An Efficient Method for Micropropagation of Red-List Herbaceous Plant Species (<i>Hedysarum cretaceum</i>)

Abstract

<em>Hedysarum cretaceum</em> Fisch<em> </em>is a critically endangered herbaceous species with high ornamental and phytomeliorative properties; thus, it needs urgent conservation and protection methods to conserve. In the present study, an efficient method for microtonal propagation of <em>H. cretaceum</em> is developed. The seed sterilization was optimized as treatment with 96% alcohol (2 min) and burned (1 sec) through a burner flame. Murashige and Skoog (MS) medium with the addition of 0.5 mg/L 6-Benzylaminopurine (BAP) (shoots per explant – 18.0) and 1.0 mg/L BAP and 0.1 mg/L Indole-3-acetic acid (IAA) (shoots per explant – 18.4) for the shoot and ⅓ MS supplemented with 0.1 mg/L IAA (rooting indicator – 62%) for roots were identified best composition for the proliferation and regeneration. Additionally, the medium for maintaining callus culture was determined – DKW with the addition of 2.5 mg/L BAP and 0.1 mg/L IAA, the possibility of direct organogenesis <em>in vitro</em> in the studied plant was also standardized. In addition, seed germination after various pre-sowing treatments was studied. It is shown that scarification makes achieving 80% germination of seeds possible. Also, sulfuric acid was used as a pre-sowing seed treatment, germination was 45%. Thus, using the protocols we developed, it is possible to maintain the <em>H. cretaceum</em> culture <em>in vitro</em> and increase seed germination using pre-sowing treatment.