Search

Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Salinity has a significant negative impact on the production of rice yield which will become severe due to recent climate changes. To cope with this increased soil salinity, we need to develop salt tolerant rice varieties that can maintaining higher yield. Rice landraces indigenous to the coastal region of Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we developed a reciprocal mapping population between a salt tolerant landrace Horkuch and a high yielding rice variety IR29 . We applied a QTL analysis framework to identify genetic loci that contributes to salt adaptive responses for two different developmental stages of salinity treatment. We identified 14 QTL for 9 traits and found most QTL are unique to the specific developmental stage. Moreover, we discovered a significant effect of the cytoplasmic genome on the QTL model for some important traits such as leaf total potassium and filled grain number. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Along with that, we detected QTL co-localization for multiple traits that highlights the constraint of multiple QTL selection for breeding program. Overall, in this study we identified multiple QTL for different physiological and yield related traits for salinity treatment for two different developmental stages of the rice plant. We detected a significant contribution of cytoplasm-nuclear genome interaction for many traits. This study also suggests the selection constraint of donor alleles due to the presence of QTL co-localization. Highlights We identified QTL for salt tolerance response for two different developmental stages of rice plant and detected a significant contribution of cytoplasm-nuclear genome interaction for few traits.

Conventional methods of transformation for crop improvement and functional studies using Agrobacterium tumefaciens are still the method of choice, even though genotype specificity in indica rice can cause poor efficiencies of cell regeneration into transgenic plants. In such cases, tissue culture-independent in planta transformation using Agrobacterium has been shown to be highly effective to produce transgenic plants. Transient transformation using both biolistics and Agrobacterium has paved the way for rapid functional assays of genes or determining the efficacy of promoter sequences. However, biolistics or RNP-mediated delivery of gene editing components using CRISPR-Cas technology is more advantageous over Agrobacterium-mediated transformation methods because the foreign components get quickly degraded after nucleotide changes have taken place. For the Agrobacterium-mediated gene editing method, at least 2–3 generations are required to select only for mutations without the gene editing components. In all cases, however, gene editing can produce improved rice and other crop varieties without the “transgenic tag.” Crop improvement has been achieved for many biotic and abiotic tolerance traits as well as yield enhancement, which are cited here. Advances in gene sequencing technologies and the management of public databases with information on many new germplasm resources mean that many novel traits can now be incorporated for tailor-made crops to defend against threats to food security from increasing population and climate change.

The production of superior rice genotypes requires that a combination of desirable traits be introduced into the background of a high-yielding, commercially successful variety. This requires that multiple tolerance QTLs be combined by pyramiding. Single nucleotide polymorphism or SNP markers have become popular because the ease and efficiency of their use make them amenable to the type of pyramiding needed. The low cost of sequencing technologies has in turn made SNP discovery very easy and information on millions of these are available in public databases. SNP markers are being used for both genetic mapping and QTL analysis in populations derived from two contrasting parents. SNPs are also the marker of choice in the analysis of diverse germplasms for genome-wide association studies, or GWAS. For breeding purposes, rapid SNP detection is being done by the use of fluorescent tags, by methods referred to as Taqman and KASP. Efficient software for SNP discovery, mapping, QTL discovery, and GWAS has made breeding applications easier. The production of mapping populations with diverse sources for the trait of interest is also making identification of the correct allele easier for eventual incorporation into breeding lines.

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T1 seeds. T0 plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T1 was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T2 generation. Transformation efficiency was found to be 30–32% at the T0 generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na+/K+, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T2 generation.

