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Micropropagation of Tricholepis Roylei Hook. F.—A Point Endemic Species of the Western Himalaya Giriraj Singh Panwar* and Bhavana Joshi

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Micropropagation of Tricholepis Roylei Hook. F.—A Point Endemic Species of the Western Himalaya Giriraj Singh Panwar* and Bhavana Joshi Panwar and Joshi Journal of Genetic Engineering and Biotechnology Journal of Genetic Engineering (2020) 18:40 https://doi.org/10.1186/s43141-020-00051-9 and Biotechnology RESEARCH Open Access Micropropagation of Tricholepis roylei Hook. F.—a point endemic species of the Western Himalaya Giriraj Singh Panwar* and Bhavana Joshi Abstract Background: Tricholepis roylei Hook. f. is a bushy annual shrub of the Asteraceae family and point endemic species of the Western Himalaya. T. roylei is a critically endangered species and factors like poor seed germination and habitat destruction are further rendering the species towards extinction. Therefore, the present investigation was designed to document the seed germination potential of the species and to establish a reproducible in vitro propagation and mass multiplication protocol for the ex situ conservation of T. roylei germplasm. Results: Seeds of T. roylei were collected from Sangdha, Himachal Pradesh, India, and were sown in different substrates under open and controlled conditions. Though the overall seed germination potential of the species was reported to be very low and maximum 45% germination was observed in cocopeat substrate after 26 days of incubation. Half-strength Murashige and Skoog (MS) medium enriched with 6-benzylaminopurine (4.4 μMl−1) and naphthalene acetic acid (1.0 μMl-1) was observed to be the optimum medium for shoot induction in shoot tip explants of T. roylei. Maximum 98.89% shoot formation was observed with 28.42 shoots per culture and 4.4-cm shoot length, respectively. The healthy shoots (4.0 cm) were transferred onto rooting media (1/4, 1/2 and full MS) and roots were developed after 8 weeks of incubation in the half-strength MS medium. Half MS augmented with 4.9 μMl−1 indole butyric acid was observed to be optimum for the root development and an average of 10.2 roots per shoot with 4.0-cm length was obtained. Rooted plantlets were successfully acclimatized under greenhouse conditions and subsequently established in the field, with a recorded survival rate of 90%. The plants acclimatized to the open environment were also planted in the wild under the habitat rehabilitation and species recovery programme. Conclusion: The seed germination study envisages that the seed germination potential of the species is very poor and might be one of the probable factors responsible for the shrinkage of T. roylei population in the wild. The standardized micropropagation protocol can be helpful for the ex situ conservation of germplasm and rehabilitation of species in the wild. Moreover, the study could be helpful in elucidating the phytochemical and molecular analysis of species. Keywords: Habitat rehabilitation, Micropropagation, Point endemic, Species recovery, Threatened * Correspondence: [email protected] Botanical Survey of India, Northern Regional Centre, Dehradun, Uttarakhand 248195, India © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Panwar and Joshi Journal of Genetic Engineering and Biotechnology (2020) 18:40 Page 2 of 8 Background Genus Tricholepis is very significant in terms of its Tricholepis roylei is a bushy, annual shrub of the Astera- medicinal potential and various species are being utilized ceae family and scattered on rocky habitat all along the as a traditional medicine for the treatment of inflamma- roadside. It is a poorly known species of the genus Tri- tion, leucoderma and skin diseases. These are also used cholepis and reported in few literatures of India [1]. Due as a nervine tonic and aphrodisiac and are used in sem- to its poor occurrence in the wild, Hajra [2] has omitted inal debility [10, 11] malaria, fever, skin grains, stomach the species from Indian flora and reckoned the species pain, blood purification and dysentery [12]. Maher et al. as extinct from the Western Himalaya. Owing to its re- [12] have also isolated two withanolide glucosides (tri- stricted occurrence (area of occupancy) and few individ- chosides A and B) from T. eburnean with antitumour, uals (very small population size) in the wild, the taxon antibacterial, antifungal, anti-inflammatory, cytotoxic, falls under the IUCN designated critically endangered hepatoprotective and immunosuppressive activities. category and is facing the risk of extinction in the Considering the lack of scientific studies and the threat- imminent future. ened and point endemic status of the species, the Moreover, due to lack of