Archana Sachdev

Characterization and Cloning of ODAP Degrading Gene from a Soil Microbe

The strain BYT-1, capable of utilizing ODAP/DAP as a sole source of nitrogen and carbon was identified as Psuedomonas stutzeri by various microbial and biochemical tests. Transformation experiments showed that the ODAP utilizing property Is encoded by the plasmid. Restriction of plasmid DNA with Pstl, followed by cloning of fragments and screening of ODAP containing medium, led to the isolation of a clone with insert size of −3.3 kb, which encoded ODAP metabolizing property. The growth and ODAP/DAP utilization by this clone (TB) was almost similar to that of the wild type strain.

Molecular Characterization of Glutenins in Wheat Varieties Differing in Chapati Quality Characteristics

Five wheat (Triticum aestivum) varieties differing in chapati quality characteristics viz. C-306, K-68, HD-2745 and HD-2735 with good and Sonalika with poor chapati quality characteristics, were selected for the characterization or distribution of glutenin genes. Polymorphism was observed when genomic DNA of wheat varieties was hybridized with a HMW glutenin probe [glutenin subunit 10 (Dy10)]. No hybridization was observed in Sonalika. PCR amplification of genomic DNA with the LMW glutenin gene-specific primers did not show any polymorphism. However, with HMW glutenin gene-specific primers a single band of ∼ 650 by was obtained in all the good chapati characteristic wheat varieties.The amplified fragment was sequenced and found to have sequence homology with HMW glutenin subunit Dx5.The deduced protein structure analysis showed that the peptide was made up of N-terminally placed (x-helices and centrally placed repetitive β-turns.

Physiological and Biochemical Analysis of Somaclones Derived from Leaf Explants of Lathyrus sativus

Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.

RAPD analysis of aromatic and non-aromatic rice (Oryza sativa L.).

Genetic variation in nine aromatic and four nonaromatic rice varieties (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) technique. Twenty six random primers were used to amplify DNA segments and 177 PCR products were obtained of which 98 were polymorphic. One primer did not show polymorphism. A dendrogram showing the genetic distances of 13 rice varieties was constructed based on RAPD data.

Lathyrus Dehydrin : A Drought Inducible cDNA Clone — Isolation and Characterization

Dehydrin like transcripts (drought inducible mRNA) accumulated in drought stressed shoots and roots of Lathyrus sativus. ABA also induced the expression of dehydrin in unstressed seedlings but to a lower extent. A cDNA expression library was prepared in lambda ZAP vector from mRNA of drought stressed shoots of Lathyrus sativus cv P-24 and screened with pea dehydrin cDNA probe. One positive clone (∼ 500 bp) was purified and characterised. The partial nucleotide sequence of the clone Lsd, contained a possible open reading frame which would encode a polypeptide with region of significant amino acid sequence similarity to dehydrin from other plant species. The genomic counterpart of Lsd cDNA was found to be present within a fragment of ∼ 3.4 and ∼ 3.2 kb in parent as well as in the somaclones of Lathyrus sativus, by using different restriction enzymes.

Enhanced nutraceutical potential of gamma irradiated black soybean extracts.

Radiation processing of soybean, varying in seed coat colour, was carried out at dose levels of 0.25, 0.5 and 1 kGy to evaluate their potential anti-proliferative and cytoprotective effects in an in vitro cell culture system. Irradiated and control black (Kalitur) and yellow (DS9712) soybean extracts were characterized in terms of total phenolics, flavonoids and anthocyanins, especially cyanidin-3-glucoside (C3G). Using an epithelial cell line, BEAS-2B the potential cytoprotective effects of soybean extracts were evaluated in terms of intracellular ROS levels and cell viability. The most relevant scavenging effect was found in Kalitur, with 78% decrease in ROS, which well correlated with a 33% increase in C3G after a 1 kGy dose. Results evidenced a correspondence between in vitro antioxidant activity and a potential health property of black soybean extracts, exemplifying the nutraceutical role of C3G. To our knowledge this study is the first report validating the cytoprotective effects of irradiated black soybean extracts.