The upsurge in global temperatures and the increasing threat of salinity are adversely affecting rice productivity in the southern coastal lands of Bangladesh. A Bangladeshi rice landrace Horkuch, adapted to these saline conditions, has long been cultivated by farmers in this region. To understand the trend and heritability of its tolerance traits, a reciprocal population of Horkuch with the high-yielding sensitive rice IR29 was generated for mapping of quantitative trait loci (QTLs) at both seedling and reproductive stages in an F2:3 strategy using double-digest restriction associated DNA. We found 29 QTLs and some QTL clusters containing survival and yield-related QTLs showing pleiotropic effects over some phenotypes. We advanced the generation up to F5 to obtain homozygosity and our current work is aimed at confirming these mapped QTLs at F5 by salt stress physiological tests at both stages as well as DNA markers linked to these QTLs. We confirmed SES QTL (survival of seedling under stress) associated with SSR marker RM25789 and two QTL clusters at chromosome 10 and 11 associated with RM26964 and RM27027, respectively. One cluster harbored yield-related filled grain number, filled grain weight, total grain number, harvest index, ionic balance and transpiration-related total sodium, total potassium and stomatal conductance QTLs. The other cluster contained total grain weight, filled grain weight and seedling root relative water content QTLs. We identified some individuals based on their physiological performance under stress as well as the presence of QTLs associated with some SSR markers that can be used for breeding and production of highly salt-tolerant cultivars with high yield.

The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F3 families from individual F2 segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F3 families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3 , SOS2 , and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3 ‐ SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 - which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture-independent Agrobacterium mediated in planta transformation method that circumvents the problems associated with tissue culture-based indica rice transformation methods. Integration of the foreign genes ( OsSOS1 ) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression ( SOS1 ) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T 1 progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice. Plant Tissue Cult. & Biotech. 25(2): 257-272, 2015 (December)

The rice (Oryza sativa L.) landrace Horkuch from Bangladesh maintains efficient photosynthesis and detoxification under salt stress and was therefore considered to be a useful donor for tolerance traits. Reciprocally crossed bi-parental mapping populations were generated from salt-tolerant Horkuch and high-yielding salt-sensitive variety IR29, in order to identify superior salt-tolerant high-yielding lines as donors. The present study reports on the phenotypic screening data of ~300 F3 segregating populations from the reciprocal cross and their parental lines in seedlings and screening of a subset at maturity stage under gradual salt stress of 12 dS m–1 for seedlings and 8 dS m–1 for mature plants. Correlation, broad-sense heritability and principal component analyses for salt tolerance as well as yield-related traits were conducted in the populations at the two developmental stages. Level of salt injury was found to be correlated with traits such as filled grain weight at maturity stage and biomass-related traits at the seedling stage. This association between yield-related and survival traits helped to identify tolerant and sensitive plants, which were predicative of agronomic performance under salt stress. Moreover, use of the reciprocal-cross population showed how cytoplasmic inheritance of specific traits such as K+ concentrations can affect characteristics of donor plants. Measurement of a large number of traits and analysis of their co-inherited interrelation can therefore help identify the best performing plants under salt stress for effective breeding strategies. The data are being utilised in mapping of quantitative trait loci, and selected progenies are being used as breeding lines for producing durable salt-tolerant, high-yielding rice varieties.

Helicases are a ubiquitous class of ATP-dependent nucleic acid unwinding enzymes crucial for life processes in all living organisms. There are six classes of helicases based on their conserved amino acid sequences. All eukaryotic RNA helicases belong to the SF1 and SF2 groups. Groups SF3–SF5 are mainly viral and bacterial DNA helicases, while SF6 includes the ubiquitous mini-chromosome maintenance or MCM group of helicases. SF3–SF6 are also characterized as hexameric ring-forming, whereas SF1 and SF2 groups are usually monomeric. The SF2 class is the largest group of helicases, including both DNA and RNA helicases with the widest range of function in replication, transcription, translation, repair, as well as chromatin remodeling. There is no clear sequence-based separation between DNA and RNA helicases. SF2 also includes both RNA and DNA helicases that are involved in biotic and abiotic stresses. While both DNA and RNA helicases play important roles in normal cellular function, the latter are more markedly involved in stress alleviation. This functional divergence was also evident in promoter sequence comparisons of the 113 A. thaliana helicases. Some DNA helicases like those from SF6 (MCM) and SF2 (CHR) are also active under stressed conditions. However, the most prominent stress-activated helicases are those with the conserved amino acid motifs, DEAD/H. Overexpression of DEAD/H helicases in many crops confers a growth advantage in the transgenic plants and has resulted in their protection against major abiotic stresses, such as salinity, drought, and oxidative stresses with minimal loss in yield potential.

