Abstract
Blepharispermum subsessile DC. is well known for its ethnomedicinal values. This plant has been used traditionally for the treatment of various diseases. Due to over-exploitation, habitat destruction, and climate change B. subsessile has been included in the list as threatened plants. Hence urgent attention is needed for the protection and conservation of this endangered plant. This plant is found in ‘Gurudangar’, Odisha, India and it is declared as Medicinal Plant Conservation Areas for in situ conservation of this plant species along with few other medicinal plants. In vitro propagation is an extremely effective alternative to overcome the limitations of conventional propagation methods and to escalate the production of B. subsessile. Keeping this in view, the present study was envisaged to develop an efficient protocol for in vitro plant regeneration of B. subsessile from cotyledonary node explant. Multiple shoots were induced from the cotyledonary node on Murashige and Skoog’s (1962) (MS) medium supplemented with different types and concentrations of plant growth regulators. A combined effect of 2.0 mg/L meta-Topolin (mT) and 1.0 mg/L indole-3-acetic acid (IAA) when augmented with MS medium was evaluated as optimum for multiple in vitro shoot regeneration from cotyledonary node. Further, in vitro nodal segments were inoculated on different plant growth regulator supplemented medium for upscaling and mT (2.0 mg/L) was found to be best for such in vitro shoot proliferation. The in vitro shoots were rooted on ½ MS medium supplemented with 0.5 mg/L indole-3-butyric acid (IBA). The in vitro regenerated plants were successfully acclimatized in the small pots containing sterile garden soil and sand (1:1) followed by the transfer to larger pots containing garden soil. The genetic fidelity of in vitro regenerated plants was assessed and ascertained by ISSR markers. The phytochemical analysis and antioxidant activity of the in vitro regenerated plants vis-à-vis mother plant were also evaluated to find out the biochemical fidelity.
Key message
A robust micropropagation protocol was developed for a threatened medicinal plant Blepharispermum subsessile DC. using axenic seedling on mT supplemented medium and evaluated their genetic fidelity and phytochemical content.
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Data availability
All data generated during this experiment are included in this article.
Abbreviations
- %:
-
-Percentage
- ºC:
-
-Degree Celsius
- ½ MS:
-
-Half-strength Murashige and Skoog’s (1962) medium
- ABTS:
-
-2, 20-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
- ANOVA-:
-
Analysis of variance
- BA:
-
-N6—Benzyladenine
- CAMP:
-
-Conservation Assessment and Management Prioritisation
- CRD:
-
-Completely randomized design
- CTAB:
-
-Cetyltrimethylammonium bromide
- DMRT:
-
-Ducan’s New Multiple Range Test
- DPPH:
-
-2, 2’- Diphenyl-1-picrylhydrazyl
- DW:
-
-Dry weight
- GA3 :
-
-Gibberellic acid
- GAE:
-
-Gallic acid equivalent
- h:
-
-Hours
- IAA:
-
-Indole-3-acetic acid
- IBA:
-
-Indole-3-butyric acid
- IC50 :
-
-Half-maximal inhibitory concentration
- ISSR:
-
-Inter Simple Sequence Repeats
- KIN:
-
-Kinetin
- mg/g:
-
-Milligram per gram
- mg/L:
-
-Milligram per Liter
- µg/mL:
-
-Microgram per milliliter
- min:
-
-Minutes
- MS:
-
-Murashige and Skoog’s (1962) medium
- mT:
-
-meta-Topolin
- NAA:
-
-α-Naphthalene acetic acid
- PCR:
-
-Polymerase Chain Reaction
- SD:
-
-Standard deviation
- TAE:
-
-Tannic acid equivalent
- WHO:
-
-World Health Organisation
- Z:
-
-Zeatin
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Acknowledgements
SKN acknowledged to University Grant Commission (UGC), New Delhi for financial support through UGC-Research Award programme. The authors express thanks to the Forest and Environment Department, Government of Odisha for their permission to visit different forest areas and use the plant/plant materials for the present study. The authors also acknowledge the help of Dr. P.C. Panda for plant identification. The help and enlightening discussion of Mr Biswa Nath Hota, Deputy Conservator of Forest (Retd.) during this work was also acknowledged.
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Funding received from UGC, Govt. of India.
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SKN conceptualized the study; supervised KM and SB. KM carried out all experiments. SKN, SB and SPP visited different forest area and collected plant samples. SKM helped in tissue culture experiments. KM and SB analysed all the data and written the manuscript. SKN edited the manuscript to its final version. All authors read and approved the manuscript for publication.
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Monalisa, K., Behera, S., Pidika, S.P. et al. Development of micropropagation protocol, assessment of genetic fidelity and phytochemical analysis of Blepharispermum subsessile DC.: a conservation concerned medicinal plant of Eastern Ghats, India. Plant Cell Tiss Organ Cult 157, 14 (2024). https://doi.org/10.1007/s11240-024-02747-z
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DOI: https://doi.org/10.1007/s11240-024-02747-z