Review
Rice transformation for crop improvement and functional
genomics
Akhilesh K. Tyagi*, Amitabh Mohanty
Centre for Plant Molecular Biology and Department of Plant Molecular Biology,Uni6ersity of Delhi South Campus,Benito Juarez Road, New Delhi110021,India
Received 20 March 2000; received in revised form 15 June 2000; accepted 16 June 2000
Abstract
Although several japonica and some indica varieties of rice have already been transformed, there is significant scope for improvement in the technology for transformation of economically important indica varieties. Successful transformation of rice employingAgrobacteriumand recent advances in direct gene transfer by biolistics, evidenced by transfer of multiple genes, have removed some of the serious impediments in the area of gene engineering. The transfer of genes for nutritionally important biosynthetic pathway has provided many opportunities for performing metabolic engineering. Other useful genes for resistance against pests, diseases and abiotic stresses have also been transferred to rice. But the limited knowledge about important target genes requires rapid progress in the field of functional genomics. Transgenic rice system can be applied to isolate new genes, promoters, and enhancers and their functions could be unravelled. The combination of novel regulatory systems for targeted expression and useful new genes should pave the way for improvement of rice and other cereals. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Crop improvement; Functional genomics; Genetic transformation;Oryza sati6a
www.elsevier.com/locate/plantsci
1. Introduction
Rice is the staple food for more than one third of world’s population. It is estimated that to feed the growing world population total food production will have to increase by 60% in the next 25 years [1]. Although the world food supply has more than doubled since the onset of the green revolution, the task of providing food for an additional 2.5 billion people would have to be accomplished without a significant increase in the area under cultivation. In fact, the problem is compounded by the loss of land to urban development, degradation of productive land, and the decreasing yield of several conven-tional varieties which contributed immensely
to-wards the green revolution. Besides, every year all over the world, crops worth several million dollars are lost due to damage caused by various biotic and abiotic stresses [2]. Although over the years plant breeders have contributed significantly to increased food production, the time has come to synchronize plant breeding with biotechnology leading to preci-sion breeding for rice improvement [3] by providing breeding lines with desired traits such as resistance against pests and pathogens, tolerance to salinity and drought as well as improved nutritional quality [4].
The rapid strides that rice biotechnology has made [5 – 8] after the recovery of the first transgenic rice plantlets in 1988 [9 – 11] is amazing. In addition, due to its small genome size and the availability of a lot of data in the form of expressed sequence tags (ESTs) and structural genomics, rice provides
* Corresponding author. Tel.:+91-11-467-3216; fax:+ 91-11-688-5270.
E-mail address:[email protected] (A.K. Tyagi).
a valuable crop system for the introduction of useful genes and to accommodate new approaches to address various fundamental problems in plant biology, such as elucidation of various principles of gene regulation and functional genomics in mono-cots [12 – 14]. In this article, work on genetic trans-formation of rice, with special emphasis on transfer of economically important traits, has been reviewed along with the potential of transgenic rice for functional genomics. Previously, related aspects have been discussed by Ayers and Park [5], Christou [6], Goff [15] and Tyagi et al. [8].
2. Development of rice transformation system
In the initial years, because of the lack of a good regeneration system as well as gene delivery meth-ods, protoplast transformation with electroporation or PEG was the method of choice. Toriyama et al. [9] and Zhang and Wu [11] recovered transgenic rice using PEG. In the same year, Zhang et al. [10] reported recovery of transgenic rice using electropo-ration. Shimamoto et al. [16] and Datta et al. [17] were the first to recover fertile transgenic plants using electroporation and PEG in japonica and indica rice, respectively. Subsequently, these meth-ods have been used widely by different groups. However, regeneration of fertile plants from proto-plasts is time consuming, laborious and highly genotype-dependent. Besides, there is the problem of somaclonal variations, multi-copy integration and regeneration of albino plants. Therefore, de-spite their use in engineering of some of the econom-ically important genes [18 – 20], these two methods have fallen out of favour and scientists prefer other methods of gene delivery.
2.1. Particle gun-mediated gene deli6ery
Soon after its availability, particle gun — also known as microprojectile bombardment or biolis-tics — was used successfully for transformation with immature embryos of rice [21]. The method was further improved by Cao et al. [22] and Li et al. [23]. Since then, biolistics has been widely used for transformation of japonica rice. Recently, trans-formation of indica and javanica rice in addition to other japonica rice has also been reported by various laboratories [6,24 – 36]. In a significant de-velopment, Chen et al. [37] reported transformation
of japonica rice with multiple genes using biolistics. They bombarded rice tissue with 14 different pUC-based plasmids and it was observed that 17% of R0 plants had more than nine target genes and 85% of R0 plants contained more than two target genes. The growth behaviour and morphology of these plants were normal: the viable seed setting percent-age reported was 63. Interestingly, integration of multiple transgenes occurred at single or two loci. This has opened up possibilities for engineering of novel biosynthetic pathways in rice. Tang et al. [36] reported transformation of rice with four genes by co-transformation using biolistics. Two out of the four genes used were economically important, viz.,
Xa 21 and GNA, providing resistance against
bac-terial blight and sap-sucking insects, respectively. Molecular analysis revealed that over 70% of the transgenic plants recovered contained all four genes. The majority of the transgenic plants were found to express these genes. The same group [38] demonstrated efficient transformation of rice using a portable and inexpensive particle bombardment device termed a particle inflow gun. They compared transformation efficiencies of three different particle bombardment devices. The transformation
efficien-cies of Dupont PDS 1000/He, electric discharge gun
and modified particle inflow gun were found to be 3 – 6%, 2 – 12% and 2 – 7%, respectively. This devel-opment may pave the way for greater access of this technology for laboratories which may not have the resources to invest in more expensive devices. The biolistics method is claimed to be genotype-indepen-dent with more than 70 rice varieties already trans-formed [6,8,30,35,39] and transformation frequency as high as in dicots has been reported in some cases. It should, however, be noted that different workers have reported variable frequency of transformation. A number of economically important genes have been transferred using this method, some of which are already undergoing field trials [40]. Further details about various explants and genes used may be found in Tyagi et al. [8].