Global increase in salinity levels has made it imperative to identify novel sources of genetic variation for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor pathway, membrane potential and cation transport. Furthermore, genes involved in regulating membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall our work has developed genetic resources and elucidated the likely mechanisms associated with the tolerance response of the Horkuch genotype.

Jute (Corchorus sp.) is one of the most important sources of natural fibre, covering ∼80% of global bast fibre production1. Only Corchorus olitorius and Corchorus capsularis are commercially cultivated, though there are more than 100 Corchorus species2 in the Malvaceae family. Here we describe high-quality draft genomes of these two species and their comparisons at the functional genomics level to support tailor-designed breeding. The assemblies cover 91.6% and 82.2% of the estimated genome sizes for C. olitorius and C. capsularis, respectively. In total, 37,031 C. olitorius and 30,096 C. capsularis genes are identified, and most of the genes are validated by cDNA and RNA-seq data. Analyses of clustered gene families and gene collinearity show that jute underwent shared whole-genome duplication ∼18.66 million years (Myr) ago prior to speciation. RNA expression analysis from isolated fibre cells reveals the key regulatory and structural genes involved in fibre formation. This work expands our understanding of the molecular basis of fibre formation laying the foundation for the genetic improvement of jute.

Haridhan rice was discovered by a farmer in his field which was planted with the widely cultivated mega-rice BR11. He noticed Haridhan’s variant characters compared to BR11, saved the seeds, planted it in 4–5 successive generations and noted the increase in yield with low input in fertilizer. Due to the overall similarity between BR11 and Haridhan, breeders were unwilling to call the latter a different genotype. This study was therefore performed to investigate the genetic similarity between BR11 and Haridhan. Polymorphism between Haridhan and BR11 using 115 SSR markers was 25 %, close to the reported 11 % for essentially derived (EDV) maize inbred lines. Similarly, 40 RAPD and 1,536 SNP markers showed polymorphism to be 16 % and 5 %, respectively. SSR polymorphic loci between the two were absent in chr 4, 6 and 9 but present in variable numbers in the rest. Some of the embedded and highly polymorphic SSRs were found in the 5′UTR of regulatory genes like Ser–Thr protein kinase and evolutionarily conserved C-terminal protein and revealed a common motif for binding TRA2-BETA1, a Serine–Arginine-rich protein involved in regulating and selecting splice sites. Genetic distance based on the Dice coefficient between Haridhan and BR11 as well as the cultivars, Lal Swarna and Guti Swarna planted in neighboring fields is 0.24, 0.26 and 0.20, respectively, compared to an average of 0.03 for EDV’s like maize inbred lines. Therefore, Haridhan could possibly have been derived from a cross of BR11 with another BR11 having outcrossed genes from the rice cultivars Lal Swarna and Guti Swarna.

Soil salinity adversely affects plant growth, development and disturbs intracellular ion homeostasis which results in cellular toxicity. The salt overly sensitive 1 (SOS1) gene is a critical component of salt tolerance in many species and encodes a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth in saline environments. In the current study, the coding sequence of SOS1 gene (3447 bp) was amplified from a Bangladeshi rice landrace, Pokkali, by applying the fusion PCR strategy. SOS1 PCR amplicons were firstly cloned into pENTR/D-TOPO and then recombined with the binary vector, pH7WG2, through LR reaction. Positive colonies were validated by PCR, restriction digestion and sequencing. Finally, the constructed vector was transformed into Agrobacterium tumeficiens, LBA4404 strain, to initiate Agrobacterium-mediated transformation with the provision to transfer the cloned SOS1 into farmer popular rice varieties. D. O. I. http://dx.doi.org/10.3329/ptcb.v23i2.17527 Plant Tissue Cult. & Biotech. 23(2): 263-273, 2013 (December)

