Artificial Seed Production and Cryopreservation by Encapsulation Dehydration for Medicinal Herb of Himalayan Region, Swertia Chirayita

Abstract

BACKGROUND: Cryopreservation of germplasm in liquid nitrogen is an ideal technique for the longer term storage of plant genetic material, including medicinal species. OBJECTIVE: To develop a somatic embryo production system for the medicinal species Swertia chirayita and to evaluate their potential for storage in liquid nitrogen (- 196˚C). MATERIALS AND METHODS: An efficient protocol of somatic embryogenesis was developed for the first time using leaves of in-vitro grown shoots of S. chirayita . Somatic embryos were then encapsulated in 3% sodium alginate, 0.85 M sucrose and 100 mM calcium chloride for synthetic seed production and subjected to cryopreservation. Marker medicinal compounds were determined by RP-HPLC analysis. RESULTS: A medium containing 1 mg/L 2,4-D+ 0.5 mg/L BAP+ 0.5 mg/L TDZ was found to stimulate the highest callus induction. Somatic embryos were recovered after 5 weeks, when cultured on the same media. Synthetic seeds were dehydrated and immersed in liquid nitrogen for 1 h. Cryopreserved synthetic seeds were successfully revived and germinated on MS media supplemented with 1 mg/L IBA+ 2 mg/L KN + 3 mg/L GA3 in which 93.3% somatic embryos differentiated into shoots. One month old in-vitro grown shoots from cryopreserved somatic embryos had similar marker medicinal compounds, such as amarogentin (4.72 ± 0.11 μg/mg) and mangiferin (14.54 ± 0.05 μg/mg), as control material. CONCLUSION: This protocol offers vast scope for multiplying material of an endangered medicinal herb and subsequent cryopreservation.