Maheshwar Prasad Sharma

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.

Difference in in vitro response and esculin content in two populations of Taraxacum officinale Weber

In vitro micropropagation has been achieved in medicinally important plant, Taraxacum officinale collected from two different regions, Kashmir (J & K) and Garhwal (Uttarakhand). Leaf segments inoculated on MS supplemented with different combinations of Indole-3-acetic acid (IAA) and Benzyladenine (BA) produced indirect regeneration. For root induction MS fortified with Indole-3-butyric acid (IBA) was used. Taraxacum officinale collected from Garhwal responded two weeks earlier and showed shoot regeneration whereas in Kashmir population only callus proliferation occurred. Esculin content was also higher in the samples from Garhwal. The content was affected by both, the hormone concentration as well as age of the cultures. RAPD of the in vitro raised regenerants confirmed genetic stability.

Immunodiagnostics for Cherry virus A and Cherry necrotic rusty mottle virus

Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft transmitted viruses in the family Betaflexiviridae, infecting cherry. It is believed that CVA causes severe symptoms and disease in combination with other stone fruit viruses in both cherry and non-cherry hosts while CNRMV infected tree show reduced growth, significant yield loss and early death. Detection of these viruses is crucial for their sanitation, indexing and certificate programmes. In this study, a polyclonal antibody was produced against recombinant coat protein (CP) of CVA and CNRMV expressed in Escherichia coli for the development serological based diagnostics. The coat protein gene of CVA and CNRMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specifically designed primer pair with restriction sites at both ends and further cloned and expressed in bacterial expression vector (pET32a). CP coding region of both the viruses was expressed separately and successfully as a fusion protein. The fusion proteins were purified and directly used for rabbit immunizations. Antibodies were purified, conjugated with alkaline phosphatase and used in DAS-ELISA. A total of 74 and 43 samples were checked for CVA and CNRMV, respectively. In this analysis, 40/74 samples were found positive for CVA and 20/43 tested samples were positive for CNRMV by DAS-ELISA, further confirmed by RT-PCR. Antibodies raised against recombinant CNRMV CP also detected the virus consistently in western blot analysis with high sensitivity and specificity. IC-RT-PCR was also developed for the detection of CVA using the produced antibody. To the best of our knowledge, this is the first report of DAS-ELISA and IC-RT-PCR based diagnostics for CVA, and first report of DAS-ELISA based diagnostics for CNRMV from India.

Correlation between genetic diversity, phenotype and Esculin content in Taraxacum officinale Weber

Attempts were made to correlate the secondary metabolite content with genetic diversity in Taraxacum . In the present study, esculin, one of the important cytotoxic coumarins was analysed in populations of Taraxacum officinale collected from Districts Baramullah (Jammu and Kashmir) and Chamoli Garhwal (Uttarakhand) respectively. The two populations differed in a number of morphological and phenotypic characters. Genetic profiling of esculin quantified individuals was done using RAPD markers for the identification of high esculin yielding genotypes.