2.2. Agrobacterium-mediated transformation
In dicots, Agrobacterium-mediated
been less successful in monocots, but in the last few years significant progress has been made in this direction in rice [41 – 45]. Early attempts to
regenerate transgenic calli from
Agrobacterium-mediated transformation were not successful [46]. Subsequently, regeneration was
achieved fromAgrobacterium-infected calli of root
explants [47] as well as immature embryos [48]. However, scientists were not convinced about the
effectiveness of Agrobacterium as a vector for rice
transformation.
In a significant development, Hiei et al. [42] reported transformation of japonica rice using
Agrobacterium. They constructed some unique vectors called ‘super-binary’ vectors which have
additional 6ir genes in the binary plasmid itself.
This modification led to achievement of high
transformation efficiency in japonica rice.
Scutella-derived calli and Agrobacterium tum
-efaciens LBA4404 (pTOK233) were found to be the most suitable explant and effective strain, respectively. Several necessary requirements for successful transformation, such as the use of acetosyringone and a temperature of 22 – 28°C during co-cultivation, were also pointed out. Molecular and genetic analyses of a large number of transgenic plants up to R2 generation together with sequence analysis of T-DNA junctions in rice were provided. Subsequently, transformation of
japonica rice by Agrobacterium was reported by
other groups [49 – 52]. Aldemita and Hodges [53] obtained transgenic indica as well as japonica rice using immature embryos. Rashid et al. [54], Mohanty et al. [55], and Khanna and Raina [56] reported successful transformation of elite indica
varieties with Agrobacterium at high efficiency.
Molecular, genetic as well as biochemical analyses of transgenic plants up to R2 progeny was reported [55]. In addition, transformation of javanica rice has also been reported [57]. Using isolated shoot apices as explant for co-cultivation, Park et al. [58] reported generation of transgenic
rice plants by Agrobacterium. Besides, Toki [59]
has reported a new binary vector (pSMABuba) for rice transformation.
In another significant development, Komari et al. [60] designed some unique plasmids that carry two separate T-DNA segments, one carrying the
non-selectable marker gene (gus) and the other
carrying the selectable gene (hph) in the same
plasmid. These vectors were employed for
generation of marker-free transgenic plants. The frequency of co-transformation with the two T-DNA was found to be greater than 47% reflecting the effectiveness of the system. The integration and segregation of T-DNAs were confirmed by molecular analysis. Notwithstanding
the recent advances made in the area of
Agrobacterium-mediated transformation of rice [61], there are already a few reports available
where Agrobacterium has been used to produce
transgenic rice with economically important genes [50,62 – 66].
3. Introduction of agronomically useful genes in rice
Considerable success has been achieved by plant breeders in developing improved rice varieties and it is imperative to argue that the success achieved with transformation techniques will supplement plant breeding programmes [3]. This becomes im-portant in view of the need to raise rice production from 560 to 850 million tons by 2025 to support additional rice consumers [1] and to manage the loss due to various biotic and abiotic stresses, such as insect pests, salinity, low temperature, water logging and drought [67].
Remarkable progress has been made since the recovery of the first transgenic rice plants just a decade ago. A number of economically important genes have been transferred to japonica as well as indica rice and scientists are now looking forward towards pyramiding of genes in rice.