Salinity is one of the most critical environmental problems, which causes plant growth retardation by disturbing intracellular ion homeostasis. The Na+/H+ antiporter plays an important role in resistance to salt stress by sequestering Na+ in exchange for H+ across the vacuolar membranes. In the current study, the coding regions of two Arabidopsis antiporters (AtNHX1 and AtNHX2) and one rice antiporter (OsNHX1) were amplified by target specific PCR. PCR amplicons were first cloned into pENTR/D-TOPO and later recombined with a destination vector (pK7WG2.0) by LR reaction. Positive clones were selected by PCR, restriction digestion (RD) and sequencing. They were then transformed into Agrobacterium tumefaciens (LBA4404 strain) for subsequent transformation of farmer popular tomato varieties.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5′ UTR with CDS and the other of 2.3 kb, including 5′ UTR, CDS, and the 3′ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5′ and 3′ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5ʹ UTR with CDS and the other of 2.3 kb, including 5ʹ UTR, CDS and the 3ʹ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5ʹ and 3ʹ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P 1.9 kb> and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P

To improve the salinity tolerance of rice, a DEAD-box helicase gene isolated from pea with a CaMV35S promoter was transformed into the Bangladeshi rice variety Binnatoa through Agrobacterium-mediated transformation. The transgenic seedlings showed significantly higher chlorophyll content, but decreased root length compared to wild type (WT) under normal physiological conditions. Their status was confirmed by polymerase chain reaction (PCR), semi-quantitative reverse-transcription PCR and Southern blot hybridization for positive integration of the transgene. The T2 progenies from three independent transformation events were characterized for salinity tolerance both at seedling and reproductive stages. Compared to the WT plants, the average decrease in chlorophyll content and dry weight of seedling leaves was lower by 20 and 12% respectively at 12 deciSiemens per meter (dS/m) NaCl stress in hydroponics. A higher leaf K+/Na+ ratio of 0.346 was maintained by the transgenic lines compared to the WT ratio of 0.157, which indicated induced ion homeostasis. At the reproductive stage, transgenic rice plants expressing PDH45 showed better fertility and produced higher grain yield by 16% compared to WT plants under continuous stress of 6 dS/m from 30 days till maturity. One of the transformed lines, PDH45-P3, outperformed the others, and replicated data in reproductive stage soil stress of 12 dS/m NaCl showed its enhanced fertility and yield by 46 and 29% over WT, respectively.

Bangladesh is home to diverse rice germplasm, including red rice, many of which are preferred for cultivation by farmers over high-yielding rice varieties due to their special characteristics, color, taste, and nutritional value. Red color of seed pericarp is unusual among modern cultivated varieties, though it is a common characteristic in wild relatives of rice, making the trait an important parameter when studying the domestication and evolution of crop plants. Diversity analysis using microsatellite markers and sequence variation of the red rice loci of the indigenous rice population in Bangladesh was therefore performed. Microsatellite fingerprinting could successfully cluster cultivars according to their specific phenotypic characteristics such as stickiness or aroma, irrespective of their pericarp color, and locate a set of unique identifiers. Sequence analysis of a portion of the bHLH transcription factor gene Rc, which controls the red pigment, confirmed the occurrence of the 14-bp deletion in white rice accessions which has been reported previously. The analysis included a group of rice cultivars which are known by the same name but produce two different colored seeds and are indistinguishable morphologically unless dehusked. Statistical and internal transcribed spacer region sequence diversity analyses established that such cultivars, although genetically different, had very low diversity, suggesting a close evolutionary relationship between them. Red and white seeds from cultivars with the same name were planted individually over two generations and dehusked to check for loss/gain of pigmentation. Red-seeded plants produced some dirty-white seeds in addition to red ones in the first generation, the proportion of which increased when planted in the successive generation. This loss in pigment was probably due to faulty transcription, since no deletion in the respective region of the genome of the dirty-white seeds was noted.

Abiotic stresses such as drought and high salinity unfavorably affect the growth and productivity of crop plants. Therefore, the development of stress-tolerant crops is essential for the affected cultivable areas. It has been shown in the current study that the overexpression of stress-responsive NAC1 (SNAC1) transcription factor (TF) significantly increases salinity and drought tolerance in a farmer-popular high-yielding, transgenic rice. The indica rice variety BRRIdhan 55, which was poorly responsive to tissue culture was transformed with the SNAC1 TF from the rice landrace Pokkali by the in planta method. Addition of acetosyringone in the Agrobacterium suspension and co-culture media increased previously reported transformation efficiencies by four-folds. Integration of foreign genes into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SNAC1) was also quantified by real-time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the law of Mendelian inheritance. Phenotypic screening was conducted at T2 and T3 seedling stages where the transgenic lines exhibited much better tolerance compared to their control non-transgenic plants at 120 mM (NaCl) salt as well as drought stress implemented by withholding water for 20 days.

