DNA methylation and methylation polymorphism in ecotypes of Jatropha curcas L. using methylation-sensitive AFLP markers.

We investigated DNA methylation and polymorphism in the methylated DNA using AFLP based methylation-sensitive amplification polymorphism (MS-AFLP) markers in ecotypes of Jatropha curcas L. growing in similar and different geo-ecological conditions. Three ecotypes growing in different geo-ecological conditions with environmental heterogeneity (Group-1) and five ecotypes growing in similar environmental conditions (Group-2) were assessed. In ecotypes growing in group-1, 44.32 % DNA was methylated and of which 93.59 % DNA was polymorphic. While in group-2, 32.27 % DNA was methylated, of which 51.64 % DNA was polymorphic. In site 1 and site 2 of group-1, overall methylation was 18.94 and 22.44 % respectively with difference of 3.5 %, while overall polymorphism was 41.14 and 39.23 % with a difference of 1.91 %. In site 1 and site 2 of group-2, overall methylation was 24.68 and 24.18 % respectively with difference of 0.5 %, while overall polymorphism was 12.19 and 12.65 % with a difference of 0.46 %. The difference of methylation percentage and percentage of methylation polymorphism throughout the genome of J. curcas at site 1 and 2 of group-1 is higher than that of J. curcas at site 1 and 2 of group-2. These results correlated the physico-chemical properties of soil at these sites. The variations of physico-chemical properties of soil at Chorwadla (site 1 in group-1 and site 2 in group-2) compared to the soil at Brahmapur (site 2 in group-1) is higher than that of soil at Neswad (site 1 in group-2). The study suggests that these homologous nucleotide sequences probably play important role in ecotype adaptation to environmental heterogeneity by creating epiallelic variations hence in evolution of ecotypes/clines or forms of species showing phenotypic/genotypic differences in different geographical areas.

Assessment of changes in DNA methylation by methylation-sensitive amplification polymorphism in Jatropha curcas L. subjected to salinity stress.

The present study assesses the changes in DNA methylation in leaf and root tissues of Jatropha curcas L., induced by salinity stress using methylation sensitive amplification polymorphism (MSAP) markers. Seedlings of 21 days (d) grown under controlled conditions were subjected to 0­100 mM salinity treatment for 24 h (1 d). Immediate changes in DNA methylation and polymorphism in methylated DNA in whole genome of both leaves and roots were assessed using 10 selective combinations of MSAP primers. In root and leaves 70.06% and 57.89% methylation was observed respectively. Similarly 67.22% and 71.21% polymorphism was observed in methylated DNA from root and leaf tissues respectively. Compared with control, the percentage of methylation and methylation polymorphism in roots of plants under different dosages of salinity was found in the order of 50 mM < 25 mM = 100 mM < 75 mM and 75 mM < 25 mM < 50 mM < 100 mM respectively. Similarly percentage of methylation and methylation polymorphism in leaves of plants treated with different levels of salinity was found in order of 75 mM < 25 mM < 50 mM < 100 mM and 50 mM < 25 mM < 100 mM < 75 mM respectively. The MSAP analysis showed that under salt stress homologous nucleotide sequences in genome from control and salt treated plants of J. curcas showed different patterns of methylation; which suggest that these fragments probably play an important role to induce immediate adaptive responses in Jatropha under salinity stress.

Molecular characterization of intra-population variability of Jatropha curcas L. using DNA based molecular markers.

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity of J. curcas germplasm. In the present study, efforts were made to analyze the genetic diversity among the elite germplasms of J. curcas, selected on the basis of their performance in field using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The plants were selected on the basis of height, canopy circumference, number of seeds per fruit, weight of 100 seeds, seed yield in grams per plant and oil content. Out of 250 RAPD (with 26 primers), 822 AFLP (with 17 primers) and 19 SSR band classes, 141, 346 and 7 were found to be polymorphic, respectively. The percentage polymorphism among the selected germplasms using RAPD, AFLP and SSR was found to be 56.43, 57.9, and 36.84, respectively. The Jaccard's similarity coefficient was found 0.91, 0.90 and 0.91 through RAPD, AFLP and SSR marker systems, respectively. Principle component analysis (PCA) and dendrogarm analysis of genetic relationship among the germplasm using RAPD, AFLP and SSR data showed a good correlation for individual markers. The germplasm JCC-11, 12, 13, 14 and 15 whose yield found to be high were clustered together in dendrogram and PCA analysis though JCC11 is geographically distinct from others. In overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and JCC 6 and JCC10 (in SSR) were found genetically diverse. Characterization of geographically distinct and genetically diverse germplasms with varied yield characters is an important step in marker assisted selection (MAS) and it can be useful for breeding programs and QTL mapping.

Assessment of genetic stability and instability of tissue culture-propagated plantlets of Aloe vera L. by RAPD and ISSR markers.

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

Plantlet regeneration from callus cultures of selected genotype of Aloe vera L.--an ancient plant for modern herbal industries.

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of L-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, L-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.