In vitro culture of Nymphaea nouchali seeds; a conservation approach for a vulnerable species

Abstract

Nymphaea nouchali var. nouchali is a non-viviparous species with a slow natural propagation by rhizomes or seeds. The plant is threatened in its natural habitats due to several reasons and is included in the vulnerable category of the National Red List of Sri Lanka. In vitro contamination-free culture method was developed to initiate mass propagation of the species. Results were validated through molecular and microscopic studies. Bacterial growth occurred in the seeds disinfected via the standard method using Clorox™. The mature seeds scarified with 75 % H2 SO4 for 60 seconds gave contamination-free cultures with optimum seed germination. Scanning Electron micrographs of mature seeds showed the rows containing trichomes running between the two poles of seeds and the sclereids between the rows of trichomes to be the potential habitats for bacteria. Light micrographs showed the thick seed coat that causes a physical dormancy. Sulphuric acid treatment was effective in degrading the trichomes completely and the seed coat partially. The highest seed germination (65.5 %) was obtained with seeds cultured/ treated with 75 % H2 SO4 on the solidified MS medium. The basal stem of the well-grown seedlings in vitro gave rise to mini rhizomes. Molecular analysis showed the close genetic relatedness within and among the isolated plant populations where the seeds were collected. The in vitro protocol developed in this study can be used for propagation of seedlings of this vulnerable species for maintaining the biodiversity by population enhancement through restoration and introduction into new habitats.