Tissue Culture Of Landoltia Punctata And Flower Induction Of Lemna Gibba

Duckweeds (Lemnaceae) belong to floating aquatic monocotyledonous plants. They usually float upon or bellow water surface, and are known as the smallest angiosperms. They are classified into5genera and37species. Their morphology are reduced to only fronds and roots, a few species even have no roots. Most duckweed species have two budding pouches by the sides of the anterior part of the frond. Duckweed usually reproduce by vegetative propagation. Frond primordia develop from the meristematic region in the budding pouches of the mother plants, and the daughter fronds grow out of the pockets of the mother fronds in short cycles, in which the daughter fronds have already contained primordia for the next generation of fronds at the time of emergence out from the pouches of the mother fronds. This reproductive mechanism allows duckweeds to grow at the exponential growth rate, and their biomass may double in2days or less under optimum conditions. Duckweed can stand for wastewater with high nitrogen and phosphorus contents and their geographical ranges span the entire globe. Their rapid assimilation of nitrogen and phosphate into biomass make them an ideal material for waste water purification, and genetic transformation and breeding research is desired for breeding of new varieties in biotechnological applications. Tissue culture and plant regeneration is the basis for genetic transformation, and flower induction is the basis for traditional hybridization breeding. However, tissue culture and flower indcution research of duckweeds are limited in a few species, and effeciency of the few known species is still low. We used Landoltia punctata DW2701and Lemna gibba SH0204for tissue culture and flower induction research in this study. The main findings are as follows:(1) A highly efficient callus induction and plant regeneration protocol was developed for L. punctata. Frond explants were prepared by removing daughter fronds from three-frond clones, and cultured dorsal-side down on agar-solidified medium. Green and fragile callus was induced from meristematic zones in the budding pouches, and no callus was observed from wounding sites. The optimum callus induction conditions were MS medium supplemented with1%sorbitol,15.0mg·L-12,4-D,2.0mg·L-16-BA, at16h day photoperiod. A high callus induction rate of98.3±2.3%was obtained. Green callus was maintained on WPM medium supplemented with2%sorbitol,1.0mg·L-12,4-D,5.0mg·L-1NAA, and0.5mg-L"1TDZ, by subculturing once every four weeks. However, when the subculture cycle was prolonged to six weeks or longer, yellow fragile embryogenic callus was obtained. The highest induction rate of yellow callus was89.3±3.5%, as a result of the subculture cycle of10weeks. The optimum plant regeneration medium was MS medium supplemented with0.5%sucrose,1%sorbitol,1.0mg·L-16-BA. On this medium, the frond induction rate reached the highest of82.1%in4weeks, and each callus produced4.4fronds. The regenerated plants rooted in Hoagland liquid medium in one week. Thus, we obtained a rapid and efficient protocol for callus induction and frond regeneration of L. punctata, which takes only9weeks from explants culture to intact plant regeneration.(2) Salicylic acid (SA) was used in the flower induction of the duckweed Lemna gibba SH0204. Flowers were induced efficiently by suitable concentrations of SA in the liquid1/2Hoagland medium, and the highest induction rate reached39.37%at20?mol-L-SA. In the meantime, SA inhibited the growth of L. gibba, and the frond mortality reached94.38%at80?mol·L-1SA. Solid medium was used in the flower induction of duckweeds, and was found to be more optimal compared to liquid medium. The highest flower induction rate reached45%on the solid medium, and the frond mortality decreased significantly. Moreover, the fronds were kept in the original locations on the solid medium, avoiding the damage of the flowers on the liquid medium by moving around, and providing convenience of tracing or operation with the flowers. Therefore, solid medium is a practically more useful medium in flower induction of duckweeds. Pollen staining revealed that the pollens viability of L. gibba SH0204is low. Electron and light microscopic observation revealed that the pollen grains were spheroid and small, with echinate-reticulate exine.