This document describes a study that developed an improved method for isolating high-quality DNA from Cinnamomum species. The standard CTAB method was modified by adding higher salt concentrations and activated charcoal to remove polysaccharides and polyphenols that can interfere with DNA extraction from these plants. The new method was tested on four Cinnamomum species and other woody plants, and yielded DNA that could be successfully amplified by PCR and other molecular techniques. This efficient DNA isolation protocol will support further molecular studies of Cinnamomum and other plants containing high levels of secondary metabolites.
The International Journal of Engineering and Science (IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
Hepatoprotective Activity of Cinnamon Zeylanicum Leaves against Alcohol Induc...IJERA Editor
Plants play an important role in the life of human, as the major source of food, as well as for the maintenance and improvement of health and for the elimination of the enemies since ages. Plants are the basic source of knowledge of modern medicine. The present study was conducted to evaluate the hepatoprotective activity of aqueos extract of aerial parts of Cinnamon zeylanicum are evaluated in alcohol induced hepatotoxicity in albino rats. Silymarin (100mg/kg) was given as reference standard. The aqueos extract of aerial parts of Cinnamon zeylanicum have shown very significant hepatoprotection against alcohol induced hepatotoxicity in albino rats in reducing SGOT, SGPT, Alkaline phosphatase (ALP) and GGT and levels of total bilirubin and total protein were investigated and showed an increase in alcohol induced rats when compared to control. The extracts of the test plant exhibited significant (p < 0.05) hepatoprotective activity against the alcohol induced liver models by improving liver function which was indicated by reduction in the levels of SGOT, SGPT, ALP, GGT, total bilirubin and total protein.
A Reliable and High Yielding Method for Isolation of Genomic DNA from Ammi MajusSandip Magdum
The developed protocol describes a cheaper, quicker and reliable method for the isolation of pure DNA from medicinal herbs, such as Ammi majus, which produces the secondary metabolites xanthotoxin and berganpectane having immense medicinal importance. Use of CTAB, liquid nitrogen and EDTA in different isolation protocols analyzed for A. majus, all were ended with polysaccharide and protein contamination with low purity of DNA (A260/280=1.3-1.6), revealed a need for method modification for the inexpensive and rapid isolation of pure DNA. Developed reliable and competent protocol isolated enough pure DNA (A260/280=1.81) without following time consuming lengthy steps and hazardous chemicals used in other protocols, which increase experimental costs, risk, and need expertise to perform. The explained protocol requires few chemicals and little time to obtain pure DNA having yield 688 μg/g of A. majus. A higher quantity of isolated DNA obtained from young fresh leaf samples than from either the callus or stem. A. majus is a pharmaceutically important medicinal herb, and the present protocol aids in the analysis and modification of its genes.
D4476, a cell-permeant inhibitor of CK1, potentiates the action of Bromodeoxy...Atai Rabby
To elucidate the mechanism of bromodeoxyuridine (BrdU) induced cellular senescence, we treated HeLa cells with D4476, a potent and specific inhibitor of casein kinase 1(CK1). We found that D4476 (10µM) treatment could arrest cell growth at G1 stage and induced cellular senescence when treated together with BrdU (10µM). However neither D4476 nor BrdU can induce cellular senescence alone, at a concentration of 10µM. These results suggest that the targets of CK1 may be involved in maintaining normal cellular process and their inactivation potentiates BrdU to induce senescence like phenomena.
Inter Simple Sequence Repeats (ISSR) markers were utilized to identify the levels of heritable varieties and patterns of the populace structure among the five populaces of Pteris biaurita, a natural fern in India. A comprehensive examination was directed in three replicates at 2013-14 seasons in the Western Ghats, South India. Five wild P. biaurita, accessions (maiden hair) were assessed for genotyping studies. Results demonstrated a pivotal discrepancy among genotypes for they were characterized in view of this uniqueness in four groups by the genetic cluster examination. In this trial, ISSR primers amplified 63 polymorphic groups. In view of the genetic identity data, genotypes were figured and differed from 0.5714 to 0.6984. The percentage of polymorphism indicated predominant genotype that may be utilized for the conservation of species. ISSR appeared to be an obliging marker for prediction of genotype inside a closed group of inter specific populace in the investigation territory
The International Journal of Engineering and Science (IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
Hepatoprotective Activity of Cinnamon Zeylanicum Leaves against Alcohol Induc...IJERA Editor
Plants play an important role in the life of human, as the major source of food, as well as for the maintenance and improvement of health and for the elimination of the enemies since ages. Plants are the basic source of knowledge of modern medicine. The present study was conducted to evaluate the hepatoprotective activity of aqueos extract of aerial parts of Cinnamon zeylanicum are evaluated in alcohol induced hepatotoxicity in albino rats. Silymarin (100mg/kg) was given as reference standard. The aqueos extract of aerial parts of Cinnamon zeylanicum have shown very significant hepatoprotection against alcohol induced hepatotoxicity in albino rats in reducing SGOT, SGPT, Alkaline phosphatase (ALP) and GGT and levels of total bilirubin and total protein were investigated and showed an increase in alcohol induced rats when compared to control. The extracts of the test plant exhibited significant (p < 0.05) hepatoprotective activity against the alcohol induced liver models by improving liver function which was indicated by reduction in the levels of SGOT, SGPT, ALP, GGT, total bilirubin and total protein.
A Reliable and High Yielding Method for Isolation of Genomic DNA from Ammi MajusSandip Magdum
The developed protocol describes a cheaper, quicker and reliable method for the isolation of pure DNA from medicinal herbs, such as Ammi majus, which produces the secondary metabolites xanthotoxin and berganpectane having immense medicinal importance. Use of CTAB, liquid nitrogen and EDTA in different isolation protocols analyzed for A. majus, all were ended with polysaccharide and protein contamination with low purity of DNA (A260/280=1.3-1.6), revealed a need for method modification for the inexpensive and rapid isolation of pure DNA. Developed reliable and competent protocol isolated enough pure DNA (A260/280=1.81) without following time consuming lengthy steps and hazardous chemicals used in other protocols, which increase experimental costs, risk, and need expertise to perform. The explained protocol requires few chemicals and little time to obtain pure DNA having yield 688 μg/g of A. majus. A higher quantity of isolated DNA obtained from young fresh leaf samples than from either the callus or stem. A. majus is a pharmaceutically important medicinal herb, and the present protocol aids in the analysis and modification of its genes.
D4476, a cell-permeant inhibitor of CK1, potentiates the action of Bromodeoxy...Atai Rabby
To elucidate the mechanism of bromodeoxyuridine (BrdU) induced cellular senescence, we treated HeLa cells with D4476, a potent and specific inhibitor of casein kinase 1(CK1). We found that D4476 (10µM) treatment could arrest cell growth at G1 stage and induced cellular senescence when treated together with BrdU (10µM). However neither D4476 nor BrdU can induce cellular senescence alone, at a concentration of 10µM. These results suggest that the targets of CK1 may be involved in maintaining normal cellular process and their inactivation potentiates BrdU to induce senescence like phenomena.
Inter Simple Sequence Repeats (ISSR) markers were utilized to identify the levels of heritable varieties and patterns of the populace structure among the five populaces of Pteris biaurita, a natural fern in India. A comprehensive examination was directed in three replicates at 2013-14 seasons in the Western Ghats, South India. Five wild P. biaurita, accessions (maiden hair) were assessed for genotyping studies. Results demonstrated a pivotal discrepancy among genotypes for they were characterized in view of this uniqueness in four groups by the genetic cluster examination. In this trial, ISSR primers amplified 63 polymorphic groups. In view of the genetic identity data, genotypes were figured and differed from 0.5714 to 0.6984. The percentage of polymorphism indicated predominant genotype that may be utilized for the conservation of species. ISSR appeared to be an obliging marker for prediction of genotype inside a closed group of inter specific populace in the investigation territory
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Abstract
Objective(s):
Gold nanoparticles (GNPs) command a great deal of attention for biomedical applications nowadays. The data about the degree of toxicity and the accumulation of gold nanoparticles in-vivo is not enough to judge.