The vacuolar Na+/H+ antiporter is known to alleviate saline stress by sequestering Na+ in both wild-type Arabidopsis and rice and when over-expressed in many transgenic plants. Here we report on the effect of the NHX1 transgene on the salt tolerance properties it confers to a rice landrace and a commercial cultivar suitable for the dry winter season, but which suffers loss due to seasonal stresses, particularly in the coastal areas. The Nipponbare Na+/H+ antiporter 1.9 kb cDNA was cloned into pCAMBIA1305.1 under the control of the CaMV35S promoter and transformed into tissue-culture-responsive rice landrace Binnatoa (BA). The best-expressing transgenic line at T2 was found to be significantly tolerant at the seedling stage and was advanced to T3. The transgene was then transferred to the tissue-culture recalcitrant farmer-popular commercial rice genotype, BRRIdhan 28 (BR28) by crossing. The data generated both from semi-quantitative RT-PCR and western blot hybridization revealed that the transgene showed similar expression in the crossbred BR28 plants and BA transgenic line. Comparative stress tolerance tests, however, revealed that the BR28 crossbred lines were significantly less tolerant than its transgenic parent BA at both seedling and reproductive stages. A single successful transgenic event may therefore not show the same performance in the recipient genetic background, if introgressed by crossing.

Good donors in breeding for salt tolerance are a prerequisite for food security under changing climatic conditions. Horkuch, a farmer-popular salt tolerant rice (Oryza sativa L.) variety from the south-west coast of Bangladesh was characterised up to maturity under NaCl stress, together with a modern variety (BRRI dhan41), a sensitive control (BRRI dhan29) and Pokkali, the salt-tolerant benchmark for rice. Horkuch had low reduction in shoot biomass, a low Na : K ratio in flag leaves, a low percent reduction in yield and good partitioning of Na in the older leaves, and maintained high levels of Ca and Mg in the flag leaves. In order to understand the physiology at the molecular level, the expression of salt-responsive genes was investigated using microarray analysis. Salt-stressed cDNA of Horkuch seedlings were hybridised with cDNA probes synthesised mainly from database sequences of Arabidopsis thaliana (L.) Heynh. The upregulated genes included transcription factors, signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants and some specific salt-induced transcripts. An increase in expression of photosynthesis-related genes as well ROS scavengers suggested that this could be the reason for the better yield performance of Horkuch. The data therefore indicate Horkuch as a potential donor alternative to Pokkali in breeding programs for salt tolerance.

Identification and analysis of regulatory sequences that control gene expression can be greatly facilitated by database-assisted bioinformatic approaches. READS (Regulatory Element Analysis DatabaSe) has been created as a web-accessible freely available database of plant non-coding regulatory sequences. It currently contains more than 300 known and putative promoters of constitutive as well as stress inducible genes belonging to diverse plants. The database has been manually curated with promoters collected mainly from scientific publications, thereafter cross-referenced with other resources (NCBI database, PubMed, PubMed Central). A user-friendly interface has been provided to allow easy access and analysis of data using different query options. A blast utility has also been provided, allowing users to search against all entries in the database. For each promoter, certain features such as expression data, GC content, core elements etc., were provided to assist in characterization of the regulatory sequences. To our knowledge, READS is the first plant promoter database that allows retrieval of sequences based on expression pattern. Thus the database can be utilized as a useful resource for identification of important putative regulatory cis-elements in promoters by analysis of upstream regions of hundreds of co-regulated or co-expressed genes. Such knowledge can also be of use for identifying minimal or stress inducible promoters for effective transgene expression. We aim to provide the most up-to-date collection of promoters of well-characterized stress inducible and constitutively expressed genes from many plant species. Hence, this resource will be updated regularly to incorporate new sequences. READs is available at http://www.pbtlabdu.net/READS/. Key words: Promoter database, stress inducible, constitutive, gene expression, cis-element D.O.I. 10.3329/ptcb.v20i2.6916 Plant Tissue Cult. & Biotech. 20(2): 211-223, 2010 (December)