3.1. Insect resistance using Bt genes
Fujimoto et al. [70] were the first to engineer
japonica rice through electroporation with
modified d-endotoxin gene (cry) from Bacillus
thuringiensis. It was found that the R2 generation of transgenic rice was more resistant to insects than wild type plants. Later, Wu¨nn et al. [71] obtained transgenic indica rice cultivar IR58
ex-pressing a synthetic cryIA(b) gene driven by 35S
promoter through particle bombardment. Insect bioassays revealed effective control of two of the most destructive pests of rice in Asia, the yellow stem borer (YSB) and the striped stem borer (SSB). Interestingly, feeding inhibition of the two
leaf folder species Cnaphalocrous medianalis and
Marasima patanis was also observed. In order to achieve high expression, Nayak et al. [72]
recon-structedcryIA(c) gene and transformed indica rice
cultivar IR64 with the synthetic gene. Insect bioassays revealed the resistant nature of trans-genic plants to the damage caused by YSB, al-though the level of expression was low. In the following years, various synthetic and modified
cryIA(b) driven by constitutive (35S, Ubi1, Act1)
as well as tissue-specific (Pepc, maize pith-specific)
promoters have been used to achieve desirable level of resistance [20,73 – 75] against SSB and YSB. Datta et al. [20] reported that a large num-ber of transgenic plants caused 100% mortality of YSB larvae. The authors suggested the use of tissue-specific promoters for minimizing the ex-pression of Bt protein in edible parts. In a signifi-cant development, Cheng et al. [62] obtained a large number of transgenic rice plants of different
varieties engineered with cryIA(b) and cryIA(c)
genes, which have been codon optimized, by
Agrobacterium-mediated transformation. South-ern, Northern and Western analyses up to R1 generation were performed to show the integra-tion, inheritance and expression of the transgenes.
Use of the Ubiquitin promoter led to high level
expression (up to 3% of total soluble protein) of transgene product. The toxic nature of the trans-genic plants to SSB and YSB was revealed by insect bioassays.
Recently, the need to revise the management
strategy forcry-dependent resistance has arisen as
reports regarding development of insect resistance
against a single cry gene were written.
Develop-ment of insect resistance for cry1A(a), cry1A(b),
cryIA(c) and cry1F has been reported. By per-forming an elaborate series of genetic crosses,
Tabashnik et al. [76] proved that just one autoso-mal gene in diamond back moth confers
cross-re-sistance against the four cry genes mentioned
above. Keeping this in view, Maqbool et al. [77]
transformed rice with a novelcry2Agene by
parti-cle bombardment. Molecular and biochemical analyses confirmed transmission of the gene to R2 progeny. In one plant line, the protein was ex-pressed to the level of 5% of total leaf protein. In other plants, the expression level was between 0.01 and 1%. Insect bioassays revealed the effectiveness of this gene in providing resistance against YSB and rice leaf folder, two of the major pests of rice. In another strategy for improvement of rice, Alam et al. [78] transformed an IRRI maintainer line IR68899B, which is used for the production of
hybrid rice with cryIA(b) gene. Southern and
Western blot analyses confirmed the integration, expression and transmission of the transgene to R2 progeny. Insect bioassays revealed enhanced resistance against YSB in the transgenic plants. The effectiveness of gene pyramiding to obtain durable resistance against insect pests has also been reported recently by Christou’s group [79,80]. They obtained transgenic indica rice plants with
insecticidal genes (cryIAc, cry2A) and the
snow-drop lectin (Gna) gene. These triple transgenic rice
plants (R0 and R1) showed significantly higher resistance to insect pests. Keeping in view that at present $8 billion is annually spent on insecticides worldwide out of which nearly $2.7 can be
re-placed by Bt technology applications, Bt research
in rice assumes great importance.
3.2. Insect resistance using proteinase inhibitors and lectins
For insect resistance, genes encoding plant proteinase inhibitors are of particular interest as they are part of the plant’s natural defence system against insect predation [81]. Hosoyama et al. [82] obtained transgenic rice through electroporation
harbouring a chimeric oryzacystatin (Oc) gene.
Later, the corncystatin (Cc) gene was introduced
into rice by Irie et al. [19]. The transgenic rice
plants were resistant to the insect pest Sitophilus
zeamais. Xu et al. [83] engineered rice with a
cowpea trypsin inhibitor (Cpti ) gene driven by the
borer and pink stem borer. Duan et al. [81]
intro-duced potato proteinase inhibitor II (PinII) gene
driven by PinII promoter for wound-inducible
ex-pression of transgene in rice. The stability of this gene (up to four generations) was confirmed by molecular analyses. The transgenic plants were found to have increased resistance to pink stem borer. These results suggest that proteinase in-hibitor genes could be used as a general strategy for the control of insect pests. Zhen et al. [84] reported that a modified cowpea trypsin inhibitor gene with ER targeting signal (KDEL) resulted in two to four times higher average proteinase in-hibitor activity than that of plants transformed
with CpTi gene. Bioassays proved the efficacy of
this modified gene as revealed by 60 – 72%
mortal-ity rate of Chilo suppressalis larvae. Another class
of proteinase inhibitors, the Kunitz family repre-sented by soybean Kunitz trypsin inhibitor, has also been engineered in rice [85]. A vector was
constructed by fusing full-length cDNA with 35S
promoter. Protoplasts isolated from japonica vari-ety ‘Nagdongbyeo’ were transformed using
PEG-mediated gene delivery. The integration,
expression and inheritance of the transgene were demonstrated up to R2. The transgene product accumulated between 0.05 and 2.5% of total solu-ble leaf protein. Insect bioassays with the progeny of the above plant lines revealed that transgenic
plants are more resistant toNilapar6ata lugensSta¨l
than control rice plants. However, one third of the plants obtained in this study were sterile.