Materials and Methods:
A total of 32 healthy male Wistar rats were randomly divided into 4 including: three GNP-treated and one control group. Groups 1, 2 and 3 received 0.5 cc of a solution containing 5, 10, and 100 ppm Au daily via intraperitoneal (IP) injection for 7 days, respectively. The control group was treated with 0.5 cc normal saline with same procedure. Then, several biochemical parameters such as serum glutamate oxaloacetat transaminase (SGOT) and serum glutamate pyrvate transaminase (SGPT) were evaluated at 2, 7 and 14 days after the last injection. After 14 days, all the rats were sacrificed and liver, lung tissues were separated and evaluated.
Results:
SGOT two days after intervention was significantly greater in the group 2 than the control group. In liver histological assessment, in group 1, basophils were observed around the central veins, in group 2 fading and no observation of central veins was seen, and in group 3 hepatic damage was noticed. The lung histological results showed severe vascular hyperemia in group 1, air sacs damage in group 2, and complete air sacs destruction in group 3.
Conclusion:
The results showed extreme changes in the histopathology of lung and liver tissues caused by spherical nanogold with 5-10 nm size in all of three treatment groups.
The HortFlora Research Spectrum (HRS), is an international-peer reviewed, open access journal that serves as a forum for the exchange and dissemination of R & D advances and innovations in all facets of Horticultural Science (Pomology, Olericulture, Floriculture, Post Harvest Technology, Plant Biotechnology, and Medicinal & Aromatic Plants etc.) and its allied branches on an international level.
HRS is officially published quarterly (March, June, September and December) every year, in English (print & online version), under the keen auspices of Biosciences & Agriculture Advancement Society (BAAS), Meerut (India).
Aims & Scope
The main objective of the journal ‘HortFlora Research Spectrum’ is to serve as a platform to promote, publish and disseminate the R & D innovations and advances in all aspects of Horticultural Sciences and allied branches of botanical sciences & technology, and to facilitate closer interaction among the academicians, researchers and entrepreneurs at global scale.
The journal HortFlora Research Spectrum (HRS), having International impact (ICV: 27.39; GIF: 0.364, IBI Factor: 2.8; NJIF: 2.14)), publishes high quality peer reviewed/refereed original research papers, review articles and research notes on all aspects of Horticultural plants’ research including agronomic management, plant nutrition, biotechnology, crop improvement, plant protection, plant physiology, cell & molecular biology, medicinal & aromatic plants, food & nutrition science, agroforestry, environmental science, plant medicinal properties, ethno-phytomedicine, technology dissemination etc.
Website: www.hortflorajournal.com
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
Detection of Genetic variation in tissue culture clones of date palm using IS...IJSRD
Date palm is a plant having high nutritional value and long life (yielding up to 100 years). Phoenix dactylifera requires 2-5 males for pollination of 100 females’ plant depending up on genetic and environment factors. Therefore paternity variation expected to very low according to PCR based techniques, Even though we have tried to find out genetic variation among tissue culture cloned plant. Tissue culture technique can be used for genetic improvement of date palm. The main purpose of this study was to evaluate the genetic variation in the tissue culture clones of date palm by using ISSR primers among mother and it’s two clones. The plant DNA was extracted and subjected to detection of genetic variation in two groups of date palm using ISSR primers. In this study ISSR primers produced monomorphic bands within group-1 and group-2. Genetic variation in tissue culture clones of date palm was not detecte by UBC primer series.
Genetic diversity in pea germplasm using RAPD MarkersShujaul Mulk Khan
Selection of the genotypes using plasmid assisted technology provides an efficient and useful tool for elaborating genetic relationships among genotypes. In present study, 48 Pea (Pisum sativum var sativum L.) genotypes obtained from different sources were analyzed through 20 RAPD, DNA markers for assessment of intraspecific DNA variations. Results revealed that significant variations were present in minor bands. Major bands also showed significant diversity. Considerable variations were also recorded in density of some common bands. Maximum and minimum genetic diversity i.e., 80% and 20% was found among 08 and 23 comparisons, respectively from banding profile. These variations can be
used further for enhancing variability, a prerequisite for crop breeding. Phylogenetic clustering (through dendrogram analysis) of genotypes revealed that genetic diversity is independent of origin of genotypes. Forty eight genotypes of pea clustered in three main groups A, B and C comprising 23, 5 and 20 genotypes, respectively. Group A1 and C1 included the most distantly related genotypes and hence can be recommended for breeding to obtain genetically diverse segregating populations.
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Abstract
Objective(s):
Gold nanoparticles (GNPs) command a great deal of attention for biomedical applications nowadays. The data about the degree of toxicity and the accumulation of gold nanoparticles in-vivo is not enough to judge.
Materials and Methods:
A total of 32 healthy male Wistar rats were randomly divided into 4 including: three GNP-treated and one control group. Groups 1, 2 and 3 received 0.5 cc of a solution containing 5, 10, and 100 ppm Au daily via intraperitoneal (IP) injection for 7 days, respectively. The control group was treated with 0.5 cc normal saline with same procedure. Then, several biochemical parameters such as serum glutamate oxaloacetat transaminase (SGOT) and serum glutamate pyrvate transaminase (SGPT) were evaluated at 2, 7 and 14 days after the last injection. After 14 days, all the rats were sacrificed and liver, lung tissues were separated and evaluated.
Results:
SGOT two days after intervention was significantly greater in the group 2 than the control group. In liver histological assessment, in group 1, basophils were observed around the central veins, in group 2 fading and no observation of central veins was seen, and in group 3 hepatic damage was noticed. The lung histological results showed severe vascular hyperemia in group 1, air sacs damage in group 2, and complete air sacs destruction in group 3.
Conclusion:
The results showed extreme changes in the histopathology of lung and liver tissues caused by spherical nanogold with 5-10 nm size in all of three treatment groups.
The HortFlora Research Spectrum (HRS), is an international-peer reviewed, open access journal that serves as a forum for the exchange and dissemination of R & D advances and innovations in all facets of Horticultural Science (Pomology, Olericulture, Floriculture, Post Harvest Technology, Plant Biotechnology, and Medicinal & Aromatic Plants etc.) and its allied branches on an international level.
HRS is officially published quarterly (March, June, September and December) every year, in English (print & online version), under the keen auspices of Biosciences & Agriculture Advancement Society (BAAS), Meerut (India).
Aims & Scope
The main objective of the journal ‘HortFlora Research Spectrum’ is to serve as a platform to promote, publish and disseminate the R & D innovations and advances in all aspects of Horticultural Sciences and allied branches of botanical sciences & technology, and to facilitate closer interaction among the academicians, researchers and entrepreneurs at global scale.
The journal HortFlora Research Spectrum (HRS), having International impact (ICV: 27.39; GIF: 0.364, IBI Factor: 2.8; NJIF: 2.14)), publishes high quality peer reviewed/refereed original research papers, review articles and research notes on all aspects of Horticultural plants’ research including agronomic management, plant nutrition, biotechnology, crop improvement, plant protection, plant physiology, cell & molecular biology, medicinal & aromatic plants, food & nutrition science, agroforestry, environmental science, plant medicinal properties, ethno-phytomedicine, technology dissemination etc.
Website: www.hortflorajournal.com
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
Detection of Genetic variation in tissue culture clones of date palm using IS...IJSRD
Date palm is a plant having high nutritional value and long life (yielding up to 100 years). Phoenix dactylifera requires 2-5 males for pollination of 100 females’ plant depending up on genetic and environment factors. Therefore paternity variation expected to very low according to PCR based techniques, Even though we have tried to find out genetic variation among tissue culture cloned plant. Tissue culture technique can be used for genetic improvement of date palm. The main purpose of this study was to evaluate the genetic variation in the tissue culture clones of date palm by using ISSR primers among mother and it’s two clones. The plant DNA was extracted and subjected to detection of genetic variation in two groups of date palm using ISSR primers. In this study ISSR primers produced monomorphic bands within group-1 and group-2. Genetic variation in tissue culture clones of date palm was not detecte by UBC primer series.
