Variability analysis in ginger (Zingiber Officinale Rosc) somaclones using molecular markers
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Date
2013
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Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara
Abstract
Ginger (Zingiber officinale Rosc.), an important spice crop grown in India, is much valued for its flavour and medicinal properties. As natural variability available in the crop is limited, somaclonal variation is being utilized for crop improvement programmes. Currently, molecular marker techniques are widely employed to detect and assess somaclonal variation in several crop species as they are stable, detectable in all tissues and are not confounded by environment, pleiotropic and epistatic effects. The present investigations on “variability analysis in ginger (Zingiber officinale Rosc.) somaclones using molecular markers” were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University during February 2012 to May 2013. The objectives of the investigations were to assess somaclonal variation in ginger at molecular level, to study the influence of genotype and mode of regeneration on somaclonal variation, to assess the extent of variability in somaclones from the original source parent cultivars and to select the variants. Two molecular marker systems viz. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were utilized for the analyses. Ginger somaclones (180 Nos.) regenerated through various modes of regeneration viz. bud culture, indirect organogenesis / embryogenesis and in vitro mutagenesis, along with two source parent cultivars (Maran and Rio-de-Janeiro) were used for the present study. The genomic DNA was extracted from somaclones using CTAB method (Rogers and Bendich, 1994) and Sigma’s GenEluteTM Plant Genomic DNA Miniprep kit. The somaclones were grouped as per genotype and mode of regeneration. DNA extracted from individual somaclones was bulked as per the procedure reported by Dulson (1998). Bulked DNA samples of the thirteen groups of somaclones along with two source parent cultivars were subjected to RAPD and ISSR analyses with selected primers. Of the 35 RAPD primers screened, twelve gave good amplification. RAPD analysis using selected primers produced 129 amplicons, 44 were polymorphic with an average of 3.66 polymorphic bands / primer and a polymorphism percentage of 34.10. In ISSR assay, twelve selected primers produced 122 amplicons, 32 were polymorphic with an average of 2.66 polymorphic bands / primer and a polymorphism percentage of 26.23. The study could identify certain specific RAPD and ISSR primers for identification of Maran and Rio-de-Janeiro cultivars and irradiated mutants from non- irradiated somaclones. The dendrograms generated based on RAPD and ISSR profiles grouped the somaclones into two separate clusters, with somaclones of Maran in first subcluster of cluster I and somaclones of Rio-de-Janeiro in second subcluster of cluster I. The regenerants from Rio-de-Janeiro calli irradiated with 20 Gy and somatic embryogenic calli irradiated with 10 Gy formed the second cluster. RAPD and ISSR marker systems showed that somaclones derived from cultivar Maran exhibited more variability than Rio-de-Janeiro. RAPD marker system was more effective for bringing out variability. The variability observed in RAPD assay was 28 per cent while in ISSR assay it was 21 per cent and in the combined it was 25 per cent. In groupwise variability analysis using bulked DNA, the groups RC20 Gy and RSe10 Gy recorded higher variability from source parent cultivar. The variability exhibited in plantwise analysis using three selected primers (OPA 28, S11 and ISSR 05) was found very high (39%) as compared to groupwise analysis (25%). The somaclone RC2Kr1031 of the callus regenerants and RSe1Kr1052 of the somatic embryo regenerants showed more variability exhibiting 59 and 53 per cent variability respectively from the source parent cultivar Rio-de-Janeiro. The extent of variability in ginger somaclones could be assessed using molecular markers and in vitro mutagenesis could be employed to widen the genetic base in ginger.
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