Chromosomal fragmentation: A possible marker for the selection of high gymnemic acid yielding accessions of Gymnema sylvestre R. Br
Ashutosh Kumar Verma1, Sunita Singh Dhawan2
1 Central Botanical Laboratory, Botanical Survey of India, AJC Bose Indian Botanic Garden, Howrah, West Bengal, India
2 Plant Biotechnology Division, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow, Uttar Pradesh, India
|Date of Submission||22-Sep-2016|
|Date of Acceptance||08-Nov-2016|
|Date of Web Publication||11-Oct-2017|
Ashutosh Kumar Verma
Central Botanical Laboratory, Botanical Survey of India, AJC Bose Indian Botanic Garden, Howrah - 711 103, West Bengal
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Gymnema sylvestre R. Br. a member of family Asclepiadaceae as mentioned in Indian Pharmacopoeia popular among the researchers because of stimulatory effect of its phytoconstituent on pancreatic cells and potential to treat Type I and II type of diabetes. Objectives: Development of cost-effective marker system for the selection of high gymnemic acid yielding accessions of G. sylvestre. Materials and Methods: Presoaked seeds of Brassica campestris treated with different dilutions of gymnemagenin and 10% leaf extract of twenty different accessions of G. sylvestre. Root tips of germinated seeds were fixed, and chromosomal studies were made by root tip bioassay method. Results: Exposure of seeds to treatment solutions promotes various types of chromosomal anomalies in root meristem, and surprisingly, direct correlation between the percentage of chromosomal fragmentation and the percentage of gymnemic acid shared by treatment solution were observed. Conclusion: Later finding may be explored for the development of a novel methodology or marker system for the selection of high active principle yielding accessions of G. sylvestre.
Abbreviations used: MI: Mitotic index; CP: Condensed prophase; CM: Clumped metaphase; MC: Metaphase cleft; FR: Fragmentation; AP: Anaphase with persistent nucleolous; LA: Laggard, BR: Bridge; BI: Bi-nucleated cell; DA: Disturbed anaphasic polarity.
Keywords: Accessions, chromosomal anomalies, Gymnema sylvestre, novel methodology
|How to cite this article:|
Verma AK, Dhawan SS. Chromosomal fragmentation: A possible marker for the selection of high gymnemic acid yielding accessions of Gymnema sylvestre R. Br. Phcog Mag 2017;13, Suppl S3:481-3
|How to cite this URL:|
Verma AK, Dhawan SS. Chromosomal fragmentation: A possible marker for the selection of high gymnemic acid yielding accessions of Gymnema sylvestre R. Br. Phcog Mag [serial online] 2017 [cited 2021 Oct 26];13, Suppl S3:481-3. Available from: http://www.phcog.com/text.asp?2017/13/51/481/216300
- An experiment was carried out using root tip bioassay method for the study of effect of different dilutions of standard gymnemic acid and 10% leaf extract of twenty different accessions of Gymnema sylvestre on root tip meristem of Brassica campestris. Various types of chromosomal anomalies were observed. Of which, percentage of chromosomal fragmentation was showed a direct (8) relationship with the percentage of gymnemic acid shared by treatment solution. This interesting result after more and more exploration and revalidation could be utilized for the development of a novel methodology for the selection of high active principle yielding accessions of G. sylvestre.
| Introduction|| |
Quality of any herbal drug is dependent on the per unit availability of active principle. Thus, there is a need for identification and characterization of high yielding accessions with respect to major therapeutic biomolecule. Gymnema sylvestre R. Br. a member of family Asclepiadaceae as mentioned in Indian Pharmacopeia as an official antidiabetic drug. Its stimulatory effect on pancreatic cells and potential to treat Type I as well as Type II diabetes attracts researchers more than the other plants used for the same disease. This species also documented as a second best-selling medicinal plant of the world market, and its based herbal formulations are more preferable over other herbal antidiabetic formulations. As per report, 85% of the demand of the same plant is primarily met with material collected from wild resources; therefore, sustainable cultivation is advocated for which elite genotypes with higher yield potential are a prerequisite factor. Thus, an effort was made for the development of cost-effective cytological marker for selection of high gymnemic acid yielding, i.e., major antidiabetic principle accessions utilizing root tip bioassay method.