Genetic diversity in pea germplasm using RAPD MarkersShujaul Mulk Khan
Selection of the genotypes using plasmid assisted technology provides an efficient and useful tool for elaborating genetic relationships among genotypes. In present study, 48 Pea (Pisum sativum var sativum L.) genotypes obtained from different sources were analyzed through 20 RAPD, DNA markers for assessment of intraspecific DNA variations. Results revealed that significant variations were present in minor bands. Major bands also showed significant diversity. Considerable variations were also recorded in density of some common bands. Maximum and minimum genetic diversity i.e., 80% and 20% was found among 08 and 23 comparisons, respectively from banding profile. These variations can be
used further for enhancing variability, a prerequisite for crop breeding. Phylogenetic clustering (through dendrogram analysis) of genotypes revealed that genetic diversity is independent of origin of genotypes. Forty eight genotypes of pea clustered in three main groups A, B and C comprising 23, 5 and 20 genotypes, respectively. Group A1 and C1 included the most distantly related genotypes and hence can be recommended for breeding to obtain genetically diverse segregating populations.
Background: Dennentia tripetalla (Pepper Fruit) belongs to the Annonaceae family and is abundant in Nigeria. Its
fruit in folklore medicine is used for treatment of varying ailments. While ample research evidence exists on the
plants fruit and seed, no current study exists on the toxicological profile of the plant leaves.
Methods: qualitative and quantitative phytochemicals and In vitro antioxidant assays were carried out using
standard methods. The acute toxicity study indicates that the LD50 was higher than 2000 mg/Kg body weight. Subchronic
toxicity studies was carried out using five groups of rats. Group 1 served as control, 2–5 received 100 mg/
Kg, 200 mg/Kg, 500 mg/Kg and 1000 mg/Kg body weight orally for 28 days.
Results: Post-administration biochemical analysis indicates there was increased weight in rats administered 100
mg/kg and 200 mg/kg while it reduced in the 500 mg/kg group. Significant elevations of liver function markers
were reported for 200 mg/kg and 500 mg/kg respectively. Serum and hepatic protein profiles remained unaltered.
Renal function analysis revealed elevated serum urea and creatinine for 200 and 500 mg/kg groups, elevated serum
Na+ and Ca+ and reduced serum Cl− for the 500 mg/Kg group. Elevated Kidney K+ and Ca+ levels, reduced Cl−
were significantly observed in 500 mg/Kg group. Significant rise in hepatic and renal lipid peroxidation was
observed in 200 and 500 mg/Kg groups. There were observed disarmament of the antioxidant defense systems
occasioned by rise and drop in tissue (hepatic, renal, testes, heart) Superoxide dismutase (SOD), Catalase (Cat),
Glutathione-s-transferase (GST), Glutathione peroxidase (GPx) activities in the test groups relative to control.
Histopathological examination indicated architectural aberrations at 500 and 1000 mg/kg.
Conclusions: It concluded that the plant had significant phytochemical and antioxidant properties of medical
interest and possessed toxic properties in rats when administered at a dose above 200 mg/Kg over a prolonged
period of time.
Effect of interaction between different plant growth regulators on in vitro s...Agriculture Journal IJOEAR
— In this paper a shoot multiplication is described for Citrus latifolia Tan. (persian lime) using nodal segment explants of young one – old – year trees by two different pathways contain with and without callusing phase. The best result for multiple shoot formation and regenerated shoot formation was 3.2 and 2.6 shoots per explants with 4.44 µM BA plus 0.053 µM NAA and 4.44 µM BA plus 0.049 µM IBA respectively. Alike shoot regeneration, shoot elongation was occurred in medium with 4.44 µM BA and 0.049 µM IBA. Micropropagated and regenerated plants are under other experiments. Abbreviation: BA – 6 benzylaminopurine; IBA – Indole acetic acid; NAA – Naphtalene acetic acid; PGRs – Plant Growth Regulators.
DNA Fingerprinting and Phylogenetic Relationship of the Genus Chlorophytum Ke...YogeshIJTSRD
Chlorophytum Ker Gawl, is a medicinally important plant genus employed since ancient time as a key component in Ayurvedic and Unani medicine. Genus represented with more than 217 species out of which 17 species have been reported from India. The main objective of this study is to evaluate molecular phylogeny of Chlorophytum species. In this study phylogenetic analysis of Chlorophytum species was carried out using AFLP marker. Total 16 selective primer combinations were scored as presence and absence of alleles for all the 17 species, resulting in total 938 allele, out of which 291 allele were found to be polymorphic. The percentage of polymorphism ranged from 18.3 in the combination E2M3 to 42 in the combination E1M1 .The phylogenetic tree is divided into two clade, each clade contains species with similar morphological characters. The extent of variations within species is discussed. Kale KA | Pohekar PV | Malode UA | Lakhe M. B "DNA Fingerprinting and Phylogenetic Relationship of the Genus Chlorophytum Ker -Gawl, from India using AFLP" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-3 , April 2021, URL: https://www.ijtsrd.com/papers/ijtsrd39932.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/39932/dna-fingerprinting-and-phylogenetic-relationship-of-the-genus-chlorophytum-ker-gawl-from-india-using-aflp/kale-ka
S M Masiul Azam, Md Shahidul Islam, Parvin Shahanaz, Md Shafiqur Rahman and Sarder Md Shahriar Alam. “Molecular Characterization of Brassica Cultivars through RAPD Markers” United International Journal for Research & Technology (UIJRT) 1.3 (2019): 41-45.
1. Accepted Manuscript
Analytical Methods
Development of An effective and efficient DNA isolation method for Cinna-
momum species
B.S. Bhau, G. Gogoi, D. Baruah, R. Ahmed, G. Hazarika, B. Borah, B. Gogoi,
D.K. Sarmah, S.C. Nath, S.B. Wann
PII: S0308-8146(15)00717-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.05.004
Reference: FOCH 17548
To appear in: Food Chemistry
Received Date: 8 January 2015
Revised Date: 28 April 2015
Accepted Date: 1 May 2015
Please cite this article as: Bhau, B.S., Gogoi, G., Baruah, D., Ahmed, R., Hazarika, G., Borah, B., Gogoi, B., Sarmah,
D.K., Nath, S.C., Wann, S.B., Development of An effective and efficient DNA isolation method for
Cinnamomum species, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.05.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
2. DEVELOPMENT OF AN EFFECTIVE AND EFFICIENT DNA ISOLATION1
METHOD FOR CINNAMOMUM SPECIES2
3
RUNNING TITLE: EFFICIENT DNA ISOLATION FROM CINNAMOMUM4
Bhau BS*, Gogoi G, Baruah D, Ahmed R1
, Hazarika G#
, Borah B, Gogoi B, Sarmah DK,5
Nath SC, Wann SB1
,6
Plant Genomics Laboratory, Medicinal Aromatic & Economic Plants (MAEP) Division,7
CSIR-Northeast Institute of Science & Technology (CSIR-NEIST),8
Jorhat 785006, Assam, India9
1
Biotechnology Division,10
CSIR-Northeast Institute of Science & Technology (CSIR-NEIST),11
Jorhat 785006, Assam, India12
#
Present address - Diagnostic Genetic Lab., Department of Anatomy.13
Assam Medical College (AMC), Dibrugarh, Assam. 785002, India14
*Corresponding Author: Tel.: +91 376 2370121; fax: +91 376 2370011.15
E-mail address: bsbhau@gmail.com; bhaubs@rrljorhat.res.in (BS Bhau)16
17
3. 1
Abstract:18
Different species of Cinnamomum are rich in polysaccharide’s and secondary19
metabolites, which hinder the process of DNA extraction. High quality DNA is the pre-20
requisite for any molecular biology study. In this paper we report a modified method for high21
quality and quantity of DNA extraction from both lyophilized and non-lyophilized leaf22
samples. Protocol reported differs from the CTAB procedure by addition of higher23
