International Journal of Botany Studies 76 International Journal of Botany Studies ISSN: 2455-541X; Impact Factor: RJIF 5.12 Received: 02-10-2020; Accepted: 16-10-2020: Published: 05-11-2020 www.botanyjournals.com Volume 5; Issue 6; 2020; Page No. 76-82 Micropropagation and clonal fidelity testing in Solanum Capsicoides All Anusree K Dharman 1 , M Anilkumar 2* 1, 2 Cell Culture Lab, Department of Botany Union Christian College, Aluva, Ernakulam, Kerala, India Abstract Solanum capsicoides All. Is a valuable medicinal plant from which the therapeutic agent ‘kantakari’ is extracted. This is used in the treatment of various ailments especially respiratory problems. The plant is diminishing in natural habitat due to over exploitation by pharmaceutical industry and this urges for protection and conservation of the plant. The objective of the study was to standardize a protocol for the direct organogenesis of Solanum capsicoides All. The explants such as cotyledon, shoot tip, and hypocotyl inoculated on MS medium supplemented with auxins and cytokinins in different concentrations and combinations initiated direct shoot buds after 15-30 days of inoculation. Shoot tips inoculated on MS medium augmented with 2.46μM 2iP produced maximum shoots with an average of 15.5. The initial cultures were subcultured on different hormonal regimes of which MS medium containing 6.97μM KIN produced an average of 57 shoots from a single cotyledon explant. The shoots thus obtained were rooted, acclimatized and transferred to field conditions. Clonal fidelity of regenerated plant was studied using ISSR marker. The polymorphism percentage ranged from 0 to 40% and the PIC value varied from 0 to 0.38 thus revealing low level of diversity. The protocol developed can thus be utilized for the mass multiplication and conservation of S. capsicoides. Keywords: solanum capsicoides, direct organogenesis, cytokinins, auxins, issr 1. Introduction Solanum capsicoides All. is an important medicinal plant of the family Solanaceae, which is commonly known as soda apple or cockroach berry. This plant is used as an alternate source of Kantakari (Solanum xanthocarpum), an important therapeutic agent which is used for the treatment of various respiratory problems [1] . The plant is also used for the treatment of ulcerated nose, toothache [2] and apoplexy [3] . Apprehension on health hazards and toxicity of synthetic drugs and antibiotics have increased the demand for herbal drugs [4] which in turn led to large scale collection of whole plants by uprooting. This over exploitation has resulted in the wiping out of many medicinally important plants from their natural habitats [5] . The medicinal plants that are diminishing and in danger of extinction due to ruthless exploitation are in need of protection and conservation as they are essential commodity of health care [6] . Conservation through ex situ method by growing the whole plants in botanical gardens or by seed storage is carried out globally [7] . Alternate method includes the application of biotechnological techniques such as tissue culture and cryopreservation [8] . Advantage of micropropagation over conventional propagation is the rapid propagation in limited time and space [9] . For mass propagation of any plant species through tissue culture technique, a protocol has to be primarily optimized since the culture requirements for each species vary. Plant tissue culture can generate variations in regenerated plants and thus the micropropagated plants may show variations from parent plants [10] . Somaclonal variation is a phenomenon which occurs in vitro that leads to the development of genetically dissimilar plants [11] . Genetic variability in regenerated plants can be attributed to the chromosomal or gene level mutations [12] . Morphological, biochemical and cytological markers have been routinely used in the assessment of clonal variability among regenerated plants. Molecular markers are heritable, stable and reproducible and hence turned out to be the most reliable method for screening genetic stability among cultures [13] . Among the various molecular markers, Inter Simple Sequence Repeats (ISSR) markers are preferred over Random Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP) etc. So ISSR markers are usually used to assess genetic stability of micro propagated plants [14] ISSR is genetically variable and generate multilocus data from the target genome in a single reaction. ISSR is highly reproducibility when compared to routine RAPD techniques and at the same time it is quick and less expensive than AFLP. The annealing temperature depends on the melting point of the primer used and is higher than that of RAPD [15] . In addition they are simple, highly polymorphic and do not require the use of radioactivity [16] . Thus the main objective of the present study was to standardize an efficient and reproducible regeneration protocol in S. capsicoides using different explants such as cotyledons, shoot tip and hypocotyl in MS medium supplemented with different concentrations and combinations of various auxins and cytokinins and to check the clonal fidelity of micropropagated plants in comparison with control plants (seed propagated) using ISSR markers. 2. Methods 2.1. Collection and Identification of Plant Material Mature, healthy plants of Solanum capsicoides All. were collected from Ernakulam District of Kerala, identified using referral herbaria and voucher specimens were deposited at Kerala Forest Research Institute (KFRI), Peechi, Kerala, with herbarium number KFRI 13056.