Zeba I. Seraj

Characterizing the Saltol Quantitative Trait Locus for Salinity Tolerance in Rice

This study characterized Pokkali-derived quantitative trait loci (QTLs) for seedling stage salinity tolerance in preparation for use in marker-assisted breeding. An analysis of 100 SSR markers on 140 IR29/Pokkali recombinant inbred lines (RILs) confirmed the location of the Saltol QTL on chromosome 1 and identified additional QTLs associated with tolerance. Analysis of a series of backcross lines and near-isogenic lines (NILs) developed to better characterize the effect of the Saltol locus revealed that Saltol mainly acted to control shoot Na+/K+ homeostasis. Multiple QTLs were required to acquire a high level of tolerance. Unexpectedly, multiple Pokkali alleles at Saltol were detected within the RIL population and between backcross lines, and representative lines were compared with seven Pokkali accessions to better characterize this allelic variation. Thus, while the Saltol locus presents a complex scenario, it provides an opportunity for marker-assisted backcrossing to improve salt tolerance of popular varieties followed by targeting multiple loci through QTL pyramiding for areas with higher salt stress.

Identification of the regeneration potential of embryo derived calluses from various Indica rice varieties

The regeneration response of mature and immature rice embryos from 15 rice varieties (Oryza sativa L. var. indica) was separately measured in terms of regeneration per cent and total number of regenerated plantlets obtained for a fixed sample size per variety, when cultured on standard MS media. The response of the rice varieties was classed as high, medium or poor, based on the above criteria. The regeneration per cent of genotypes showing high, medium and poor response ranged from 67 to 97, 38 to 63 and 0 to 36 respectively. The variation in the regeneration response amongst the defined groups was found to be statistically significant in terms of regeneration per cent and total plantlets for a constant sample size when subjected to one way analysis of variance and Duncans multiple range test. Only small influences on regeneration frequency were found when the most and least responsive genotypes, Binnatoa and BR-23 were cultured on N6 and MS media with additional or different supplements.

Plant regeneration in a jute species (C. capsularis) and its possible relationship with glyoxalase-I

SummaryThe addition of 3 mg/l of nordihydroguaiaretic acid (NDGA) to BAP and tyrosine fortified MS medium was essential to obtain organogenic callus from the hypocotyl segments of two varieties (D-154 and CVL-1) of Corchorus capsularis — one of the two jute species. When the organogenic callus, which is rich in large starch granules, was transferred to MS basal medium, it differentiated into single or multiple shoots usually in the first subculture and sometimes in the second. The activity of glyoxalase-I of the organogenic callus was found to be significantly lower than that observed in the nonorganogenic callus initiated on MS medium supplemented with 2,4-D, tyrosine, BAP or just BAP and tyrosine. This suggests an inverse relationship between differentiation and the level of glyoxalase-I activity in the two varieties of C. capsularis jute.

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na+/H+ Antiporter Gene Compared to 1.9 kb Coding Region with 5′ UTR in Transgenic Lines of Rice

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.

In silico predicted mycobacterial epitope elicits in vitro T-cell responses

Epitope-based vaccines permit the selection of only a specific subset of epitopes to induce the necessary immune response, thus providing a rational alternative to conventional design approaches. Using a range of immunoinformatics tools, we identified a novel, contiguous 28 amino acid multi-epitope cluster within the highly conserved secretory protein Ag85B of Mycobacterium tuberculosis, the causative agent of TB. This cluster, named Ep85B, is composed of epitopes which bind to three HLA Class I and 15 Class II molecules, and harbors the potential to generate 99% population coverage in TB-endemic regions. We experimentally evaluated the capacity of Ep85B to elicit T-cell immune responses using whole blood cells and, as predicted, observed significant increases in populations of both CD4+ and memory CD4+ CD45RO+ T-cells. Our results demonstrate the practical utility of an epitope-based design methodology - a strategy that, following further evaluation, may serve as an additional tool for the development of novel vaccine candidates against TB and other diseases.

Physiology and gene expression of the rice landrace Horkuch under salt stress

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.

Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309

The green plant regeneration ability from anthers of BR-7, a high yielding indica cultivar, Binnatoa (BA), a salt tolerant indica land race and IR-43 was tested in N6, M8, He2 and R2 media. The response was calculated on the basis of number of anthers producing green plants. The number of green plants per responding anther was also recorded. The response of BR-7 and BA was poor compared to the indica cultivar IR-43 in three of the media that were tested. In N6 medium, green plant regeneration of BA and BR-7 was respectively 10-fold and 100-fold less than the japonica cultivar Taipei 309 (T-309). No anther-derived green regenerant was obtained from another salt tolerant indica land race, Rajashail (RAJ). The N6 medium was selected to test green plant regeneration frequency from anthers obtained from the F1 crosses of T-309 × BR-7, T-309 × BA, T-309 × RAJ and T-309 × BR-7 AC regenerants backcrossed with BA. Our objective was to combine the salt tolerant trait of BA and the high yield of BR-7 in a single line. The intermediate crossing step with T-309 was performed to increase the green plant regenerability of the anthers. All F1 progeny from the crosses with T-309 showed significantly increased callus induction compared to the indica parent although the values were lower than the midparent means. Green plant regeneration compared to their respective indica parents either increased or decreased but never approached the level of T-309.

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through Agrobacterium-mediated transformation of PgNHX1 from Pennisetum glaucum

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na+/H+ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.

Pollen tube growth in crosses between Porteresia coarctata and Oryza sativa

SummaryComparative study with fluorescence microscopy revealed that the pollen tubes of the self-pollinated rice variety BR-9 reached the ovary within 75 min after pollination. In P. coarctata it took 150 min. In the cross between P. coarctata and BR-9, pollen tubes reached the ovary in 165 min but seed setting was not observed. In the cross between BR-9 and P. coarctata, small and deformed pollen tubes were formed and they failed to grow through the stylodium.

Interactive effects of nitric oxide and glutathione in mitigating copper toxicity of rice (Oryza sativa L.) seedlings.

Nitric oxide (NO) and glutathione (GSH) are 2 vital components of the antioxidant system that play diverse roles in plant responses to abiotic stresses. Recently, we have reported that exogenous supply of both these molecules reduced copper (Cu) toxicity in rice seedlings. Individual as well as co-treatment of sodium nitroprusside (SNP: a NO donor) and GSH with Cu significantly mitigated the adverse effects of Cu, evident in the reduced level of oxidative markers such as H2O2, superoxide (O2·−), malondialdehyde (MDA), and proline (Pro). GSH content and most of the antioxidative and glyoxalase enzymes were up-regulated upon Cu stress, indicating their responses were co-related with the level of stress. Our results indicated that direct ROS scavenging, reduced Cu uptake, and the balanced antioxidative and glyoxalase systems, at least in part, successfully executed NO- and GSH-mediated alleviation of Cu toxicity in rice seedlings. In addition, the combined effect of adding SNP and GSH together was more efficient than the effect of adding them individually. Here, we are speculating that 1) GSH and Pro could be used as potential markers for copper stress, and 2) adding SNP and GSH might produce S-nitrosoglutathione (GSNO) which could be a source of bioactive NO and may affect many regulatory processes involved in Cu-stress tolerance. We further note that the combined effect of adding SNP and GSH was pronounced in inhibiting the uptake and translocation of Cu in rice seedlings.