concentration of salt and activated charcoal to remove the polysaccharides and polyphenols.24
Wide utility of the modified protocol was proved by DNA extraction from different woody25
species and 4 Cinnamomum species. Therefore, this protocol has also been validated in26
different species of plants containing high levels of polyphenols and polysaccharides. The27
extracted DNA showed perfect amplification when subjected to RAPD, restriction digestion28
and amplification with DNA barcoding primers. The DNA extraction protocol is reproducible29
and can be applied for any plant molecular biology study.30
Key Words: Cinnamomum, DNA isolation, CTAB, activated charcoal, DNA, PCR reaction,31
polyvinyl pyrrolidone32
33
4. 2
1. Introduction34
The Cinnamomum Schaeffer belongs to the family Lauraceae that comprises about35
250 species of trees and shrubs of tropics and sub-tropics (Willis, 1973; Leela 2008).36
Cinnamomum genus (cinnamon) is a very popular spice throughout the world. Out of 2637
species distributed in different parts of India (Hooker, 1885) 12 species are recorded from the38
North-Eastern Part of India (Kanjilal et al., 1940; Deb, 1983; Nath & Baruah, 1984).39
Cinnamon has a broad range of historical uses in different countries of the world and40
different cultures. The species C. zeylanicum Blume originates from Ceylon, being also41
native to Southeast India, are source of cinnamon bark and leaf and their essential oils. Its42
unique qualities are flavor, slightly sweet, pleasant, warm and bitter, besides being strongly43
aromatic (Thakur et al., 1989; Devi et al, 2007; Mir et al, 2004; Rana et al, 2012). Almost44
every part of the cinnamon spice is considered as a remedy for many diseases like45
respiratory, gastritis, menstrual problems, poor circulation, leucorrhea, digestive, diarrhea,46
nausea, gynecological aliments (vaginal yeast infection), dysentery (Chakraborty and Das,47
2010; Begum et al, 2013). Due to economic and ethno-botanical importance world wide, as48
well as, wide variability reported among the species of Cinnamomum, identification based on49
morphological characteristics alone is not sufficient. Cinnamomum needs a thorough insight50
studies that will prove more beneficial to the mankind. In this context, molecular techniques51
as well as several novel DNA-markers (RAPD, RFLP, SSR, ISSR etc.) in the field of biology52
have not only helped us to establish genetic relationship between the members of different53
taxonomic categories but also helped to study close genetic relationship among inter and54
intra-specific genetic variations. DNA based methods also have application in biological55
adulterant detection and authentication of a wide range of food and agricultural commodities56
(Dhanya and Sasikumar, 2010). Identification of true cinnamon from adulterant species based57
on physical traits is very difficult, and the situation is all the more difficult once the58
5. 3
commodity loses its physical form (e.g., powder). Incidentally, powdered bark is more59
frequently used as food flavor and in medicine.60
Plant biochemical profile are immensely influenced by the place of origin, processing61
of the plant tissue; age and tissue type and season (Xie and Leung, 2009; Bressan et al. 2014),62
while place of origin, processing of the plant tissue; age, tissue type and season have no63
influence on the DNA. Chemical markers are further prone to errors, since they need to be64
specific for the species, stable during storage and modification processes and should65
represent therapeutically relevant compound.66
In recent years, although immense progresses in molecular techniques have been67
made but, there are few successful reports available for isolation of pure nucleic acid from68
plant species containing high level of secondary metabolites (Doyle and Doyle 1990;69
Abeysinghe et al. 2009; Sharma and Purohit 2012; Sahu et al. 2012; Swetha et al. 2014).70
These secondary metabolites like polysaccharides and polyphenols severely affect the71
isolation procedure by interacting irreversively with nucleic acid and interfere with the72
function of enzymes in subsequent analysis (Demeke and Adams, 1992, Loomis 1974).73
Polysaccharides, due to their chemical properties, co-precipitate with genomic DNA, giving74
solutions a viscous, glue-like appearance (Porebski et al. 1997) and are known to inhibit75
proper functioning of the enzymes. Phenolics, such as terpenoids and tannins, undergo quick76
oxidation upon their release from leaf tissue and irreversibly bind to the phosphate backbone77
of DNA, characterized by the browning of leaf material (Sahu et al. 2012; Maliyakal, 1992).78
Both contaminants prevent the use of DNA for molecular biology purposes, such as PCR,79
restriction digests, or sequencing by inhibiting the action of polymerases or endonucleases80
(Khanuja et al. 1999). Although there are so many established protocols to isolate nucleic81
DNA from plant tissues, but with high content of secondary metabolites, very few are82
successful and reproducible.83
6. 4
A fast, simple, cost-effective and reliable method is a pre-requisite for any DNA84
extraction and subsequent downstream application. It has been reported that DNA extraction85
from tissues past the budding stage is problematic and also unstable under long-term storage86
(Lodhi et al., 1994). However, in cases when only older tissues are available, a proper87
procedure for selective extraction of DNA is needed. Many protocols have been used in plant88
DNA isolation, but because of the chemical heterogeneity of the species most of them could89
be applied to a limited number of species or even closely related species (Weishing et al.,90
1995). Cinnamomum plant is rich in polyphenols and secondary metabolites, which are major91
hurdles to precipitate purified and high quantity nucleic acids. Another problem that could92
arise during plant DNA isolation is the requirement of liquid nitrogen for crushing the plant93
material as reported in most of the protocols and involvement of lengthy procedure (Ouenzar94
et al., 1998). In the present study most of above-mentioned concerns have been addressed.95
The present investigation is therefore undertaken to standardize a protocol to extract intact96
high quality DNA from Cinnamomum sp. through testing the suitability of the available97
methods and making necessary modification for the purpose.98
2. Material and methods99
Fresh tender leaves of C. impressinervium Meissn., C. zeylanicum Blume and C.100
tamala Nees. & Eberm. were collected in zip lock bags from the pre-identified plots of101
experimental botanical garden of CSIR-NEIST and Assam Agricultural University, Jorhat,102
Assam, India. Keeping in view the criteria for the collection, fresh leaves were collected from103
the similar environmental conditions for DNA isolation studies. For comparing DNA104
concentrations in old and young leaves, the plant material was collected from the same plant.105
The collected leaf material was cleaned with distilled autoclaved water and external moisture106
from the leaves was allowed to air dry. Part of the sample was used fresh and the other half107
was lyophilized for 48 h at -110 °C and then kept in airtight bags till extraction.108
7. 5
2.1. DNA extraction109
For DNA extraction, 400 mg of the lyophilized leaf samples were grounded to110
powder in sterile mortar with silica powder and transferred powdered samples directly to the111
preheated extraction buffer (1.4 M NaCl, 100 mM Tris–HCl [pH 8.0], 20 mM EDTA [pH112
8.0], and 0.2% β-mercaptoethanol, 2% CTAB) and carefully mixed by hand and incubated at113
55 °C for 1 hr. with periodic shaking to avoid aggregation. Another set of experiment was114
conducted parallerly by taking 400 mg of fresh leaf sample and grind the sample to fine115
