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 1000He, 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 transforma-
tion is the most popular technique as it generates transgenics at relatively high efficiency with trans-
ferred DNA not showing any major rearrange- ments. In addition, frequency of single copy gene
integration is relatively high. This method has
been less successful in monocots, but in the last few years significant progress has been made in
this direction in rice [41 – 45]. Early attempts to regenerate
transgenic calli
from Agrobacterium-mediated transformation were not
successful [46]. Subsequently, regeneration was achieved from Agrobacterium-infected calli of root
explants [47] as well as immature embryos [48]. However, scientists were not convinced about the
effectiveness of Agrobacterium as a vector for rice transformation.
In a significant development, Hiei et al. [42] reported transformation of japonica rice using
Agrobacterium. They constructed some unique vectors called ‘super-binary’ vectors which have
additional 6ir genes in the binary plasmid itself. This modification led to achievement of high
transformation
efficiency in
japonica rice.
Scutella-derived calli and Agrobacterium tum- efaciens LBA4404 pTOK233 were found to be
the most suitable explant and effective strain, respectively. Several necessary requirements for
successful transformation, such as the use of acetosyringone and a temperature of 22 – 28°C
during co-cultivation, were also pointed out. Molecular and genetic analyses of a large number
of transgenic plants up to R2 generation together with sequence analysis of T-DNA junctions in rice
were provided. Subsequently, transformation of japonica rice by Agrobacterium was reported by
other groups [49 – 52]. Aldemita and Hodges [53] obtained transgenic indica as well as japonica rice
using immature embryos. Rashid et al. [54], Mohanty et al. [55], and Khanna and Raina [56]
reported successful transformation of elite indica varieties with Agrobacterium at high efficiency.
Molecular, genetic as well as biochemical analyses of transgenic plants up to R2 progeny was
reported [55]. In addition, transformation of javanica rice has also been reported [57]. Using
isolated shoot apices as explant for co-cultivation, Park et al. [58] reported generation of transgenic
rice plants by Agrobacterium. Besides, Toki [59] has reported a new binary vector pSMABuba for
rice transformation.
In another significant development, Komari et al. [60] designed some unique plasmids that carry
two separate T-DNA segments, one carrying the non-selectable marker gene gus and the other
carrying the selectable gene hph in the same
plasmid. These
vectors were
employed for
generation of marker-free transgenic plants. The frequency of co-transformation with the two
T-DNA was found to be greater than 47 reflecting the effectiveness of the system. The
integration and segregation of T-DNAs were confirmed by molecular analysis. Notwithstanding
the
recent advances
made in
the area
of Agrobacterium-mediated transformation of rice
[61], there are already a few reports available where Agrobacterium has been used to produce
transgenic rice with economically important genes [50,62 – 66].
3. Introduction of agronomically useful genes in rice