The snowdrop (Galanthus ni6alis) lectin (GNA)
gene has also been used for obtaining resistance against insect pests in rice. Transformation of rice with the gene driven by the phloem-specific su-crose synthase promoter of rice (RSSGNA) through electroporation as well as with maize
ubiquitin promoter drivingGNA gene (UBIGNA)
by biolistics has been achieved [86,87]. Western blot analysis revealed the presence of 12-kDa band in both types of transgenic rice plants correspond-ing to the standard GNA polypeptide. Semi-quan-titative estimates of expression levels of GNA from Western blots revealed 0.01 – 0.25% of total soluble protein for RSSGNA plants and up to 2% of total soluble protein for UBIGNA plants. In-sect bioassay showed that by expressing lectin GNA, transgenic rice plants can be partially pro-tected against brown planthopper. Immunolocal-ization studies revealed the phloem tissue-specific
expression of rice sucrose synthase 1 promoter driven GNA [88].
3.3. Resistance against 6iruses
Hayakawa et al. [89] engineered the coat protein
(Cp) gene of rice stripe virus into two japonica rice
varieties by electroporation of protoplasts result-ing in significant levels of resistance against the virus in the transgenic plants. Huntley and Hall [90] obtained transgenic rice with four different constructs containing regions of RNA-2, RNA-3 and capsid protein genes derived from the brome mosaic virus (BMV). When challenged with virion RNA, protoplasts obtained from transgenic plants or cell lines showed up to 95% reduction in the accumulation of viral RNA. To achieve resistance against rice dwarf virus, Zheng et al. [91]
intro-duced the outer coat protein gene (S8) of this
virus into rice.
Rice tungro disease may cause an estimated US$ 343 million annual loss in crop in South East Asia alone. Two viruses, rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (RTBV), are known to be causative agents. RTBV is a double stranded virus and RTSV is a positive-sense, single stranded RNA virus. Of these, RTBV causes severe disease symptoms. These viruses are
transmitted by green leafhopper (GLH) Nepho
-tettix 6irescens. To obtain resistance against
RTBV, it is necessary to understand the molecular biology of the viral cycle. Therefore, the RTBV genome has been characterized and mutant viral proteins have been created which can act as com-petitive inhibitors of viral functions [92]. Further, transgenic indica rice plants have been produced expressing RTBV proteins which serve as precur-sors to viral coat protein and reverse transcriptase [92]. Element which provides specificity to the RTBV promoter has also been analyzed [93,94]. Sivamani et al. [95] reported detailed analysis of transgenic plants with three different coat protein
genes, Cp1,Cp2, andCp3 of RTSV. Northern blot
analysis revealed the presence of detectable levels of mRNA in all except three lines. However, no signal was obtained in Western blot analysis prob-ably because of the low amount of transgene
product. Inheritance of the Cp gene was analyzed
infection. One noteworthy feature of the study was that no cumulative effect on the resistance was observed when these genes were expressed to-gether. This is contrary to the popular belief that pyramiding of these genes would help.
Pinto et al. [96] constructed transformation vec-tors by fusing either full length rice yellow mottle virus (RYMV) cDNA encoding RNA-dependent RNA polymerase or C-terminal deletions of this
to the 35S promoter. Transgenic plants were
re-generated following gene delivery by biolistics. Some of the transgenic plants obtained were
highly resistant against different isolates of
RYMV up to R3 progeny. Northern analysis of these resistant plants revealed the presence of very low levels of RYMV transcript. In comparison, the plants having partial resistance had high level of RYMV transcript. A post-transcriptional gene silencing of the RYMV transgene in the resistant lines has been implicated as the basis of RYMV resistance mechanism. This is the first report of homology-dependent resistance in rice.
Mun˜oz et al. [97] reported transformation of Costa Rican indica rice with coat protein gene of rice hoja blanca virus (RHBV) under the control
of the rice Actin promoter together with MAR
sequences. Thebargene was used for the selection
of transformed tissues. Molecular analysis showed the presence of RHBV coat protein gene in low copy number.
3.4. Herbicide resistance
Genes for herbicide resistance have also been
introduced in rice. The bar gene is advantageous
as it serves the dual purpose of selectable marker gene as well as conferring resistance to the herbi-cide, phosphinothricin (PPT). Christou et al. [21] and Datta et al. [98] were able to engineer several
rice cultivars to express the bar gene. The
trans-genics were resistant to high doses of the commer-cial formulations of PPT. A field study, conducted
for 3 years, with 1.12 or 2.24 kg/ha glufosinate
showed significant improvement in the perfor-mance of transgenics [40].
3.5. Resistance against fungal pathogens
Transgenic rice plants, expressingbargene, were
not infected byRhizoctonia solani[99] and showed
decreased symptoms of rice blast disease,
follow-ing bialaphos treatment [100], probably because of the sensitivity of pathogens to the herbicide. The
rice chitinase gene Chi11 under the control of the
constitutive 35S promoter was used to transform
rice protoplasts to obtain rice plants resistant to
the sheath blight pathogen R. solani [18].
Simi-larly, a basic chitinase gene from rice could be employed to engineer resistance to pathogens [101]. Nishizawa et al. [65] introduced two rice
chitinase genes Cht-2 and Cht-3 into japonica rice
under the control of an enhanced 35S promoter.