powder with the help of liquid nitrogen (Table 1). Both the grounded samples were then116
incubated in preheated extraction buffer at 55 °C for 1 hr. The extraction buffer was117
standardised by changing the concentration of one variable at one time i.e. activated charcoal,118
CTAB and Polyvinylpyrrolidone (PVP). Therefore twelve extraction buffers were prepared,119
where 0.5%, 0.7%, 1% and 3% activated charcoal were mixed with 2% CTAB and 2% PVP120
separately, 1%, 2%, 3% and 4% CTAB was mixed with 2% PVP and 0.7% activated charcoal121
separately and 1%, 2%, 3% and 4% PVP was mixed with 2% CTAB and 0.7% activated122
charcoal separately (Table 2). The remaining component of the extraction buffer were kept123
constant i.e. 1.4 M NaCl, 100 mM Tris–HCl [pH 8.0], 20 mM EDTA [pH 8.0], and 0.2% β-124
mercaptoethanol. After incubation of samples in different extraction buffers, the supernatant125
was extracted twice with chloroform: isoamyl-alcohol (24:1 v/v) after centrifugation at126
10,000 rpm at room temperature. To the supernatant extracted pre-chilled ethanol was added127
in equal volume to precipitate the DNA. The sample was centrifuged at 14,000 rpm for 5 min128
and the supernatant was discarded. The DNA pellet was air-dried for 2 h and then suspended129
in 100 µl of 10 mg Ribonuclease A (Sigma R642) in TE/RNase A buffer and incubated at 37130
°C for 30 min. To the incubated solution add equal volume of chloroform-isoamyl alcohol131
(24:1) and centrifuge at 12,000 g for 10 min. DNA was precipitated by the addition of cold132
isopropanol (0.5 Volume) and 100 µl of 2M NaCl to the solution followed by centrifugation133
8. 6
at 10000 rpm at 5 °C. The resulting DNA pellet was washed with 70% ethanol and allowed to134
air dry. The DNA pellet was re-suspended in 50 µL TE buffer and stored at 5 °C for further135
studies.136
To find out the wide applicability of the standardized protocol, 4 (Androgrephis137
paniculata, Litsea cubeba, Azadirachta indica, Cinnamomum camphora) different plant138
species were used for DNA extraction. In addition to this, our standardized DNA extraction139
method was compared with other four conventional methods (Doyle and Doyle 1987; Lee et140
al. 2010; Abeysinghe et al. 2009; Križman et al. 2006) for DNA extraction from C. tamala141
leaves.142
2.2. Quantification of DNA143
The quantity and purity of the extracted DNA was evaluated by using144
biospectrophotometer (Eppendorf, Germany) using an aliquot of 3 µL of DNA sample from145
the stock. The concentration of the extracted DNA was determined using the absorbance at146
260 nm and the purity of the DNA sample was evaluated by the A260/A280 ratio. The purity of147
the DNA bands was also confirmed by 0.8% agarose gel electrophoresis. The bands were148
observed, documented and analyzed using a gel doc system (G: BOX, Syngene, U.K.).149
2.3. Restriction Digestion150
To check the quality of the extracted Cinnamomum DNA, single enzyme restriction151
digestion was also carried out. Single restriction digestion was done using 5 units of EcoR I152
and Hind III (Thermo Scientific, Lithunia) separately. Briefly, the reaction mixture was153
prepared by adding 10 µL of extracted DNA, 15 µL of 2X assay buffer, 10 µL of BSA, and 3154
µL of restriction enzyme (EcoR I and Hind III). Reactions were carried out at 37 °C for 2, 4155
and 6 hrs and the digested products were resolved on 1% agarose gel and visualized through156
Ethidium bromide staining.157
2.4. RAPD Analysis158
9. 7
The DNA quality was confirmed by RAPD technique using decamer primers RPI 12159
(5´ACGGCAACCT 3´) and RPI 15 (5´AGCCTGAGCC 3´) (Bangalore Genei, Bangalore).160
The stock DNA was diluted to 10 ng/µL. The RAPD PCR amplification volume was 25 µL161
containing 1.5 µL of 10X Taq Buffer A with 15 mM MgCl2 (GeNeiTM
), 2.5 mM dNTP162
(Fermentas, Lithunia), 1 µL of Primer (Bangalore Genei), 1.5 µL of 1U/µL Taq DNA163
Polymerase, and the volume was adjusted by adding double distilled autoclaved water. The164
amplification was carried out in a thermal cycler (Veriti® Thermal Cycler, Applied165
Biosystems) using a program configured with a denaturation step of 5 min at 94 °C followed166
by 35 cycles of 30 s at 94 °C, 30 s at 36 °C, and 1 min at 72 °C. The program ended with one167
final extension cycle at 72 °C for 8 min. The amplified product was observed using a gel doc168
system (G: BOX, Syngene, U.K.).169
2.5. DNA Barcoding170
DNA amplification using four sets of DNA barcoding primer for Cinnamomum171
species was done. Four different DNA samples are taken from each of the three different172
Cinnamomum species viz. C. impressinervium, C. zeylanicum and C. tamala. The 4 sets of173
forward and reverse primers were:174
(a) ITS5a-5´-CCTTATCATTTAGAGGAAGGA-3´175
ITS4-5´ -TCCTCCGCTTATTGATATGC-3´) [Kress et al.2005]176
(b) rbcL1F (5´ATGTCACCACAAACAGAAAC-3´)177
–rbcL724r (5´TCGCATGTACCTGCAGTAGC- 3´) [Kress et al.2005]178
(c) rbcL1F (5´-ATGTCACCACAAACAG-3´) &179
rbcL724r (5´-ATGTACCTGCAGTAGC- 3´) [Modified by National Botanical Research180
Institute-NBRI]181
10. 8
(d) trnH (5´-CGCGCATGGTGGATTCACAATCC-3´) –182
psbA (5´GTTATGCATGAACGTAATGCT- 3´)[Kress et al.2005]183
PCR amplification was performed on a Veriti 96 well thermal cycler (Applied184
Biosystems) as follows: 95 °C for 1 min, followed by 35 cycles of 95 °C for 30 s, T °C for 30185
s and 68 °C for 1 min, followed by an elongation step at 68 °C for 5 min. All the PCR186
conditions were the same for all the primer-pairs except the annealing temperature (T) for187
different primer pairs as follows: 53 °C-ITS5a, 53 °C-ITS4, 50 °C for trnH-A, 68 °C for188
psbA, 54 °C for rbcL1F and 58 °C for rbcL-724. Agarose gel (1%) was used for189
electrophoresis of PCR-products. Gel images were obtained using (G: BOX, Syngene, U.K.)190
imaging system.191
3. Results and discussion192
Isolation of high quality of DNA from medicinal and food plant samples is always193
challenging and foremost requirement for molecular biology studies. However, isolation of194
DNA has always not been achievable from many medicinally and economically important195
plants due to the presence of phenolic compounds and secondary metabolites (Sharma and196
Purohit, 2012). Various challenges were encountered during the DNA extraction from197
different accessions of C. tamala, while following the protocol of Doyle and Doyle (1987),198
due to the presence of the high concentration of polysaccharides and phenolic compounds in199
the leaf tissue. The original protocol of Doyle and Doyle and modified CTAB method when200
used did not yield any DNA. Highly viscous, sticky and brownish pellets were difficult to201
handle and colour indicated contamination by phenolic compounds as earlier reported in202
Dimorphandra mollis (Moreira and Oliveira, 2011). The efficiency of the protocol reported203
here was compared against some of the most commonly used plant DNA extraction protocols204
i.e. Doyle and Doyle (1987), Lee et al. (2010), Abeysinghe et al. (2009) and Križman et al.205
11. 9
(2006). These protocols were tested for DNA extraction from non-lyophilized (0.4 gm) and206
lyophilized leaves (0.2 gm) of C. tamala. The DNA isolated was quantified using a207
spectrophotometer at the absorbance of 260 and 280nm. Among different concentrations of208
CTAB (1%, 2%, 3% and 4%), extraction buffer having 2% CTAB gave the best result with a209
DNA yield of 227.56 ± 9.34 ng/mg sample (Table 1) and produced a clear DNA band on the210
agarose gel (Fig. 1a, b). There was no significant difference among fresh or lyophilised leaf211
samples on the quality and quantity of the DNA when used in modified new method (Table212
1). The protocol reported by Abeysinghe et al. (2009) and Lee et al. (2010) yielded very low213
concentration of DNA. Protocol by Križman et al. (2006), which though yielded good214