| Materials and Methods|| |
The experiment was conducted with water presoaked seeds of Brassica campestris L., different aqueous dilutions (0.01%–0.08%) of gymnemic acid standard (Gymnemagenin, Sigma-Aldrich) and 10% aqueous leaf extract of different accessions (Gs1-Gs20) of G. sylvestre. Quantification of gymnemic acid in leaves of mentioned accessions was carried out by high-performance liquid chromatography method. The aqueous leaf extract was prepared by heating (70°C) 10 g decoction in 100 ml distilled water for 6 h followed by filtration process. Volume of filtrate was made up to 100 ml by adding distilled water. For treatment, presoaked seeds were divided into 26 lots of 50 seeds each. Five lots were treated with different dilutions of gymnemic acid standard, twenty lots with aqueous leaf extracts of different accessions, and remaining lot was maintained in distilled water as a control. The duration of treatment is about 6 h. Treated seeds were washed with distilled water thoroughly and kept in Petri-dishes on moist blotting paper for germination. Radicle from germinated seed was fixed in Carnoy's fluid (3 alcohol: 1 acetic acid) between 1:20 and 1:25 pm and preserved in 90% alcohol. Cytological preparations were made by the following hematoxylin squash procedure. Random scoring was made from the ten different microscopic fields in ten root tips for determination of the frequency of mitotic index and chromosomal aberrations. Pooled data were statically analyzed using on STATISTICA (version 6.0, Statsoft Inc., Oklahoma, USA) software.
| Results and Discussion|| |
Exposure of B. campestris seeds to different dilutions of standard gymnemic acid and crude leaf extracts of G. sylvestre accessions promotes decrease in mitotic index (MI) and various types of chromosomal anomalies, namely, condensed prophase (CP), clumped metaphase (CM), metaphase cleft (MC), fragmentation (FR), anaphase with persistent nucleolus (AP), disturbed anaphasic polarity (DA), laggard (LA), bridge (BR), binucleated cell (BI), etc. For prior case, decrease in mitotic indices was found dose depended [Table 1]. Range of chromosomal aberrations noted higher for the later case [Table 2]. Surprisingly, chromosomal FR showed a positive correlation with gymnemic acid percentage present in treatment solution [Figure 1],[Figure 2],[Figure 3]. Reductions in mitotic index have been attributed to inhibition of DNA synthesis at S-phase and formation of irregular and disorganized phragmoplast. Stickiness/clumping have been resultant of entanglement of interchromosomal chromatin fibers or defective functioning of specific type of nonhistone proteins involved in chromosomal separation and segregation. The persistence of nucleolus can be assumed, the extension of heterochromatin activity up to the stage of division or due to disturbance of equilibrium between nuclei and the nucleolus. Lagging chromosome indicates a complete failure of spindle apparatus or formation of acentric chromosome, during exchange or delayed terminalization. Treatments also induced clastogenic chromosomal aberrations in B. campestris represented by chromosomal FR and bridge at ana-telophase. Formation of ring chromosome and chromosomal bridges may be the result of reunion of broken chromosome ends. The formation of binucleated and multinucleated cells in treated material may be due to inhibition of cytokinesis process and other metabolic disorders. Binucleate cells are otherwise interpreted as a consequence of inhibited cell cycle, in which chromosome DNA is replicated but not distributed in usual way. Higher range of mitotic anomalies in second treatment probably due to the presence of various types of cytotoxic metabolites in the leaf extract.
|Table 1: Mitotic index and different chromosomal anomalies in root tip of Brassica campestris treated with different dilutions of standard gymnemic acid (gymnemagenin) (average±standard error)|
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|Table 2: Percentage of different chromosomal anomalies in Brassica campestris is root tip treated with leaf extracts of different accessions of Gymnema sylvestre (average±standard error)|
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|Figure 1: Relative association of chromosomal fragmentation and different dilutions of standard gymnemic acid (gymnemagenin)|
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|Figure 2: Percentage of gymnemic acid in different accessions of Gymnema sylvestre|
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|Figure 3: Percentage of chromosomal fragmentation after treatment with leaf extracts of different accessions of Gymnema sylvestre|
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| Conclusion|| |
Above results suggested that gymnemic acid and its allied biomolecules possibly responsible for chromosomal FR. Direct correlation between the percentage of gymnemic acid in treatment solution and percentage of chromosomal FR also validate above result. This astonishing result of aforesaid experiment may be utilized for the development of novel methodology or marker system for identification of high active principle yielding accessions of G. sylvestre.
Authors of manuscript are grateful to Director, CSIR CIMAP, Lucknow, India, Director, Botanical Survey of India, Kolkata, India and Head Department of Botany, University of Lucknow, India, for providing all necessary facilities and support for executing the experiments.
Financial support and sponsorship
First author specially grateful to Science and Engineering Research Board, India, for providing financial support during this Postdoctoral Research work by sanctioning research grant under SERB Young Scientist Scheme (File No: SB/YS/LS 115/2013 date 30.10.2013).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]