In transgenic plants, the Cht-2 product was
targeted extracellularly, whereas theCht-3 product
accumulated intracellularly. Transgenic plants ex-pressing both the genes constitutively were found to show enhanced resistance to two races (007.0
and 333) of the rice blast pathogen, Magnaporthe
grisea. With a similar objective, Stark-Lorenzen et al. [102] employed a stilbene synthase gene from grapevine, under the control of its own promoter, to raise transgenic rice plants. The accumulation of stilbene synthase mRNA in response to
inocula-tion with Pyricularia oryzae as well as wounding,
elicitor treatment and UV irradiation was ob-served in R2 plants. Plants expressing this gene
showed resistance against P. oryzae. Kim et al.
[103] reported transgenic rice plants expressing the
maize ribosome inactivating protein gene, Rip b
-32. Southern hybridization revealed that 30% of the regenerated plants had a single transgene
in-sert. Transmission of the Rip b-32 as well as
selectable marker (bar) gene was observed up to
R2 progeny. Gene silencing was observed in some of the transgenic plants having multiple copies of transgene. Expression levels of the protein were 0.5 – 1% of total soluble leaf protein. However,
when R2 transgenic plants were challenged withR.
solani and M. grisea, there was no significant reduction in disease severity as compared to con-trol suggesting that normal processing of b-32 protein may be required for in planta antifungal activity. To achieve resistance against sheath blight disease, transformation with thaumatin-like
protein (PR-5) gene (Tlp) under the control of35S
promoter has also been attempted in Chinsurah BoroII, IR72 and IR51500 [39]. Transformation was achieved by PEG-mediated transformation of protoplasts as well as gene delivery to immature embryos by biolistics. Southern hybridization data revealed transmission of the transgenes up to R2
showed the presence of 23-kDa TLP-D34 protein. Insect bioassay revealed that several transgenic plant lines were having limited infection compared to the control.
3.6. Resistance against bacterial diseases
The rice Xa21 gene which confers resistance to
blight pathogen, Xanthomonas oryzae was cloned
by Song et al. [104]. Transgenic rice plants har-bouring the cloned gene displayed high levels of resistance. The gene has been found to be effective against several isolates [105]. An elite indica rice cultivar IR72 has also been transformed with the
Xa21 gene [32] and transgenic plants from R1
generation were found to be resistant to bacterial blight. However, in some of the lines gene silenc-ing was observed. Zhang et al. [106] reported production of transgenic elite indica rice IR64, IR72 and Minghui 63, a restorer line for Chinese
indica hybrid rice with Xa21 gene. Bioassays
re-vealed that a number of lines up to R2 generation were resistant against the bacteria. R3 generation of one line of IR72 was also found to be resistant. However, since the plants analyzed include ho-mozygous as well as heterozygous plants, efficacy of these transgenic plants would be clear only after the field trials, which are being performed.
3.7. Engineering of rice for abiotic stress tolerance
To engineer rice for tolerance to abiotic stresses, Xu et al. [107] introduced a late embryogenesis
abundant (LEA) protein gene H6a1 from barley
using biolistics method. Use of rice Actin1
pro-moter led to high level constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic plants and the second generation showed increased tolerance to water deficit and
salinity. Another gene, Cor47, has also been
shown to confer resistance against several abiotic
stresses [108,109]. Yokoi et al. [50] employedAra
-bidopsiscDNA for glycerol-3-phosphate
acyltrans-ferase (Gpat) under the control of maizeUbi1 gene
promoter to obtain transgenic plants with high levels of unsaturated fatty acids and chilling toler-ance of photosynthesis.
The codA gene for the enzyme choline oxidase
from Arthrobacter globiformis, which can catalyze conversion of choline to glycine betaine in a single
step, fused to a rice RbcS transit peptide
(chl-COD) for targeting codA gene product to the
chloroplast was employed for transformation of japonica rice [63]. In the other construct (cyt-COD), no targeting signal was used and it was expected to enhance glycine betaine in the cytosol. Additionally, for better processing of RNA, rice
Sod gene intron was incorporated in both the
constructs. It was reported that cyt-COD and chl-COD plants accumulated glycine betaine at a
level of 1 and 5 mmol per gram fresh weight,
respectively. Both types of transgenic plants were tolerant to low temperature as well as salinity stress as revealed by PSII activity. Since the glycine betaine concentration achieved was not significant for osmotic adjustment, the authors suggest that a possible mechanism for glycine be-taine action could be the stabilization of the struc-ture of large protein complexes. This gene has also been transferred to an elite indica rice variety, ‘Pusa Basmati 1’, and a preliminary study with R0 plants showed effectiveness of this gene for en-hancing salinity tolerance (Mohanty and co-work-ers, unpublished).
The effectiveness of superoxide dismutase (Sod)
gene in providing salt tolerance has also been
examined [110]. A yeast mitochondrial Mn-Sod
gene, activity of whose product is not inhibited by
H2O2, fused to the chloroplast targeting signal of
glutamine synthase gene and 35S promoter was
used for transformation by electroporation into protoplasts of the japonica variety ‘Sasanishiki’. Studies with R2 homozygous lines revealed in-creased total SOD activity and enhanced tolerance to salt stress in these transgenic plants. It is obvi-ous that much more work is needed to engineer such complex traits as abiotic stress tolerance [111 – 114].
