Isolation and expression analysis of salt stress-associated ESTs from contrasting rice cultivars using a PCR-based subtraction method

Salt stress adversely affects the growth of rice plants. To understand the molecular basis of salt-stress response, four subtracted cDNA libraries were constructed employing specific NaCl-stressed tissues from salt-tolerant (CSR 27 and Pokkali) and salt-sensitive (Pusa basmati 1) rice cultivars. An efficient PCR-based cDNA subtraction method was employed for the isolation of the salt-stress responsive cDNA clones. In all, 1,266 cDNA clones were isolated in the course of this study, out of which 85 clones were end-sequenced. Database search of the sequenced clones showed that 22 clones were homologous to genes that have earlier been implicated in stress response, 34 clones were novel with respect to their function and six clones showed no homology to sequences in any of the public database. Northern analysis showed that the transcript expression pattern of selected clones was variable amongst the cultivars tested with respect to stress-regulation

Apomixis: an enigma with potential applications

Apomixis has been the focus of research in plant sciences in recent years with lot of scope for crop improvement. It results in clonal progeny without fertilization, having maternal genetic constitution. The impact of introducing apomixis in crop plants could be significant mainly for its use in fixation of hybrid vigour. Because of epigenetic barriers, introgression of apomixis from a close relative to a sexual crop plant by conventional plant breeding methods could not generate expected results. Recent developments in plant molecular biology and biotechnology can help in developing potential strategies. This article summarizes various aspects of apomixis research that are being followed in India and abroad

directional genome walking using pcr

We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a ..

Expression of a rice chitinase gene enhances antifungal potential in transgenic grapevine (Vitis vinifera L.)

To enhance the antifungal potential of grapevine, transgenic plants were generated by transferring rice chitinase gene under a maize-ubiquitin promoter along with its first intron into the leaf disc-induced somatic embryos via Agrobacterium mediated transformation. After co-cultivation for 2 days with recombinant Agrobacterium, somatic embryos were transferred onto WPM medium containing BAP 1.5 μM and NAA 0.1 μM supplemented with 25 mg/L hygromycin. Secondary or tertiary embryos were selected and the antibiotic resistant transgenic plantlets were analyzed. The integration and stability of the transgene were confirmed by PCR, RT-PCR, Southern blotting and by Western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. These analyses indicated that the foreign gene was translated into the protein of expected molecular weight that showed chitinase activity. Following in vitro inoculation of powdery mildew (Uncinula necator), the transgenic plants showed delayed onset of the disease and smaller lesions. The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.

Functional validation of a novel isoform of Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice

Salt stress is an environmental factor that severely impairs plant growth and productivity. We have cloned a novel isoform of a vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that contains 5 transmembrane domains in contrast to AtNHX1 and OsNHX1 which have 9 transmembrane domains. Recently we have shown that PgNHX1 could confer high level of salinity tolerance when overexpressed in Brassica juncea. Here, we report the functional validation of this antiporter in crop plant rice. Overexpression of PgNHX1 conferred high level of salinity tolerance in rice. Transgenic rice plants overexpressing PgNHX1 developed more extensive root system and completed their life cycle by setting flowers and seeds in the presence of 150 mM NaCl. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high saline areas

Encapsulated somatic embryos of grape (Vitis vinifera L.): An efficient way for storage and propagation of pathogen-free plant material

Cotyledonary-stage somatic embryos (5-7 mm in length) originating from leaf explants of grape (Vitis vinifera L.) cv. Pusa seedless were encapsulated individually in 2 % alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7 % agar medium containing B5 macrosalts (half strength), MURASHIGE and SKOOG microsalts (full strength), 3 % sucrose and 2.9 μM gibberellic acid. The percentage of germination of ESEs was higher than that of nonencapsulated somatic embryos (NSEs) of the same size on the same medium. The percent germination of ESEs increased (69.2 ±2.8) on medium supplemented with quarter strength B5 macrosalts. Of the germinating ESEs, 36 % developed into plantlets. Abscisic acid at 0.004 and 0.02 μM had no significant influence on the frequency of germination and plantlet development, however resulted in a 4-week delay in germination. Transferring the embryos onto the full-strength B5 medium containing sucrose and ABA (0.04 μM) for 4-6 weeks prior to encapsulation resulted in extended storage of up to 90 d without loss of the germination potential and the capacity to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil.