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Rice is likely the most important food crop in the world. Almost half of the
world?s population depends on rice as their staple food. Therefore, to meet
the needs of the growing world population, conventional breeding methods
need to be combined with recent achievements in rice biotechnology. Rice
genetic transformation has taken rapid strides since the first transgenic
rice plant was produced 15 years ago. During the last 10 years, tremendous
progress has been made to develop a high frequency, routine, and
reproducible genetic transformation protocol for rice, either through direct
DNA transfer or Agrobacterium-mediated transformation technology. Using
these technologies, a number of agronomically important traits and increased
rice nutritional value have been achieved1,2,3. In the last five years, the
focus has shifted to use rice as a model monocotyledon system, similar to
the use of Arabidopsis as a model for dicotyledons. Additionally, rice was
the first crop plant that had its genome sequenced4. Besides effecting its
own improvement, sequencing the rice genome opens up the possibility of
improving other cereals such as maize and wheat, as there is significant
conservation of genes among cereals, July 2005 by Shavindra Bajaj and
Amitabh Mohanty.

This article summarizes highlights of the progress in transgenic rice
technology over the last five years, with particular emphasis on agronomic
and nutritional traits1. Due to space constraints, we have cited recent
reviews covering the advancements in rice biotechnology1,2,3. The readers
are referred to the references in these reviews for further reading.
Finally, because transgenic rice field trials are being initiated, we have
also discussed related biosafety concerns.

Rice transformation

Rice transformation is efficiently mediated either through Agrobacterium or
particle bombardment transformation. Although both systems have their own
merits and limitations, Agrobacterium--mediated transformation has an
advantage over particle bombardment because it is a relatively simple and
inexpensive method of transformation. Further, transformation achieved
through Agrobacterium produces fewer transgene integration copies. Recently,
efforts have been made to produce transgenic rice devoid of elements that
are otherwise important for transformation, such as selectable markers or
vector backbones, but do not impart any value to the trait1. Several new
reporter genes and technologies for producing selectable marker-free plants
have been applied in rice. Interestingly, both negative and positive
selection technologies have been studied using transgenic rice. These
selection systems and recent advances in selectable marker-free technologies
not only provide an alternative to antibiotic- or herbicide-based selection
systems but also open up opportunities for multigene engineering1. New
methods of increasing gene expression and reducing gene silencing have also
been tested in transgenic rice. Of them, the use of Matrix Attachment
Regions (MARs) sequences is very significant. Use of these sequences
flanking the transgene cassette results in higher gene expression and
increasing stability of integrated genes in transgenic plants1.

Introduction of agronomically important genes in rice

As discussed above, rice production needs to be enhanced to meet the demands
of an increasing population. One way to increase production is to reduce the
yield loss caused by various abiotic and biotic factors. Transgenic rice has
been produced that is tolerant to various diseases and tolerant to extreme
environmental conditions.

Insect resistant rice has been produced expressing various Bt genes, as well
as non-Bt genes such as the mannose-specific lectin gene, gna. Improvements
in existing systems, such as the use of tissue-specific or wound-inducible
promoters to drive expression of insect resistance genes, which reduce
biomass loss and delay resistance among the target insect populations, would
help in developing a second generation product for prolonged insect
tolerance. Several field trials have been initiated for transgenic rice
containing genes of agronomic interest, such as insect resistance, involving
not only varieties of breeding interest but also agronomically important
hybrid rice1. Furthermore, stacking of genes in transgenic rice has produced
plants that are tolerant to multiple stresses. It is expected that China
will be the first country to commercially release insect resistant
transgenic rice.

Isolation of resistance genes in rice has led to the creation of transgenic
rice plants expressing their own resistance genes and highly tolerant to
fungal and bacterial diseases. The isolation of the Xa21 and recently Xa25
rice genes has led to the production of rice resistant to bacterial
blight1,2,3. Field trials of transgenic rice expressing genes for bacterial
resistance, herbicide tolerance, as well as to assess the gene flow of
transgenic rice using the herbicide tolerance gene as a marker, have also
been initiated1.

Rice has also been engineered to withstand different abiotic stress
conditions, such as drought, heat, cold, salinity, and mineral deficiency.
Of these, tolerance to salt/drought is the most studied using transgenic
rice. It has been suggested that the effect of each individual gene
conferring abiotic stress tolerance would be rather small; therefore, a
multigene approach or activation of multiple genes through transcriptional
activation of master regulatory genes can confer much greater tolerance5.
The overexpression of regulatory genes, such as the Ca++-dependent protein
kinase (CDPK) gene, OsCDPK17, or stress responsive MAPK gene, OsMAPK5,
resulted in the activation of stress-responsive genes and provided tolerance
to salt/drought. Overexpression of genes involved in ion or water transport,
such as Na+/H+ antiporter, Na+-ATPase, and aquaporins, also resulted in
varying degrees of tolerance to abiotic stresses. Finally, overexpression of
genes leading to accumulation of osmolytes or compatible solutes, such as
trehalose, proline, LEA proteins, glycine betaine, or polyamines, also
resulted in tolerance to various abiotic stresses1. Significant progress has
also been made to produce transgenic rice that can grow in mineral deficient
conditions.