available literature, it is still present investigation aimed to study the seed germin- obscure whether the species is neo-endemic (i.e. taxa ation potential of the species in different substrates and that have evolved relatively recently and may be re- to standardize micropropagation protocol for the mass stricted in their distribution because they have not had multiplication and ex situ conservation of T. roylei yet time to disperse further) or paleoendemic (i.e. taxa germplasm by using shoot tip explants. that have a long evolutionary history and usually are re- stricted by barriers to dispersal or by extensive extinc- Methods tion in the remaining areas where they were distributed Collection of seeds and evaluation of seed germination in the past) [3]. Lack of occurrence of the taxon in any percentage botanical literature (state or country flora) and herbaria Mature seeds of T. roylei were collected from Sangdha, envisages that the species is neo-endemic in nature and Himachal Pradesh (2300 masl, 30° 24 N, 78° 17 E), in appears very young in the evolutionary time scale. November 2018. The plant material was identified by Due to its point endemic nature and roadside habitat, the first author with the help of a herbarium specimen the species is much prone to anthropogenic disturbances available in the BSD and live specimen growing in the such as massive mining and widening of roads prevalent Experimental Botanical Garden of Botanical Survey of in the area. Besides this, the Himalaya is one of the most India, Dehradun, India. Seed germination was studied in fragile ecosystems on the earth and steep slopes, poor both conditions, i.e. field and controlled conditions. Ini- soil binding and heavy monsoon rains further make it tially, the mature healthy seeds were segregated into two vulnerable for habitat destruction, cloud burst, landslide lots and seeds of one lot were washed in running tap and other natural calamities [4]. Plant species endemic water for 30 min. Thereafter, seeds were surface steril- to the Himalayan ecosystem, particularly the point en- ized with 0.1% HgCl2 (Himedia Laboratories, Mumbai, demic one, are much prone to such factors and might India) for 5 min and seeds were thoroughly rinsed with get washed away if any eventuality occurs, which leads autoclaved double-distilled water. Seeds of one lot were towards the extinction of taxa. The situation is further sown in soil, sand and cocopeat and placed in polyhouse aggravated by the poor seed germination in T. roylei re- at 25 °C. Another lot of disinfected seeds were sown in vealed by the recruitment of few seedlings in the wild filter paper and half- and full-strength Murashige and during a personal visit of the area. Skoog medium [13] and were kept in the seed germin- The increasing anthropogenic activities in the fragile ator (CHM-16, REMI, India) at 25 °C temperature, 50% Himalayan ecosystem are a key driver of the loss of bio- relative humidity and 16/8-h light and dark photoperiod. diversity in the region [5]. Habitat destruction and over- exploitation have resulted in the extinction of umpteen Initiation of aseptic culture number of plant species and losing a species has grave Young meristematic shoot tip explants of T. roylei were ecological consequences [6]. All these activities in the re- collected from the Experimental Botanical Garden of the gion have led to a considerable depletion of the species Botanical Survey of India, Dehradun, and washed in run- from the wild. The poor seed germination and continu- ning tap water for 30 min. It was followed by shaking in ous habitat degradation may render the species towards Tween-20 solution (Himedia Laboratories, Mumbai, extinction in the imminent future. Thus, it is pertinent India) for 20 min on a rotatory shaker at 100 rpm. Each to conserve the taxon and in vitro technique offers a sig- treatment is followed by thorough rinsing in running tap nificant alternative for the rapid mass multiplication and water to remove the detergent completely. Thereafter, conservation of threatened and endemic species germ- shoot tip explants were surface sterilized with 70% etha- plasm [7–9]. nol, 6% NaOCl (Merck & Co., United States) and 0.1% Panwar and Joshi Journal of Genetic Engineering and Biotechnology (2020) 18:40 Page 3 of 8 HgCl2 (Himedia Laboratories, Mumbai, India). After compost-enriched soil after 4 weeks and maintained in each treatment, explants were thoroughly rinsed with the greenhouse. autoclaved double-distilled water. Properly disinfected All the experiments were conducted in triplicates and explants were inoculated onto Murashige and Skoog each set of experiment was carried out with 20 seeds/ex- (MS) medium enriched with different plant growth regu- plants.
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