Manoj Kumar Panda

Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis

Callus cultures initiated from shoot base explants of Curcuma aromatica Salisb. were maintained on Murashige and Skoog (MS) media supplemented with 2 mg dm−3 2,4-dichlorophenoxyacetic acid alone or with 0.5 mg dm−3 kinetin. Plantlets were regenerated from 60 and 180-d-old callus on MS media supplemented with 3 mg dm−3 benzyladenine and 0.5 mg dm−3 α-naphthalene acetic acid. Approximately 8–10 plantlets were produced after 30–40 d of culture per 50 mg of callus inoculated. Out of 113 regenerants analyzed 85 plants were exclusively diploid and 28 were predominantly diploid revealing presence of polyploid nuclei. Frequency of polyploid cells were more in regenerants obtained from 180-d-old callus then from 6-d-old callus which might be attributed to the ageing of callus.

Micropropagation of Zingiber rubens and assessment of genetic stability through RAPD and ISSR markers

Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5–5.0 mg dm−3), indole-3-acetic acid (IAA; 0.5–2.0 mg dm−3), kinetin (KIN; 1.0–3.0 mg dm−3), naphthaleneacetic acid (NAA; 0.5–1.0 mg dm−3) and adenine sulphate (ADS; 80–100 mg dm−3). MS basal medium supplemented with 3 mg dm−3 BA and 0.5 mg dm−3 IAA was optimum for shoot elongation. The elongated shoots (1–2 cm) were transferred to multiplication medium containing 2 mg dm−3 BA, 1 mg dm−3 IAA and 100 mg dm−3 ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed.

Genetic Stability of Micropropagated Ginger Derived from Axillary Bud through Cytophotometric and RAPD Analysis

A protocol was developed for the in vitro propagation of ginger (Zingiber officinale) cv. Suprava using dormant axillary buds from unsprouted rhizomes. The dormant axillary buds embedded in the rhizome nodes were induced to sprout when cultured on MS medium supplemented with 6-benzyladenine (BA) alone (1 - 6 mg/l) or with a combination of BA (1 - 6 mg/l) and indole-3-acetic acid (IAA) (0.5, 1 mg/l). In vitro sprouted buds were transferred to the multiplication medium containing various combinations of auxins and cytokinins. MS basal medium supplemented with BA (1 mg/l), IAA (1 mg/l) and adenine sulfate (100 mg/l) was found optimum for the in vitro multiplication of shoots producing (8.2 ± 0.2) shoots from a single explant within 30 days of culture. The multiplication rate remained unchanged in subsequent subcultures. Rooting of shoots occurred in the same multiplication media. Upon transfer of the in vitro culture to ex vitro in pots, 96% of plants survived and established successfully under natural conditions. Tissue culture-raised plantlets of ginger could be conserved in vitro through subculturing at an interval of 4 months. The genetic stability of micropropagated clones was evaluated at regular intervals of 6 months up to 24 months in culture using cytophotometric estimation of 4C nuclear DNA content and random amplified polymorphic DNA (RAPD) analysis. Cytophotometric analysis revealed a unimodal distribution of the DNA content with a peak corresponding to the 4C value (23.1 pg), and RAPD analysis revealed monomorphic bands showing the absence of polymorphism in all fifty regenerants analyzed, thus confirming the genetic uniformity among in vitro grown somaclones of Z. officinale. This study is of commercial significance as axillary bud explants are available throughout the year for initiating a fresh culture of the elite ginger cv. Suprava to be used as a source of true-to-type disease-free planting material thereby minimizing the adverse effect of repeated subculturing from the same explant source.

Genetic diversity and gene differentiation among ten species of Zingiberaceae from Eastern India.

In the present study, genetic fingerprints of ten species of Zingiberaceae from eastern India were developed using PCR-based markers. 19 RAPD (Rapid Amplified polymorphic DNA), 8 ISSR (Inter Simple Sequence Repeats) and 8 SSR (Simple Sequence Repeats) primers were used to elucidate genetic diversity important for utilization, management and conservation. These primers produced 789 loci, out of which 773 loci were polymorphic (including 220 unique loci) and 16 monomorphic loci. Highest number of bands amplified (263) in Curcuma caesia whereas lowest (209) in Zingiber cassumunar. Though all the markers discriminated the species effectively, analysis of combined data of all markers resulted in better distinction of individual species. Highest number of loci was amplified with SSR primers with resolving power in a range of 17.4–39. Dendrogram based on three molecular data using unweighted pair group method with arithmetic mean classified all the species into two clusters. Mantle matrix correspondence test revealed high matrix correlation in all the cases. Correlation values for RAPD, ISSR and SSR were 0.797, 0.84 and 0.8, respectively, with combined data. In both the genera wild and cultivated species were completely separated from each other at genomic level. It also revealed distinct genetic identity between species of Curcuma and Zingiber. High genetic diversity documented in the present study provides a baseline data for optimization of conservation and breeding programme of the studied zingiberacious species.

Assessment of genetic diversity among some elite cultivars of ginger (Zingiber officinale Rosc.) using isozyme and protein markers

Protein and isozyme markers were used to characterize 20 elite cultivars of ginger. Four different isozymes (acid phosphatase, esterase, peroxidase and polyphenolic oxidase) were analyzed to assess the genomic relationship among 20 varieties of ginger. Buffer soluble protein was isolated and resolved in SDS-PAGE. A total of 108 types of proteins were resolved, out of which 33 were unique. A total of 85 bands were detected for 4 isozymes, among them only 3 were found to be unique. The dendrogram constructed based on pooled data (protein and isozyme) divided the varieties into two major clusters each containing 10 members, respectively. The varieties Singjhara and Waynad local were found to be the most closely related while Singjhara and Sleeva local were distantly apart. The present study gave an indication of usefulness of the isozyme and protein markers for genetic discrimination between different varieties of ginger. The result of the present investigation will help in future breeding program.