3.8. Engineering of rice for nutritional quality
Improvement of the nutritional quality of rice is a very important consideration keeping in view its importance as food for a large number of people worldwide. Shimada et al. [115] produced trans-genic rice plants with antisense construct of rice
Waxy gene coding for granule-bound starch
syn-thase under the control of35Spromoter. A
b-phaseolin of the common bean (Phaseolus 6ul -garis L.). With the ultimate aim of increasing the lysine content of rice grain, the gene was driven by 5.1- or 1.8-kb promoter fragment of rice seed
storage protein glutelin (Gt1) gene. This resulted
in the expression of three glycoforms of 51, 48 and 45, in the endosperm. The antisense strategy was also applied to suppress the expression of the maturing rice seed 14 – 16-kDa allergen gene [117]. The seeds of transgenic plants showed much lower amounts of this protein and its transcripts, as compared to the parental wild type rice, for at least three generations.
Most interestingly, to confer the capability of
producing precursor (b-carotene) of vitamin A in
rice endosperm, Burkhardt et al. [29] engineered rice with the cDNA coding for phytoene synthase from daffodil, the first of the four specific enzymes
involved inb-carotene (provitamin A) biosynthesis
in plants. In the endosperm of these transgenic plants, phytoene synthase accumulation was ob-served indicating that engineering of provitamin A biosynthesis pathway is possible in non-photosyn-thetic, carotenoid lacking tissue. Recently, the
same group reported Agrobacterium-mediated
transformation of rice with all the genes necessary for the accumulation of provitamin A in trans-genic rice seeds [66]. The genes transferred were
daffodil phytoene synthase, Erwinia phytoene
de-saturase and Narcissus lycopene b-cyclase.
Glutelin promoter was used for endosperm-spe-cific expression and transit peptide sequences were used for targeting the products in endosperm plas-tids. The seeds of R0 transgenic rice showed
re-markable accumulation of b-carotene providing
yellow colour to seeds. It is expected that the homozygous derivative would be capable of
providing enough b-carotene to fulfil the
nutri-tional requirements.
In yet another significant development, Goto et
al. [64] reportedAgrobacterium-mediated
transfor-mation of the japonica variety ‘Kitaake’ with
soy-bean ferritin gene. They used rice Glu-B1
promoter for endosperm-specific expression of the transgene. Southern hybridization analysis re-vealed the presence of independent transformation events in different lines. Transgene expression was confirmed by RT-PCR. A polyclonal antibody raised against this protein detected a band of 28-kDa size by Western analysis. This protein
accumulated up to 0.01 – 0.3% of the level of
total soluble protein in seeds and the iron content of transgenic rice seeds increased up to threefold over control. The group led by Potrykus also reported their efforts to counter iron deficiency, which is the most prevalent micro-nutrient defi-ciency, by a three pronged approach [118,119].
They engineered rice with a ferritin gene from P.
6ulgaris to increase iron content. As the phytate
present in the gut inhibits absorption of iron, they engineered rice with a heat stable phytase gene from Aspergillus fumigatus. The sulphur-rich metallothionin-like protein exhibits resorption en-hancing effects, hence they also engineered rice with one such gene from rice. Preliminary studies revealed that transgenic plants have twofold higher iron content. Different transgenic plants harbouring these genes are being crossed to com-bine these useful traits [119]. These work may pave the way for reducing iron deficiency, which affects
30% of the total world population.
3.9. Other engineered genes
Several other examples of the introduction of genes into rice with variable objectives are avail-able. Interestingly, engineering of a proteinase
in-hibitor gene for oryzacystatin (Oc), IDD86, in rice
also resulted in resistance against nematodes [34]. Although the level of expression was low (up to 0.2% of total soluble protein), it resulted in 55% reduction in egg production by the nematode
Meloidogyne incognita. Capell et al. [120] reported production of transgenic rice with oat arginine
decarboxylase (Adc) cDNA keeping in view the
role played by polyamines in several plant pro-cesses including stress. This led to morphological abnormalities in tissues and plants producing high
level Adc transcripts accompanied with high level
of putrescine. Tissues expressing very high level of
Adc mRNA failed to differentiate. The effect was
more prominent when tissues were exposed to light. Kisaka et al. [121] reported transformation of japonica rice by electroporation of protoplast
withRgp1 gene, a small GTP binding protein from
‘Nip-ponbare’ with an intact maize gene encoding
C4-specific phosphoenolpyruvate carboxylase (Pepc).
