Cymbopogon is an important genus of family Poaceae, cultivated mainly for its essential oils which possess high medicinal and economical value. Several cultivars of Cymbopogon species are available for commercial cultivation in India and identification of these cultivars was conceded by means of morphological markers and essential oil constitution. Since these parameters are highly influenced by environmental factors, in most of the cases, it is difficult to identify Cymbopogon cultivars. In the present study, Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers were employed to discriminate nine leading varieties of Cymbopogon since prior genomic information is lacking or very little in the genus. Ninety RAPD and 70 ISSR primers were used which generated 63 and 69 % polymorphic amplicons, respectively. Similarity in the pattern of UPGMA-derived dendrogram of RAPD and ISSR analysis revealed the reliability of the markers chosen for the study. Varietal/cultivar-specific markers generated from the study could be utilised for varietal/cultivar authentication, thus monitoring the quality of the essential oil production in Cymbopogon. These markers can also be utilised for the IPR protection of the cultivars. Moreover, the study provides molecular marker tool kit in both random and simple sequence repeats for diverse molecular research in the same or related genera.
Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.
Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial. Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m 2 s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA 3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA 3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.
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