In Vitro Propagation of Aconitum chasmanthum Stapf Ex Holmes: An Endemic and Critically Endangered Plant Species of the Western Himalaya

Abstract

Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum.

In Vitro Propagation of Aconitum violaceum Jacq. ex Stapf through Seed Culture and Somatic Embryogenesis

“…Cold scarification is the most efficient strategy for breaking dormancy in Ranunculaceae [35][36][37]. The seeds stratified at 4 • C for 4-10 days enhanced the germination rate in A. chasmanthum and A. nagarum when cultured on the MS basal medium [22,25]. Similarly, A. violaceum seeds also require chilling treatment for successful germination.…”

“…The seeds of A. violaceum Jacq. ex Stapf were collected in the month of August (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) as soon as the follicle burst for seed dispersal, from the villages of Khawous and Numsuru (3220 m asl to 3340 m asl; 34 • 15.905 N to 34 • 12.705 N; 75 • 96.040 E to 75 • 96.720 E) in Ladakh, India. The plant specimen was identified and authenticated by the Center for Biodiversity and Taxonomy (CBT), Department of Botany, University of Kashmir, Hazratbal-Srinagar.…”

“…A. violaceum faces a great threat of extinction in its natural habitat due to various factors, such as physiological seed dormancy of >5 months [4,21,22], and long-term burying of seeds beneath snow (more than five months). Due to its specific ecological requirement, seed germination and seedling establishment in A. violaceum are quite challenging.…”

“…Therefore, uprooting whole plants due to collection, overgrazing, and premature harvesting, alongside construction of high-altitude roads, dams, cemented water channels, and human settlements, also contribute to the decline of this species from its natural habitat. Beetles and aphids are another potential hazard to this species as they consume the flowers and other reproductive portions of the plant, thereby reducing the species' sexual potential [22]. In addition, the species has a unique niche, which may limit its area of occupancy and dispersion [4].…”

“…When properly applied, mass propagation methods may help to reduce the extinction risk of vulnerable species by using procedures such as in vitro propagation [22]. In vitro regeneration techniques have been established for some Aconitum species such as A. chasmanthum Stapf ex Holmes [22], A. ferox [23], A. heterophyllum [24], A. nagarum [25], and A. vilmorinianum [26], but no such attempts have been made for A. violaceum. Giri et al [24] regenerated complete plantlets from the somatic embryos of leaf and petiole explants in Aconitum heterophyllum.…”