Physiological and Biochemical Analysis of Lathyrus sativus Somaclones Derived from Root and Internode Explants

Low ODAP somaclones from root and internode explants were evaluated for physiological and biochemical parameters. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. All the somaclones during development had substantially lower ODAP content in leaves compared to parent P 24. Amongst somaclones, Bio R 202 had the maximum leaf area, biomass as well as photosynthetic rate. Harvest index and seed yield were highest in Biol 222 whereas ODAP content was the least in it.

Molecular characterization of inositol pentakisphosphate 2-kinase (GmlPk1) from soybean and its expression pattern in the developing seeds

The principal storage compound of seeds, phytic acid, poses several problems for agriculture due to its antinutritional effects making the development of low phytate crops a prime priority in research. Soybean, with its high nutritive value, wide acceptability and varied usage, but possessing high levels of phytate can be a major target of low phytate research. To this effect, Inositol pentakisphosphate 2-kinase (IPK1) can prove to be critical player given its key role in catalyzing the terminal step of phytate biosynthesis. In this paper, we report the expression profile of GmIPK1 across different tissues and seed development stages, cloning of 1371 bp nucleotide sequence representing the full length cDNA of GmIPK1 and in silico analysis for characterization. Amino acid sequence of GmIPK1 showed much similarity with that of Phaseolus vulgaris and Cicer arietinum. It also showed the presence of the characteristic Ins_P5_2-kinase domain required for catalytic activity. Thus this study provides an initial idea on the molecular characterization of GmIPK1 enzyme for its further in detailed study and subsequent utilization in generating low phyate soybean with enhanced mineral bioavailability.

Developmental Expression of Microsomal Omega-6 Desaturase Gene (fad2-1) in Soybean Seeds

Fad2-1 gene encoded microsomal omega-6 desaturase plays a major role in controlling the conversion of oleic acid to linoleic acid within storage lipids during seed development. A 180 bp region (1241–1422) specific to fad2-1 was amplified and its sequence confirmed. Northern blot analysis using the labelled amplicon as probe indicated that the fad2-1 expression was induced during early stages of embryo development and peaked in the mid-maturation stages. Tissue specificity of fad2-1 transcripts was confirmed by the complete absence of fad2-1 transcripts in the vegetative tissues of leaf, stem and root. Developmental profile of fatty acids showed increased levels of linoleic acid during the mid-maturation stages which coincided with the enhanced expression of fad2-1 during the same period.

Exploring the role of Inositol 1,3,4-trisphosphate 5/6 kinase-2 (GmITPK2) as a dehydration and salinity stress regulator in Glycine max (L.) Merr. through heterologous expression in E. coli

Phytic acid (PA) is implicative in a spectrum of biochemical and physiological processes involved in plant stress response. Inositol 1,3,4, Tris phosphate 5/6 kinase (ITPK), a polyphosphate kinase that converts Inositol 1,3,4 trisphosphate to Inositol 1,3,4,5/6 tetra phosphate, averting the inositol phosphate pool towards PA biosynthesis, is a key regulator that exists in four different isoforms in soybean. In the present study, in-silico analysis of the promoter region of ITPKs was done and among the four isoforms, promoter region of GmITPK2 showed the presence of two MYB binding elements for drought inducibility and one for ABA response. Expression profiling through qRT-PCR under drought and salinity stress showed higher expression of GmITPK2 isoform compared to the other members of the family. The study revealed GmITPK2 as an early dehydration responsive gene which is also induced by dehydration and exogenous treatment with ABA. To evaluate the osmo-protective role of GmITPK2, attempts were made to assess the bacterial growth on Luria Broth media containing 200 mM NaCl, 16% PEG and 100 μM ABA, individually. The transformed E. coli BL21 (DE3) cells harbouring the GmITPK2 gene depicted better growth on the media compared to the bacterial cells containing the vector alone. Similarly, the growth of the transformed cells in the liquid media containing 200 mM NaCl, 16% PEG and 100 μM ABA showed higher absorbance at 600 nm compared to control, at different time intervals. The GmITPK2 recombinant E. coli cells showing tolerance to drought and salinity thus demonstrated the functional redundancy of the gene across taxa. The purity and specificity of the recombinant protein was assessed and confirmed through PAGE showing a band of ∼35 kDa on western blotting using Anti- Penta His- HRP conjugate antibody. To the best of our knowledge, the present study is the first report exemplifying the role of GmITPK2 isoform in drought and salinity tolerance in soybean.