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 O
2
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 from Allium 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 B 10 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 EnSpm-Idspm 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 maize Ac-Ds as 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
hygromycin resistant plants, since Ac element was
originally cloned between the promoter and the hph coding 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 6000 Ac element 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 containing Ds element was further crossed with a transgenic plant carrying Ac trans-
posase gene under the control of
35
S promoter and germinal transposition of Ds 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 Tto
1
and demonstration of its autonomous transposition through reverse tran-
scription [145]. A transposable element, Tag
1
, from Arabidopsis thaliana has also been analyzed
in rice [146]. It was observed that the transcription and excision behaviour of Tag
1
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 or EnI system already demonstrated for their
utility in Arabidopsis [150,151] containing either gus or Gfp as 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