Of the regenerated plants 10% showed
abnor-mal morphology such as growth retardation,
al-binism, narrow leaves, infertility, etc. The
transgene was inherited up to R3 progeny in a Mendelian fashion and active enzyme was present in these generations. Western blot analysis re-vealed the presence of protein of expected size (106 kDa). The level of expression was very high as the transgene product accumulated up to 12% of total soluble protein. This is in fact the highest level of transgene expression reported in monocots to date. The probable reasons for such high expres-sion could be the use of an intact monocot gene together with its introns in another monocot driven by its own promoter as well as the strength
of Pepc promoter. They also concluded that high
level of expression is positively correlated with transgene copy number, which is contrary to con-temporary data that high copy number results in gene silencing. These transgenic plants resulted in
reduced sensitivity of photosynthesis to O2
inhibi-tion, which could be positively correlated with PEPC activity in these plants. Sung-In et al. [123] reported transformation of rice with an
antimicro-bial protein (Asa-AMP) gene fromAllium sati6um.
They used MAR sequences from chicken lysozyme for position-independent expression of the transge-nes. Molecular analysis revealed the positive effect of MAR in obtaining low copy number transgen-ics and reduced variability of transgene expression.
4. Rice and functional genomics
Rice with its small size (450 Mb), relatively
well developed genetics, rapidly progressing EST/
cDNA database, and progress in structural ge-nomics along with transgenics, provides an ideal system for functional genomics of a crop plant [15,124]. Given the synteny in grasses, especially among rice and wheat [125,126], long range co-lin-earity of markers could pave the way for isolation of important genes in rice which, in turn, could be employed for cereal genomics. Although much progress has been made in the area of model dicot
plants such as Arabidopsis with sequencing of its
genome nearing completion [127], recent studies have revealed that findings obtained from such model plants cannot be simply applied to highly
diverse species, such as rice [128,129]. The Rice Genome Program (RGP) in Japan has up to the
present catalogued 15 000 non-redundant ESTs
in rice. They have also constructed a genetic map with 2275 DNA markers [130]. In the second phase, the program has adopted the objective of sequencing the complete genome with global par-ticipation. It may, however, be mentioned that
only 25% of ESTs show significant homology to
known genes and the function of most of the genes remains to be determined.
Functional genomics allows analysis of the func-tion of genes at genomic scale and incorporates high throughput technology such as micro-arrays for mRNA expression and gene tagging by inser-tional mutagenesis, entrapment or activation of genes [131]. Tagging of genes requires insertion of gene tags on genome on a wide scale and transgen-ics play a crucial role for such a purpose. Inser-tional mutagenesis by T-DNA has already been used to produce more than 30 000 independent
inserts in Arabidopsis and several tagged genes
have been isolated [132]. Although such an effort in rice is not in practice, its feasibility cannot be ruled out with improvement in transformation
fre-quency. It has been estimated that B10% of the
inserts in Arabidopsis genome are likely to
gener-ate a visible phenotype change and there is a need for a more powerful or complementing technology to assess the function of remaining genes. One such technology is entrapment tagging which in-volves activation of transgenes by regulatory se-quences associated with native genes and includes enhancer, promoter and gene trapping [133]. Transgenes for entrapment can be delivered di-rectly as part of T-DNA insertions at multiple sites. Alternatively, entrapment vectors can be constructed by making use of transposons like
Ac-Ds or En/Spm-I/dspm from maize which can
be activated in heterologous transgenic plants after genetic transformation [134 – 136]. It has been found that activation of transgene in promoter trap vector could be as high as 30%. Still another approach makes use of over-expression of native genes by transgene (enhancer) and is known as activation tagging [133].
With a view to develop maizeAc-Dsas a system
for gene tagging in rice, Izawa et al. [137] and
Murai et al. [138] introduced Ac element into rice
and demonstrated its transposition by recovering
originally cloned between the promoter and the
hphcoding region, as well as molecular analysis of
transposed elements and empty donor sites. En-hanced variability of phenotypes with progeny plants has also been reported [139,140]. Analysis of the behaviour of 559 plants of four transgenic rice families for three successive generations (R5 – R7) revealed that 18.9% plants contain newly
trans-posed Ac insertions [140]. It seems transposons
show a preference for protein coding genes. A
combination of PCR and 6000Acelement
contain-ing rice plants was used to evaluate the utility of the system for functional analysis. Transposition of
Ds element from plasmid or viral vector
trans-fected into rice protoplasts, in the presence of Ac
transposase, was also demonstrated [141,142]. A
transgenic plant containingDselement was further
crossed with a transgenic plant carryingAc
trans-posase gene under the control of35Spromoter and
germinal transposition ofDs was observed at high
frequency in R2 progeny [143]. But, frequency of
Ds transposition declined in subsequent
genera-tions even in the presence of transposase which could be overcome in certain lines by employing protoplast regeneration. The utility of such a sys-tem for gene tagging has also been demonstrated [144]. Another effort for gene tagging by transpo-sons in rice involved the introduction of tobacco
retrotransposon Tto1 and demonstration of its
autonomous transposition through reverse
tran-scription [145]. A transposable element, Tag1,
from Arabidopsis thaliana has also been analyzed in rice [146]. It was observed that the transcription
and excision behaviour ofTag1 was similar in rice
and Arabidopsis. However, unlike Arabidopsis, the excision was tissue-specific in rice. Interestingly, however, rice also has its own retrotransposons, some of which can be specifically activated, during tissue culture and regeneration, to carry out inser-tional mutagenesis in rice [147] and gene tagging [148].
