Cassava is one of the most important sources of energy. To meet the growing demand, genetic improvement is of utmost importance. Its cross-pollinating nature limits the opportunity of exploitation of hybrid vigor and demands the development of homozygous lines through doubled-haploid technologies. The problems in callus-mediated embryogenesis, such as longer processing time and genetically unstable nature, can be overcome by direct embryogenesis. Conditions to produce embryos directly from microspores in cultured anthers were optimized. The optimum stress pretreatment condition was 40 °C for 6 h after culturing the anthers into the induction medium. For proembryo formation, 2% sucrose and 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg/l 1-naphthaleneacetic acid were optimum. Globular embryos were formed by subculturing proembryos into the medium with 0.5 mg/l 2,4-D and 5 mg/l 6-benzylaminopurine after two weeks of culturing. Light microscopy of cultured anthers demonstrated the formation of multicellular structures and their further development into proembryos. Microscopic studies showed proembryos emerging through the damaged anther wall. Monoallelic banding in simple sequence repeat (SSR) analysis indicated homozygous or haploid states in some of the originated embryos. The conditions optimized in this study were effective in the early development of direct embryos after two weeks of culture initiation. This is the first report of the formation of direct embryos in cultured anthers of cassava.

Nymphaea nouchali var. nouchali is a nonviviparous 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 TM . The mature seeds scarified with 75 % H 2 SO 4 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 % H 2 SO 4 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.