, 2: multiple resistance in rice against bacterial blight, yellow stem
167―173 borer and sheath blight. Theor Appl Genet, 2002, 106: 1―8
64 Lee S I, Lee S H, Koo J C, et al. Soybean Kunitz trypsin inhibitor 81 Datta K, Koukolikova-Nicola Z, Baisakh N, et al.
(SKTI) confers resistance to the brown planthopper (Nilaparvata Agrobacterium-mediated engineering for sheath blight resistance of
lugens Stal) in transgenic rice. Mol Breed, 1999, 5: 1―9 indica rice cultivars from different ecosystems. Theor Appl Genet,
65 Irie K, Hosoyama H, Takeuchi T, et al. Transgenic rice established 2000, 100: 832―839
to express corn cystatin exhibits strong inhibitory activity against 82 Nandakumar R, Babu S, Kalpana K, et al. Agrobacterium-mediated
insect gut proteinases. Plant Mol Biol, 1996, 30: 149―157 transformation of indica rice with chitinase gene for enhanced
66 Vain P, Worland B, Clarke M C, et al. Expression of an engineered sheath blight resistance. Biol Plantarum, 2007, 51: 142―148
cysteine proteinase inhibitor for nematode resistance in transgenic 83 Nishizawa Y, Nishio Z, Nakazono K, et al. Enhanced resistance to
rice plants. Theor Appl Genet, 1998, 96: 266―271 blast (Magnaporthe grisea) in transgenic Japonica rice by
67 Alfonso-Rubi J, Ortego F, Castanera P, et al. Transgenic expression constitutive expression of rice chitinase. Theor Appl Genet, 1999,
of trypsin inhibitor CMe from barley in indica and japonica rice, 99: 383―390
4064 www.scichina.com | csb.scichina.com | www.springer.com/scp | www.springerlink.com