quantity of DNA, the quality was compromised. The DNA extracted using Križman et al215
(2006) protocol was very viscous and full of mucilage. The Comparative data of DNA216
concentration and absorbance ratio using numerous earlier standardised methods were217
tabulated in table 1. In this communication, few modifications were done to the existing218
protocol of Križman et al (2006) to develop a standardize protocol for high output DNA219
extraction from Cinnamomum sp. Fresh leaf and lyophilized leaf samples yielded similar220
quantity of DNA (Fig.1b and Table 1). This finding was in accordance with the result of221
Chen and Ronald (1999).222
Fresh and young leaf materials are the first choice to obtain good-quality DNA in223
plants (Moreira and Oliveira, 2011). However, mature leaves contain higher quantities of224
polyphenols and polysaccharides, which make it very difficult to isolate DNA of good225
quality. However, even young leaves for the molecular studies is quite challenging for226
species like Cinnamomum. Overcoming this issue using the present optimized protocol227
yielded better quality DNA even from the mature leaf samples. No fragmentation due to228
shearing of DNA during extraction procedure was seen in any of samples and results were229
reproducible. The absence of smears further substantiates the high purity of extracted DNA.230
12. 10
It has been reported previously that shearing of DNA during extraction can directly or231
indirectly interfere with the enzymatic reactions. The average yield of the DNA through this232
modified protocol was found to be 227.56 ± 9.34 ng/µl and the A260/A280 value was found to233
be 1.80 ± 0.09 (Table 1) ensuring that the DNA samples were free from contamination of the234
secondary metabolites and chemicals used during the extraction procedure and were235
amplifiable in PCR reactions. The spectrum of DNA isolated from different species of236
Cinnamomum indicated the ratio of wave of absorbance at wavelength (λ) 260 nm and 280237
nm is 1.82. The gel electrophoresis (0.8% agarose) before RNase treatment indicates the238
presence of impurity (Fig. 1aM), which could be removed after RNase treatment. Picture of239
agarose gel electrophoresis after RNase treatment shows clear intact bands, which proves that240
high molecular weight DNA without degradation was obtained (Fig. 1aL).241
Review and literature suggested the use of 2% CTAB in the extraction buffer as it242
help to disrupt the cell membrane (Saravanaperumal et al., 2012, Bressan et.al. 2014, Doyle243
et al., 1987). By incorporating different concentration of CTAB (e.g., 1%, 2%, 3%, and 4%)244
in the present experiment, 2% CTAB showed better DNA in respect of quality and quantity245
in comparison to other concentration of CTAB. The results showed that at 2% CTAB DNA246
yield was found to be 169.3±13 ng/µl and A260/A280 ratio was 1.81±0.06. Likewise with247
1% CTAB, DNA yield was 85.46±11.88 ng/µl and A260/A280 ratio was 1.38±0.16, with 3%248
CTAB, DNA yield was 169.3±20.43 ng/µl A260/A280 ratio was 1.26±0.12 and 4% CTAB,249
DNA yield was 91.46±3.75 ng/µl A260/A280 ratio was 1.41± 0.05 (Table 2).250
Polyvinylpyrrolidone (PVP) is an important agent to remove the polyphenols by251
forming complex hydrogen bonding with polyphenols and efficiently separate it from DNA252
(Kit and Chandran, 2010). In the extraction buffer, all the components were kept constant and253
PVP concentration was changed to see its effect on the extracted DNA. In our experiment, we254
have used 1%, 2%, 3%, and 4% PVP respectively (Table 2). Khanuja et al., (1999) also used255
13. 11
different concentration of PVP for plants having high content of secondary metabolites like256
polyphenol and polysaccharides. In the present investigation, addition of 2% PVP yielded the257
optimum quality and quantity of DNA. . In our results, approximately 141.73±9.86 ng/µl and258
A260/A280 ratio 1.65 ±0.11was obtained when 2% PVP was used. Likewise at 1% PVP259
produced 92.56 ±6.9 ng/ µl and A260/A280 ratio 1.52±0.09, 3% PVP produces 85.1±5.9 ng/µl260
and A260/A280 ratio 1.32 ±0.04, and at 4% PVP produced 96.1±14.29 ng/ µl and A260/A280261
ratio 1.50 ±0.07 (Table 2). Lade et al. (2013) mentioned that the quality of DNA get declined262
when the PVP concentration was increased and therefore it confirms as our result was263
accurate.264
The intact and high quality of genomic DNA (Fig. 1b, c) obtained could be ascribed265
to the use of a higher concentration of PVP (2.5%) with lower molecular weight (10,000)266
rather than 40,000 (Table 2). A number of workers (Couch and Fritz, 1990; Chaudhry et. al.,267
1999) have recommended the use of PVP with molecular weight of 10,000 at 2% (w/v) to268
address the high concentration of phenolics present in the plant tissue. PVP with low269
molecular weight has less tendency of precipitating with the nucleic acids as compared to270
PVP with high molecular weight thus yielding sufficient amount of polyphenol free DNA271
(Zhang and Stewart, 2000; Križman et al., 2006).272
The principal modification that proved to be fruitful in extraction procedure of DNA273
from Cinnamomum sp. was the use of activated charcoal, high concentration of CTAB274
(2.5%) and precipitation of the DNA under the influence of high salt (2M NaCl). The275
activated charcoal binds with the resinous substances and thereby settles along with the276
debris in the interference layer between the buffer and Chloroform. In addition to PVP,277
activated charcoal also plays a pivotal role as it can absorb resinous matter and coloured278
impurities in the aqueous phase (Bi et al., 1996). Various literatures suggested the use of279
activated charcoal with PVP in the extraction buffer freshly help to remove the polyphenol280
14. 12
more efficiently. Križman et al., (2006) obtained high quality DNA by using 0.5 % activated281
charcoal in the extraction buffer. In the present investigation, 0.7% (w/v) of activated282
charcoal proved to be sufficient in achieving high quality DNA. The incorporation of283
activated charcoal in the extraction buffer before sample get incubated in the water bath284
greatly enhanced the concentration of DNA, and the most appropriate reason for this might285
be by preventing irreversible interaction of DNA with polyphenols since it comes in contact286
with charcoal than DNA (Bi et.al., 1996). In our experiment 0.5% activated charcoal yielded287
148.2±10.9 ng/µl and A260/A280 ratio 1.66 ±0.04, 0.7% activated charcoal yielded 162.8288
±10.35 ng/µl and A260/ A280 ratio 1.83 ±0.05, 1% activated charcoal yielded 122.1±18.68289
ng/µl, and A260/A280 ratio 1.58 ±0.06 and 3% activated charcoal yielded 122±26.09 ng/µl290
and A260/A280 1.44±0.11 ratio (Table 2).291
Wide utility of the protocol was tested by extracting DNA from different plant species292
(Table 3). Using this protocol, DNA was extracted from Androgrephis paniculata, Litsea293
cubeba, Azadirachta indica and Cinnamomum camphora and Cinnamomum tamala. All294
these four species yielded high quality and quantity of DNA (Table 3). Androgrephis295
paniculata, Litsea cubeba, Azadirachta indica, C. camphora and C. tamala yielded296
501.66±76.53 ng/µl, 439.4±18.53 ng/µl, 341.36±30.18 ng/µl, 317.4±25.97 ng/µl and297
568.6±42.73 ng/µl of DNA respectively. This protocol also worked well for different species298
of Cinnamomum (C. tamala, C. impressinervium, C. zeylanicum). Using this protocol good299
quality and quantity of genomic DNA was extracted from all the species under study, which300
are otherwise very difficult species for DNA extraction (Fig. 1c). The DNA extracted from C.301
tamala, C. impressinervium, and C. zeylanicum was 246.82 ng/µl, 190.21 ng/µl and 257.80302
ng/µl respectively.303
Complete digestion with both the restriction enzyme (EcoR1 and Hind III) confirmed304
