The Comprehensive In Vitro Propagation and Genetic Homogeneity Analysis of Cryptocoryne crispatula var. yunnanensis: Addressing the Conservation Concerns for an Endangered Species in China and the Mekong Basin

This study focused on the propagation of the endangered aquatic plant species Cryptocoryne crispatula var. yunnanensis, threatened by hydropower dam construction in Thailand and classified as endangered in China. The objective was to propagate the species using shoot explants. A sterilization method with a 45.45% success rate involved treating shoots with 0.01% HgCl2 for 90 minutes and 5% commercial bleach (6% NaOCl) for 20 minutes. The study investigated the effect of Murashige and Skoog (MS) medium, supplemented with 0.5 mg/L NAA and 1-4 mg/L cytokinins (BA, kinetin, and TDZ), on shoot initiation and proliferation. TDZ was found to be more effective than BA and kinetin in enhancing shoot growth. The optimal shoot induction, averaging 7.14 shoots per explant, occurred in MS medium with 0.5 mg/L NAA and 3 mg/L TDZ. A medium of 0.5 mg/L NAA and 1 mg/L TDZ significantly increased shoot proliferation, yielding an average of 23.75 shoots per explant. The most successful ex vitro rooting and acclimatization method involved 1X vitamin stock MS medium with 0.5 mg/L IBA, followed by transfer to plastic pots with a 1:1 sand and vermiculite mix, achieving a 73.33% survival rate and an average of 6.31 roots per explant. Genetic uniformity and stability of the propagated clones were verified using ISSR markers. This protocol enhances the conservation efforts for C. crispatula var. yunnanensis by supporting its multiplication and preservation in synthetic habitats.