Nutritional enhancement of rice

Because rice is the staple food for many poor people in developing
countries, an increase in the nutritive value of rice could be highly
valuable. It is estimated that about three million children of preschool age
suffer from eye defects due to vitamin A deficiency and about 500,000 go
blind and some may die. In this context, the recent landmark development of
provitamin A-enriched rice, popularly known as ?Golden Rice?, has immense
significance. The entire ?-carotene biosynthesis pathway has been engineered
into rice endosperm in a single transformation step; the genes for phytoene
synthase (psy) and lycopene ?-cyclase (?-lcy) originated from the daffodil
and the gene for phytoene desaturase (crt1) was of bacterial origin6. Since
then, several programs have been initiated to introduce these genes for
provitamin A biosynthesis into popular rice varieties. Recently, this
technology has been improved further by replacing daffodil psy with maize
psy. The transgenic rice plants termed ?Golden Rice 2? showed an increase in
total carotenoids of up to 23-fold compared to the original Golden Rice, and
displayed a preferential accumulation of ?-carotene7. It is expected that
?Golden Rice? could provide 50% of the RDA for vitamin A. Similarly,
transgenic rice has also been produced that is rich in iron, which would
also be a very useful supplement for women and children in developing
countries, half of whom are thought to be anemic. Rice has also been
transgenically improved to contain greater quantities of various amino
acids, such as glycine, lysine, tryptophan, cysteine, and methionine.
Similarly, improvements in starch biosynthesis and oil quality have also
been addressed1.

Biosafety of transgenic rice

Transgenic crops have been grown commercially for several years in both
developed and developing countries. These crops are released for cultivation
after extensive biosafety studies as per the regulations in individual
countries and the guidelines recommended for the safe release of GMOs by
international institutions such as the Food and Agriculture Organization
(FAO), World Health Organization (WHO), Organization of Economic Cooperation
and Development (OECD), and International Life Science Institute (ILSI).
Initial studies on transgenic insect resistant Bt rice indicated minimal
inpact on non-target organisms.

No safety evaluation precedence exists for rice that has been modified for
agronomic traits and nutrient content. However, only certain issues may be
important when analyzing a specific trait. For example, pollen-mediated gene
flow is an important issue when evaluating the biosafety of herbicide
resistant rice. The weedy rice (red rice), which often grows within a
cultivated rice field, can acquire the gene for herbicide tolerance through
cross-fertilization. However, for ?Golden Rice?, a food safety evaluation
would likely be of more concern than pollen-mediated gene flow.

Overall, the most important issues related to rice are: a) crop to wild gene
flow; b) food safety of transgenic rice; and c) impact on non-target
organisms. Several studies have been conducted around the world to study
gene flow from transgenic rice to non-transgenic cultivated rice or to wild
rice. Similarly, food safety studies of transgenic rice have begun to
emerge. These studies are important especially for nutrient-rich rice and
are an essential prerequisite for its safe release. Initial studies on
transgenic insect-resistant Bt rice1 indicated minimal impact on non-target
organisms.

Conclusions and future directions

Transgenic rice technology has moved beyond proof of concept and reached a
stage where it can supplement existing breeding methods to improve
production. Furthermore, transgenic rice with improved nutritional qualities
such as ?Golden Rice? should be available to consumers in the next 7?8 years
and could be adopted very quickly, mainly due to the commitment shown by
governments of developing countries such as India to move this technology
forward.

The availability of a complete rice genome sequence has opened up a sea of
opportunities, not only for rice but also for the plant community as a
whole. Rice research in a post-genomics era will likely change our approach
towards problem solving in biology. The research will evolve from a
single-gene based approach to more holistic genome- and proteome-wide
translational research. The availability of large resources of mutants and
full-length cDNA sequences could be utilized for large scale functional
genomics studies and other "OMICS"-based studies, such as proteome-wide
protein and localization studies, with the ultimate goal of mapping and
analyzing complete biological networks.

Thus, the above discussion suggests that rice biotechnology could witness
dramatic changes in the coming years, both in terms of the commercial
release of transgenic rice containing the existing gene resource and the
discovery of new genes by utilizing the advances in rice genomics.

References

1. Bajaj S & Mohanty A. (2005) Recent advances in rice biotechnology ?
towards genetically superior transgenic rice. Plant Biotechnol. J. 3,
275-307

2. Tyagi AK, Mohanty A, Bajaj S, Chaudhury A & Maheshwari SC. (1999)
Transgenic rice: A valuable monocot system for crop improvement and gene
research. Crit. Rev. Biotechnol. 19, 41-79

3. Tyagi AK & Mohanty A. (2000) Rice transformation for crop improvement and
functional genomics. Plant Sci. 158, 1-18

4. Sasaki T, Matsumoto T, Antonio BA & Nagamura Y. (2005) From mapping to
sequencing, post-sequencing and beyond. Plant Cell Physiol. 46, 3-13

5. Bajaj S, Targolli J, Liu LF, Ho T-HD & Wu R. (1999) Transgenic approaches
to increase dehydration-stress tolerance in plants. Mol. Breed. 5, 493-503

6. Ye XD, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, & Potrykus I.
(2000) Engineering the provitamin A (?-carotene) biosynthetic pathway into
(carotenoid-free) rice endosperm. Science 287, 303-305

7. Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright
SY, Hinchcliffe E, Adams JL, Silverstone AL, & Drake R. (2005) Improving the
nutritional value of Golden Rice through increased pro-vitamin A content.
Nat. Biotechnol. 23, 482-487

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