Recently, Chin et al. [149] developed an Ac-Ds
based gene trapping system for rice by employing
Agrobacterium-mediated gene delivery. As much as
80% of Ds elements were excised from original
T-DNA sites in the presence of Ac transposase
activity, provided in trans, and 8% of transposed
Ds elements expressed GUS activity in various
tissues of the inflorescence, which is claimed to be
as good as in Arabidopsis. Other groups have also
developed promoter trap, enhancer trap or gene
trap vectors based on two component Ac-Ds
sys-tem orEn/Isystem (already demonstrated for their
utility in Arabidopsis [150,151]) containing either
gusor Gfpas reporter gene [152 – 154] and
demon-strated their utility for rice. The next step involves generation of large number of rice plants contain-ing tagged genes to help find out pattern of expres-sion or function of tagged genes. To perform ‘gain of function’ mutagenesis of rice, transgenics with gene activators are also being produced [154].
The potential of gene tag systems seems to be tremendous as substantiated from results in rice and Arabidopsis [131] and most such systems would require efficient genetic transformation of rice varieties having superior traits. Transgenic systems will pave the way not only for producing tagged lines but also for proving the function of isolated genes and promoters. The activity of sev-eral regulated promoters has already been evalu-ated in rice (Table 1). These efforts along with the International Rice Genome Sequencing Project (IRGSP) involving researchers from ten countries [173] and the enormous amount of data being generated [174] are leading to accurate information on rice genome, its colinearity and divergence with related crops, and rapidly evolving gene families and their functions [175].
5. Conclusions and prospects
Significant progress has been made in developing appropriate techniques for rice biotechnology. This has allowed a better understanding of the biology of rice at molecular level along with its interactions with insects, pathogens and various environmental stresses [8,176]. A stage has been reached where field trials of a number of genetically modified traits in rice are being performed. It is becoming obvious that a combination of transgenics and genomics holds great promise for future develop-ments. We are also entering the era of molecular farming where speciality chemicals are being pro-duced in transgenic plants [177,178]. Engineering the whole biosynthetic pathway is also not too distant. Rice with obvious advantages can play an important role in the coming years. Use of single gene encoding a transcription factor, either CBF1
or DREB1A of Arabidopsis, has been shown to
Table 1
Expression patterns of regulated promoters in transgenic ricea
References Promoter Patterns of expression
rolC Vascular tissue-specific [155]
In root caps, scutellum, endosperm, anthers, filaments, pollen and
Maize AdhI [156,157]
anaerobically induced in roots
RiceCab Light-inducible in leaves. stem and floral organs, absent in root [158] [159]
RTBV Phloem-specific
Cells of vascular bundle of leaves and root, leaf sheath parenchyma, [94] epidermis, root cap, endosperm and embryo of seeds, low activity in anthers and pollen
[160] WheatHistone Cell division-dependent activity in meristematic cells of shoots, roots and
H3 young leaves. Cell division-independent expression in anther cell wall, pistil, coleoptile and mature embryos
Inducible by wounding, methyljasmonate and abscisic acid. Specific
PotatoPin II [161]
expression in the vascular tissues of leaves and roots
Expression in mesophyll cells of leaf blade and leaf sheath, light-inducible
RiceRbcs and [162,163]
maize Pepc
BarleyLtp2 Aleurone layer-specific [164]
[165] Light- independent but cell type-specific expression
PineCab
In endosperm and vascular tissues of stem, young lemma and palea, low [166] RiceWaxy
(Wx) expression in anthers
In scutellar epithelium and aleurone layer. Aleurone-specific expression is GA
Ricea-AmylA [167]
inducible
[168]
CVMV Vascular tissue-specific
[49] Tapetum-specific
RiceOsg6b
In the presence of elicitor (Nod factors) expression observed in cortical
Alfalfa [169]
parenchyma, endodermis and pericycle of roots
ENOD12
[170] Endosperm-specific expression due to GCN4 motif
RiceGlu-B1
Seed-specific expression
Wheat [171]
Tapuro-b
RiceRa8 Tapetum, endothecium and connective tissue of anther [52]
SpinachPetH Low expression in leaf and green anthers, very low expression in roots [172], Mohanty and Tyagi (unpublished)
aa-Amy, a-amylase gene IA; AdhI, alcohol dehydrogenase 1 gene;CVMV, cassava vein mosaic virus; ENOD12, nodulation
factor 12 gene; Glu-B1, glutelin gene B1; Ltp2, lipid transfer protein gene 2; Osg6h, a tapetum specific gene; PetH, a photosynthesis-related gene;PinII, proteinase inhibitor II gene;rolC, a gene fromAgrobacterium;Tapuro, puroindoline b gene.
the same time the information available from se-quencing efforts [173] as well as genome-wide ex-pression [181] should be put to use employing functional genomics. If successful, these concerted efforts of genome sequencing, functional genomics and bioinformatics [182] put together can benefit the farmer by increasing productivity or reducing crop loss [183]. It is hoped that in the coming years, rice biotechnology will lead the way for achievement of a sustainable agriculture and food security.
Acknowledgements
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