The DREB and CBF transcription factors play a critical role in plant development and abiotic stress responses and, therefore, represent attractive targets for a molecular plant breeding approach. In this study, the rice OsDREB2A gene was isolated and expressed under the control of a stress-inducible promoter (4ABRC) to improve the abiotic stress tolerance of japonica rice variety TNG67. T2 and T3 transgenic lines over-expressing OsDREB2A were found to have improved survival rates under severe drought and salt stress conditions relative to non-transgenic rice plants or rice plants transformed with the empty vector control. OsDREB2A expression was found to be markedly induced by drought and ABA treatment. The results indicate that the induced over-expression of OsDREB2A driven by the 4ABRC promoter in engineered rice plants may protect cells during stress.

The everyday deteriorating environmental factors such as extremes of temperature (high and low), water availability (drought and flooding), ion or physiological pH (salinity and alkalinity), UV light and anoxia pose deleterious effects on the survival and overall yield of the plants. Since, the abiotic stresses are multigenic as well as quantitative in nature, it is far more difficult to understand the response of the plants towards these stresses. Intensive efforts are being made worldwide employing physiological, biochemical and molecular tools to raise plants with improved suitability towards environmental stresses. With the advances in recent years towards identifying the genes that are regulated under stress, and sequencing of the whole plant genomes, research is being taken up actively to understand the molecular basis of abiotic stress responses and to manipulate these processes via genetic engineering. Employing transgenic technology, functional validation of various target genes, involved in diverse processes such as signal transduction, transcriptional regulation, ion homeostasis and antioxidant defense for various abiotic stresses has been attempted in various model systems. Some of these efforts have been extended to crop plants such as rice, maize, Brassica, wheat etc. This chapter presents a brief description of the transgenic studies that have been attempted with a view to understand the role of various genes which were indicated to be important, and transfer of some of these genes to crop plants for enabling them to survive under stress conditions.

To identify minimal effective promoters for driving abiotic stress-inducible transgene expression in rice, we selected promoter elements of three stress-responsive genes, viz. rab16A coding for dehydrin, OsABA2 coding for zeaxanthin epoxidase, and a gene coding for a hypothetical protein (HP1) based on the presence of ABA-, salt- and drought-responsive cis-acting elements. These were translationally fused to the gusA reporter gene and introduced into rice to study their effect on heterologous gene expression. The OsABA2 promoter was found to be the most effective and desirable promoter among the three in terms of driving a low constitutive transgene expression under normal conditions and high induction in response to ABA, salt and drought stress, the highest being a 12-fold induction in response to ABA. The rab16A and HP1 promoters resulted in high levels of constitutive expression. While induction of GUS activity was generally two- to threefold for all the treatments in roots for both the promoters, induction in leaves was generally insignificant, the exceptions being rab16A in response to continuous salt stress and HP1 in response to water deficit. It was also observed that the three promoters, in general, resulted in lower constitutive expression, but higher induction in roots as compared to leaves.

We have isolated and characterized the 5' region of the rice actin2 gene (OsAct2), which contains 793 bp of sequence upstream of the OsAct2 transcription initiation site, 58 bp of the first non-coding exon, 1736 bp of the 5' intron and the first 8 bp (non-coding sequence) of the second exon. It was found that the 5' region of OsAct2 is an efficient gene regulatory region for driving the constitutive expression of foreign genes in transgenic rice. In situ histochemical results indicated that OsAct2::GUS (GUS, beta-glucuronidase) gene expression in transgenic rice plants is high in sporophytic and gametophytic tissues. It was demonstrated that a 2.6-kb upstream sequence of the OsAct2 translation initiation codon contains all of the 5' regulatory elements necessary for high-level gus expression in transgenic rice tissues. OsAct2 promoter activity was significantly enhanced by the deletion of a 1590-bp segment from the central region of the first intron. The +96 to +274 region of the intron negatively regulates gus expression in leaves. To identify regulatory elements within the OsAct2 promoter, nested truncations of the promoter region were made and fused to gus. The results showed that the region from -1 to -376 was sufficient for promoter activity. In addition, two OsAct2-based expression vectors for use in monocot transformation were developed to promote the high-level expression of foreign genes.

We present a simple and rapid method for screening second-generation transgenic rice plants (T1) to identify homozygous plants. The plasmid (pfd11) used for rice transformation contains a partially deleted cytochrome c gene (cyc) for comparing with the endogenous cyc for copy number. After polymerase chain reaction (PCR) amplification of a segment of the cyc in transgenic rice DNA followed by agarose gel electrophoresis, two specific bands are obtained. The upper band represents the endogenous cyc, and the lower band represents the partially deleted cyc in the transgene. The first-generation plants (T0) that harbor a single copy of the transgene are selected based on the fact that the density of the lower band is half as dense as the upper band. Next, only plants harboring a single copy of the transgene are advanced to the second generation (T1). The same PCR procedure is used again, and homozygous T1 plants are easily identified from samples in which the intensity of the two bands is the same.

For plant transformation, a selectable marker gene is generally required for proliferation of cells that harbor the input gene, while inhibiting the growth of non-transformed cells. The most commonly used selection systems for rice involve the use of an antibiotic or an herbicide. We report here a novel selection system for rice based on the use of 200mM NaCl for selection and regeneration at the cell culture stage. Plasmids containing cDNA encoding salt tolerance genes OsDREB2A and AtSOS1 were prepared. Both plasmids support high transformation efficiencies under high-salt conditions. This salt selection system can be conveniently used to rapidly generate salt-tolerant transgenic plants free of genes that confer antibiotic or herbicide resistance.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134 285 F 2 plants in the field. Over 2 800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.

Glycine betaine (GB) is a compatible solute that is also capable of stabilizing the structure and function of macromolecules. Several GB-producing transgenic rice lines were generated in which the Arthrobacter pascens choline oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI) gene promoter that is considered to be constitutive. This comparison led to interesting observations that suggest complex regulation with respect to GB synthesis and plant growth response under stress. In spite of the use of the well-studied stress-inducible promoter, the highest level of GB accumulation (up to 2.60 micromol g(-1) DW) in the SIP lines grown under saline conditions was not as high as in the UBI lines (up to 3.12 micromol g(-1) DW). Therefore, the use of an ABA-inducible promoter was not more beneficial for de novo production of GB. Interestingly, saline growth conditions enhanced GB accumulation by up to 89% in the SIP lines, whereas up to 44% increase was seen in a UBI line. In all these cases the GB levels were many-fold below the range reported for plant species that produce GB naturally. In spite of lower GB concentrations, statistically greater levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress protection observed in SIP plants cannot be totally explained by the increase in the GB content.

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T5) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.

We investigated the effect of tobacco Rb7 Matrix Attachment Region (MAR) sequences on transgene expression in a japonica rice variety TNG67. Six plasmids were constructed and used to transform rice. For each plasmid, over 30 independent transgenic plant lines containing a β-glucuronidase (GUS) gene expression cassette, either by itself, or flanked by a full-length or a half-length tobacco Rb7 MAR sequence were generated, out of which 15 lines were randomly selected and used for detailed analysis. Quantitative GUS activity assay of the transgenic plant lines showed an average 77-fold increase in GUS activity when the Gus gene was flanked by the full-length MAR sequence. The MAR sequence also minimized variations in Gus gene expression in independently produced transgenic plants harboring the same plasmid. The half-length MAR sequence was unable to stimulate Gus gene expression but did retain the ability to reduce variations in the level of expression among different transgenic plants.