Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly ...Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl<sub>2</sub>, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.展开更多
Embelia ribes Burm f., also known as Vidanga or Baibidanga, belonging to the family of Myrsinaceae, is an important but vulnerable medicinal woody climber. Recent survey and observations of E. ribes in different aspec...Embelia ribes Burm f., also known as Vidanga or Baibidanga, belonging to the family of Myrsinaceae, is an important but vulnerable medicinal woody climber. Recent survey and observations of E. ribes in different aspects like distribution, population structure, growth habit, climate, natural regeneration and seedling ecology in Karnataka have been thoroughly discussed. This is the first report on artificial regeneration through seeds of diverse origins in ex-situ and in situ conditions and field planting of them in its natural environment. Field planting of in vitro and nursery raised seedlings showed better field performance in terms of survival and growth in its natural growing areas only. Current studies and observations have shown that this species has low ecological gradient and is a “habitat specialist”. E. ribes being a threatened species with small populations coupled with low ecological gradient and virtually no natural regeneration, a modified approach of quasi in situ conservation where in in-situ raising of seedlings of diverse origin with an aim to enrich the diversity of existing population was attempted with considerable success.展开更多
Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extr...Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.展开更多
文摘Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl<sub>2</sub>, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.
文摘Embelia ribes Burm f., also known as Vidanga or Baibidanga, belonging to the family of Myrsinaceae, is an important but vulnerable medicinal woody climber. Recent survey and observations of E. ribes in different aspects like distribution, population structure, growth habit, climate, natural regeneration and seedling ecology in Karnataka have been thoroughly discussed. This is the first report on artificial regeneration through seeds of diverse origins in ex-situ and in situ conditions and field planting of them in its natural environment. Field planting of in vitro and nursery raised seedlings showed better field performance in terms of survival and growth in its natural growing areas only. Current studies and observations have shown that this species has low ecological gradient and is a “habitat specialist”. E. ribes being a threatened species with small populations coupled with low ecological gradient and virtually no natural regeneration, a modified approach of quasi in situ conservation where in in-situ raising of seedlings of diverse origin with an aim to enrich the diversity of existing population was attempted with considerable success.
文摘Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f;verbanaceae), Black Rosewood (Dalbergia latifolia f;Fabaceae) Ben Teak (Lagerstroemia lanceolata f;Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.