the purity of DNA (Fig. 2a). High purity DNA is required for PCR and other PCR-based305
15. 13
techniques, such as random amplified polymorphic DNA (RAPD), micro- and macro-satellite306
analyses, restriction fragment length polymorphism (RFLP) and amplified fragment length307
polymorphism (AFLP) used for genome mapping and DNA fingerprinting (Sharma and308
Purohit 2012). The DNA extracted by this method yielded reproducible and scorable bands309
proving its suitability for PCR applications using RAPD, which proves that, there is no310
contamination of PCR inhibitory products (Fig. 2b). The problem that mainly arises in DNA311
extraction are due to the presence of agents like higher contents of polyphenolic compounds,312
resins, latex, Polysaccharides and tannins present in the cell as secondary metabolites usually313
co-precipitate with DNA and interfere with the activity of the DNA polymerase enzyme. The314
presence and the concentration of these compounds vary considerably from plant to plant.315
This protocol is mainly designed for the Cinnamomum sp., which contains very high316
concentration of polysaccharide, but it could be also used for such similar plants successfully.317
Effective DNA barcoding depends on the quality of the biological material. Following318
this simple sampling protocol will ensure proper preservation of biological samples for DNA319
studies. DNA barcoding, using a short gene sequence from a standardized region of the320
genome, is a species identification tool which would not only aid species discovery but would321
also have applications ranging from large-scale biodiversity surveys to identification of a322
single fragment of material in forensic contexts. To fulfill this vision a universal, relatively323
cheap, scalable system needs to be in place. We used rbcL + matK and trnH-psbA primer324
combination to check the quality of Cinnamomum sp DNA isolated using our modified325
protocol (Fig. 2c). All the primer combination and in all the Cinnamomum sp they showed326
the amplification proving the high quality of the DNA isolated. The ITS1 subset produced a327
consistently smaller amplicon with fewer artifactual amplification products and exhibited328
higher levels of sequence divergence relative to ITS2 and was therefore selected for further329
trials against the other loci. A set of 3 different forward and reverse primers for ITS1 were330
16. 14
then evaluated in all possible combinations on the 4 test species, and a consensus primer pair331
was chosen and applied to the entire taxon set for the empirical experiment.332
Based on these findings, it can be concluded that this protocol provides nuclear DNA333
that has little or no visible coloration; possesses a spectrophotometric A260/A280 value >334
1.8, has an intact DNA or at least the mean fragment length more than 10 kb. Moreover, the335
protocol can be used to isolate DNA from young plant leaves as well as younger tissues336
including seedlings, and it works well with frozen tissue, which is suitable in conditions337
when liquid nitrogen is not available. The protocol may also be applied to other medicinal338
plants with mature tissues rich in polysaccharides and polyphenolic compounds.339
Acknowledgements340
The authors are thankful to the Dr. D. Ramaiah, Director, CSIR-North East Institute of341
Science & Technology, Jorhat, Assam, India, for consistent support and encouragement to342
carry out this work and CSIR, Govt. of India, New Delhi for financing the network project343
(BSC-0117). BSB & SCN are thankful to Department of Biotechnology, Government of344
India for research grant under twinning project.345
346
17. 15
References347
Abeysinghe, P. D., Wijhesinghe, K. G. G., Tachida, H., & Yoshda, T. (2009). Molecular348
characterisation of cinnamon (Cinnamomum verum Presl) accessions and evaluation of349
genetic relatedness of cinnamon species in Sri Lanka based on trnL intron region,350
intergenic spacers between trnT-trnL, trnL-trnF, trnH-psbA and nuclear DNA ITS.351
Research Journal of Agriculture and Biological Sciences, 5 (6), 1079–1088.352
Begum, M., Begum, M., Ahmed, S., & Akter, T. (2013). Studies on the effectiveness of353
tejpata (Cinnamomum tamala Nee) leaf extract on dried kachki (Corica soborna) fish354
preservation in laboratory condition. American Journal of Food Science and355
Technology, 1(3), 14-17.356
Bi, I. V., Harvengt, L., Chandelier, A., Mergeai, G., & du Jardin, P. (1996). Improved RAPD357
amplification of recalcitrant plant DNA by the use of activated charcoal during DNA358
extraction. Plant Breeding, 115, 205-206.359
Bressan, E. A., Rossi, M. L., Gerald, L. T., & Figueira, A. (2014). Extraction of high-quality360
DNA from ethanol-preserved tropical plant tissues. BMC Research Notes, 7 (1), art. no.361
268.362
Chakraborty, U., & Das, H. (2010). Antidiabetic and antioxidant activities of Cinnamomum363
tamala leaf extracts in stz-treated diabetic rats. Global Journal of Biotechnology &364
Biochemistry, 5(1), 12-18.365
Chaudhry, B., Yasmeen, A., Husnain., & Riazuddin, S. (1999). Mini-scale genomic DNA366
extraction from cotton. Plant Molecular Biology Reporter, 17, 1–7.367
Couch, J. A., & Fritz, P. J. (1990). Isolation of DNA from plants high in polyphenolics. Plant368
Molecular Biology Reporter, 8(1), 8–12.369
Deb, B. D. (1983). The flora of Tripura State, Today and tomorrow’s Printers and370
Publications, New Delhi, Vol 1.371
18. 16
Demeke, T., & Adams, R. P. (1992). The effects of plant polysaccharides and buffer372
additives on PCR. Biotechniques, 12, 332-334.373
Devi, S. L., Kannappan, S., & Anuradha, C. V. (2007). Evaluation of in-vitro antioxidant374
activity of Indian bay leaf, Cinnamomum tamala (Buch. -Ham.) T. Nees & Eberm375
using rat brain synaptosomes as model system. Indian Journal of Experimental376
Biology, 45, 778-784.377
Dhanya, K. & Sasikumar, B. (2010). Molecular marker based adulteration detection in traded378
food and agricultural commodities of plant origin with special reference to spices.379
Current Trends in Biotechnology and Pharmacy, 4(1), 454-489.380
Diadema, K., Baumel, A., Lebris, M., & Affre, L. (2003). Genomic DNA isolation and381
amplification from callus culture in succulent plants, Carpobrotus species (Aizoaceae).382
Plant Molecular Biology Reporter, 21, 173a-173e.383
Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of384
fresh leaf tissues. Phytochemical Bulletin, 19, 11-15.385
Doyle, J. J., & Doyle, J. L. (1990). Isolation of Plant DNA from fresh tissue. Focus, 12, 13-386
15.387
Hooker, J. D. (1885) Flora of British India, Wilium Claws and Sons Ltd., London.388
Kanjilal, U. N., Kanjilal, P. C., De, R. N., & Das, A. (1940). Flora of Assam, Govt. of389
Assam Publication, 1940.Vol 4.390
Khanuja, S. P. S., Shasany, A. K., Darokar, M. P., & Kumar, S. (1999). Rapid isolation of391
DNA from dry and fresh samples of plants producing large amounts of secondary392
metabolites and essential oils. Plant Mol Biol Report, 17, 1-7.393
Kress, W. J., Wurdack, K. J., Zimmer, E. A., Weigt, L. A., & Janzen, D. H. (2005). Use of394
DNA barcodes to identify flowering plants. Proceedings of the National Academy of395
Sciences, USA, 102, 8369 – 8374.396
19. 17
Križman, M., Jakše, J., Baričevič, D., Javornik, B., & Prošek, M. (2006). Robust CTAB-397
activated charcoal protocol for plant DNA extraction. Acta Agriculturae Slovenica,398
87(2), 427-433.399
Lee, S. C., Chiou S. J., Yen, J. H., Lin, T. Y., Hsieh, K. T., & Yang, J. C. (2010). DNA400
barcoding Cinnamomum osmophloeum Kaneh. based on the partial non-coding ITS2401
region of ribosomal genes. Journal of Food and Drug Analysis, 18, 128–135.402
Leela, N. K. (2008). Cinnamon and Cassia. In: V. A. Parthasarathy, B. Chempakam, & T. J.403
Zachariah (Eds.), Chemistry of Spices (pp.124). Oxford shire, UK: CAB International.404
