Ramin Hosseini

Corms as a source of explants for the successful clonal propagation of Crocus cancellatus

The genus Crocus comprises plants with a potential to be developed as a new ornamental crop but to date, there are not many reports on in vitro propagation of many members of this genus. The present study involves in vitro propagation of Crocus cancellatus with ornamental and horticultural value. Two different types of corm explants (apical and basal halves of corms) were cultivated onto Murashige and Skoog’s (MS) medium supplemented with different levels of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). One to five cormlets emerged from every responding explant through direct organogenesis. Apical halves of corms were more highly responsive than basal halves and produced a maximum multiplication rate with 3.45 ± 0.06 cormlets per explant in 95.33 ± 2.33% of the explants in MS medium supplemented with 3% sucrose and 2 mg L−1 NAA and 1 mg L−1 BAP. The effect of cold storage temperature on in vitro cormlets sprouting was studied. Cormlets stored at 4°C for 8 weeks had more statistically significant positive effects on cormlets sprouting from the controls. In vitro rooting of cormlets was induced on MS medium without plant hormones.

A QUICK, EFFICIENT, AND COST-EFFECTIVE METHOD FOR ISOLATING HIGH-QUALITY TOTAL RNA FROM TOMATO FRUITS, SUITABLE FOR MOLECULAR BIOLOGY STUDIES

Tomato (Solanum lycopersicum L.) is the primary model for the study of fleshy fruits, and research on this species has elucidated many aspects of fruit physiology, development, and metabolism. However, for advancing such studies at molecular biology levels, the RNA isolation from fruit tissues is often essential. The RNA isolation from tomato fruits is complicated because of the presence of high levels of polysaccharides, polyphenolics, pigments, and secondary metabolites and also the varying water content during development. Here, we present an optimized protocol for the isolation of total RNA from the fruit tissues at different developmental stages. In comparison to the previous methods described for the RNA isolation from tomato fruit, this method has the advantages that it does not involve the use of guanidine salts, lyophilizers, and commercial reagents, reduces the time and cost of extraction, overcomes the high water content problem, and promotes RNA quality by inhibiting RNA degradation and minimizing the gDNA, polyphenolic and polysaccharide contaminations. Using this method, high yields of high-purity and intact RNA samples were obtained as confirmed by the spectrophotometric readings and the electrophoresis on denaturing agarose gels. The isolated RNA was employed as a robust template for cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), and temporal gene expression analysis. The functionality of the isolated RNA was further demonstrated through cloning full-length cDNAs encoding β-galactosidase proteins by RT-PCR and sequencing.

Molecular charcterization of tatD DNAse gene from Ralstonia paucula RA4T soil bacterium

Ralstonia paucula strain RA4T, a gram negative, non-spore forming, motile bacterium having positive catalase and oxidase test, was isolated from surface soil. Twin arginine translocation protein type D (TatD) is shown to be located in cytoplasm and exhibits magnesium-dependent DNase. A tatD DNase gene was isolated and cloned from Ralstonia paucula RA4T genome. Nucleotide sequence analysis of the gene revealed 813 nucleotides encoding a protein of 270 amino acid residues. The tatD gene showed a high similarity to homolog gene from Ralstonia pickettii strain 12D. The deduced polypeptide sequence of TatD DNase from R. paucula RA4T had a typical catalytic site, HHPLDEHRHDP, and its calculated molecular mass and predicted isoelectric point were 29616 Da and 5.33, respectively. The deduced amino acid sequence showed a high degree of similarity to TatD DNase isoforms from Ralstonia genus and other sources. Predicted three-dimensional structure of TatD confirmed the presence of active site and theoretical function as DNase.