Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata

Abstract

The starch-rich duckweed Landoltia punctata is a valuable aquatic plant in wastewater purification, bioenergy production, and many other applications. A highly efficient callus induction and plant regeneration protocol is desirable so that biotechnology can be used to develop new varieties with added value and adaptation. We studied both known and unknown factors that influence callus induction in L. punctata and obtained almost 100 % induction rate in 30 days. The optimum medium for callus induction was MS basal medium supplemented with 1 % sorbitol, 15 mg/L 2,4-D, and 2 mg/L 6-BA. Green fragile callus was induced from the meristematic region in the budding pouches. The optimum photoperiod for callus induction was 16-h day, and the optimum explant orientation was dorsal side down on the medium. The optimum medium for callus subculture was WPM basal medium supplemented with 2 % sorbitol, 4 mg/L 2,4-D, and 0.5 mg/L TDZ. Green callus could be maintained by subculture once every 4 weeks. However, when the subculture cycle was prolonged to 6 weeks or longer, yellow fragile embryogenic callus was obtained. The optimum plant regeneration medium was MS medium supplemented with 0.5 % sucrose, 1 % sorbitol, and 1.0 mg/L 6-BA with frond regeneration rates of approximately 90 %. The regenerated fronds rooted in Hoagland’s liquid medium in 1 week. The callus induction and frond regeneration protocol was tested for its efficiency in geographically distinct strains 5502, 8721, and 9264. Thus, we obtained a rapid and efficient protocol for callus induction and frond regeneration of L. punctata, which takes only 9 weeks.