Lodhi, M. A., Ye, G. N., Weeden, N. F., & Reisch, B. I. (1994). A simple and efficient405
method for DNA extractions from grapevine cultivars and Vitis species. Plant406
Molecular Biology Reporter 12: 6-13.407
Loomis, M. D. (1974). Overcoming problems of phenolics and quinones in the isolation of408
plant enzymes and organelles. Methods in Enzymology, 31,528-544.409
Maliyakal, J. E. (1992). An efficient method for isolation of RNA and DNA from plants410
containing polyphenolics. Nucleic Acids Research, 20, 2381.411
Mir, S. R., Ali, M., & Kapoor, R. (2004). Chemical composition of essential oil of412
Cinnamomum tamala Nees et Eberm. leaves. Flavour and Fragrance Journal, 19(2),413
112 – 114.414
Moreira, P. A., & Oliveira, D. A. (2011). Leaf age affects the quality of DNA extracted from415
Dimorphandra mollis (Fabaceae), a tropical tree species from the Cerrado region of416
Brazil. Genetics and Molecular Research, 10(1), 353–358.417
Nath, S. C., & Barua, I. C. (1994). A Rare Cinnamomum (C. sulphuratum Nees) Discovered418
in Assam. Journal of Economic and Taxonomic Botany, 18, 211-212.419
Ouenzar, B., Hartmann, C., Rode, A., & Benslimane, A. (1998). Date palm DNA mini420
preparation without liquid nitrogen. Plant Molecular Biology Reporter, 16, 263-269.421
20. 18
Porebski, S., Bailey, L. G., & Baum, B. R.(1997). Modification of a CTAB DNA extraction422
protocol for plants containing high polysaccharide and polyphenol components. Plant423
Molecular Biology Reporter, 15, 8-15.424
Rana, V. S., Langoljam, R. D., Verdeguer, M., & Blázquez, M. A. (2012). Chemical425
variability in the essential oil of Cinnamomum tamala L. leaves from India. Natural426
Product Research, 26(14), 1355- 1357.427
Sahu, S. K., Thangaraj, M., & Kathiresan, K. (2012). DNA Extraction Protocol for Plants428
with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid429
Nitrogen and Phenol. ISRN Molecular Biology, 1-6.430
Sharma, P., & Purohit, D. S. (2012). An improved method of DNA isolation from431
polysaccharide rich leaves of Boswellia serrata Roxb. Indian Journal of Biotechnology,432
11, 67-71.433
Swetha, V. P., Parvathy, V. A., Sheeja, T. E., & Sasikumar, B. (2014). Isolation and434
amplification of genomic DNA from barks of Cinnamomum spp. Turkish Journal of435
Biology, 38, 151-155.436
Syamkumar, S., Jose, M. & Sasikumar, B. (2005). Isolation and PCR Amplification of437
Genomic DNA from Dried Capsules of Cardamom (Elettaria cardamomum M.). Plant438
Molecular Biology Reporter, 23, 417a–417e.439
Thakur, R. S., Puri, H. S., & Husain. (1989). Major medicinal plants of India. CIMAP440
Publication, Lucknow, 182-184.441
Weishing K., Nybom, H., Wolff, K., Meyer, W. 1995. DNA isolation and purification. In:442
DNA fingerprinting in plants and fungi. Florida: CRC Press. pp. 44–59443
Willis, J. C. (1973). A Dictionary of Flowering Plants and Ferns (8th
Edn. Revised by Airy444
Shaw, H.K), Cambridge Univ. Press, Cambridge.445
21. 19
Xie, P. S., & Leung, A. Y. (2009). Understanding the traditional aspect of Chinese medicine446
in order to achieve meaningful quality control of chinese materia medica. Journal of447
Chromatography A. 1216, 1933-1940.448
Zhang, J. & Stewart, J. M. (2000). Economical and rapid method for extracting cotton449
genomic DNA. Journal of Cotton Science, 4(3), 193–201.450
451
452
453
22. 454
455
Fig. 1a. Genomic DNA isolated from plant young leaves (C7, C25, C32) and mature leaves (C7 M, C25456
M, C32M) resolved under 0.8% agarose gel. Lane1 shows the uncut λ DNA457
458
459
460
Figure 1a: Genomic DNA preparation of Cinamomum tamala resolved by electrophoresis461
(0.8% agarose) using the standardize new protocol without RNase treatment (M) and with462
RNase treatment (L):463
(λ: uncut λ DNA, lane 1-8: extract of 8 accessions of Cinamomum tamala)464
465
466
467
Fig 1b Genomic DNA isolated from plant young leaves (C7, C25, C32) and mature leaves (C7 M, C25468
M, C32M) resolved under 0.8% agarose gel. Lane1 shows the uncut λ DNA469
470
471
472
473
474
Fig.1c Genomic DNA of 3 different species of Cinnamomum species C. impressinervium (C-475
24, C-27, C-32, C-55); C. zeylanicum( AAU-A, AAU-B, AAU-C, AAU-7) and C. tamala (476
C-92, C-100, C-101, C-106) resolved by electrophoresis (0.8% agarose) using the standardize477
new protocol . λ: uncut λ DNA.478
479
23. 480
Fig. 2a Restriction digestion of genomic DNA isolated from 3 different species of481
Cinnamomum with EcoR1 (A) and HIND-III(B) for 2hrs (lane 1-3), 4hrs (lane 4-6) and 6hrs482
(lane 7-9) respectively. C.zeylanicum: AAU-6; C. impressinervium: C-32 and C. tamala: C-483
238.484
L is DNA ladder485
486
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
24. 487
Fig.2b RAPD Profile of DNA isolated from leaf of three different Cinnamomum species viz.488
C. impressinervium (C-24, C-27, C-32, C-55);C. zeylanicum( AAU-A, AAU-B, AAU-C,489
AAU-7) and C. tamala ( C-92, C-100, C-101, C-106) (L-100bp ladder)with primer:(A)RPI-490
12 (B)- RPI-15491
492
493
494
495
496
497
498
Fig. 2c. DNA amplification using Barcoding primer for three different Cinnamomum sp. viz.499
C. impressinervium (C-24, C-27, C-32, C-55) C. Zeylanicum( AAU-A, AAU-B, AAU-C,500
AAU-7) C. tamala ( C-92, C-100, C-101, C-106 ) using (A) ITS5a-ITS4 primer (B) rbcL1F-501
rbcL724r primer (C) rbcL1F- rbcL724r (modified by NBRI) primer (D) trnH- psbA primer502
503
504
25. 23
505
Table 1. Comparrison of diffent DNA isolation methods for yield and purity of the DNA isolated506
using fresh and lypholised leaves of Cinnamomum tamala.507
Average yield of 3 DNA extractions and standard deviation508
Note: - NL: non- lyophilised; L: lyophilised; -ve: negative.509
510
511
512
513
514
515
LANE
METHOD Doyle &
Doyle
Doyle
&
Doyle
Shih-
Chieh
Lee
Shih-
Chieh
Lee
Modified
method
using
charcoal
Modified
method
using
charcoal
Krizma
n
Krizma
n
Abeysing
he
Abeysing
he
Leaf Sample NL L NL L NL L NL L NL L
DNA Concentration
(ng/µl)
-ve -ve 307.3 ±
13.35
334.7 ±
5.9
212.13 ±
7.82
227.56 ±
9.34
174.83
± 10.91
195.1 ±
12.01
315.7 ±
19.97
320.5 ±
19.24
A260/A280 -ve -ve 1.10 ±
0.05
1.12 ±
0.05
1.75 ±
0.09
1.80 ±
0.09
1.57 ±
0.04
1.70 ±
0.11
1.2 ±
0.15
1.17 ±
0.12
26. 24
Table 2. Effect of different concentrations of Charcoal, CTAB and PVP on DNA quantity and quality516
of Cinnamomum tamala517
Average yield of 3 DNA extractions and standard deviation518
519
520
521
LANE
CTAB
concentration
2% 2% 2% 2% 1% 2% 3% 4% 2% 2% 2% 2%
Charcoal
concentration
0.5% 0.7% 1% 3% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7%
PVP
concentration
2% 2% 2% 2% 2% 2% 2% 2% 1% 2% 3% 4%
DNA
concentration
(ng/µl)
148.2
±
10.9
162.8 ±
10.35
122.1±
18.68
122±
26.09
85.46±
11.88
169.3±
13.00
169.3±
20.43
91.46±
3.75
92.56±
6.9
141.73±
9.86
85.1±
5.9
96.1±
14.29
A260/A280 1.66
±
0.04
1.83 ±
0.05
1.58 ±
0.06
1.44±
0.11
1.38±
0.16
1.81±
0.06
1.26±
0.12
1.41±
0.05
1.52±
0.09
1.65±
0.11
1.32±
0.04
1.50±
0.07
27. 25
TABLE 3: The concentration and purity of the DNA samples isolated using modified new method.522
Lane 1 Androgrephis paniculata. Lane 2 cLitsea cubeba. Lane 3 Azadirachta indica. Lane 4523
Cinnamomum camphora. Lane 5 Cinnamomum tamala.524
525
526
Average yield of 3 DNA extractions and standard deviation527
528
529
530
531
LANE
Plant Species Androgrephis
paniculata
Litsea cubeba Azadirachta
indica
Cinnamomum
camphora
Cinnamomum
tamala
DNA
Concentration
(ng/µl)
501.66±76.53 439.4±18.53 341.36±30.18 317.4±25.97 568.6±42.73
A260/A280 1.76±0.07 1.70±0.02 1.64±0.10 1.67±0.11 1.73±0.03
28. 26
High Lights532
• Polysaccharide and secondary metabolites in Cinnamomum hinder DNA extraction.533
• Developed DNA extraction protocol proved better then earlier protocols.534
• An efficient protocol has been developed for extraction of DNA mature leaves of535
Cinnamomum.536
• Extracted DNA was successfully amplified and digested.537
• Extracting DNA from different plant species proved wide utility of the protocol.538
539
540