# Direct somatic embryogenesis, genetic fidelity assessment and GC-MS analysis of regenerated Crinum asiaticum L. Plants

## TL;DR

This study develops a reliable method to grow Crinum asiaticum plants in a lab, ensuring genetic stability and producing valuable medicinal compounds.

## Contribution

A novel in vitro propagation method for Crinum asiaticum with confirmed genetic fidelity and phytochemical consistency.

## Key findings

- Direct somatic embryogenesis achieved 95.83% efficiency using MS medium with 2.7 µM NAA and 4.4 µM BAP.
- Genetic fidelity confirmed via cytological, flow cytometric, and SCoT marker analyses showed no ploidy changes.
- GC-MS analysis revealed bioactive compounds like tazettine and beta-sitosterol in both in vivo and in vitro plants.

## Abstract

Crinum asiaticum L. is an important reservoir of phytocompounds containing galanthamine, lycorine, tazettine and others with diverse pharmacological uses. Due to high commercial demand for these promising compounds in pharmaceutical sector, an efficient in vitro micropropagation protocol optimization study was conducted via direct somatic embryogenesis in C. asiaticum. The regenerated plants were subject to genetic fidelity assessment; and the phytochemical composition was analysed and compared with donor plants. In this investigation, the bulb-scales were used as explants onto media containing different PGRs for various regeneration processes.

In media containing BAP and NAA, somatic embryos were formed directly on bulb-scale explant surfaces with the highest (95.83%) being at MS medium + 2.7 µM NAA + 4.4 µM BAP. The occurrence of somatic embryos at different stages was confirmed by histological and scanning electron microscopic (SEM) analysis. The embryos were later converted to shoots on 2.2–8.8 µM BAP augmented MS medium, with highest germination percentage of 75 ± 7.22 at 4.4 µM BAP. These regenerated plants were successfully transferred to medium containing NAA, IBA or IAA for rooting and the best rooting response (91.67% rooting frequency, 7.67 mean root numbers/shoot and 7.5 ± 0.6 cm average root length) was noted at 5.4 µM NAA. The plants were transferred to greenhouse with pretty good growth and survival. The genetic fidelity of tissue cultured plants was checked through cytological, flow cytometric and SCoT marker-based PCR technique. The root tips of in vitro raised and mother plants showed 2n = 44 chromosome numbers, and the flow cytometric histograms revealed similar fluorescence peaks with nuclear 2 C DNA content of 31.79 and 31.51pg, respectively, displaying no change in ploidy level. Six SCoT primers based genetic homogeneity study showed 42 scorable, monomorphic bands, confirming true-to-type regenerated plants. Finally, the GC-MS based metabolite profiling of in vivo and in vitro raised plants were conducted, which exhibited a wide range of bioactive compounds like tazettine, squalene, gamma-tocopherol, beta-sitosterol, glycidyl palmitate, glycidyl oleate of pharmacological significance.

The current study presents an effective method for genetically stable clonal propagation of C. asiaticum for extraction of compounds like tazettine, squalene, beta-sitosterol for pharmaceutical applications.