Pre-adaptation to Cu during Plant Tissue Culture Enhances Cu Tolerance and Accumulation in Begonia (Begonia evansiana Andr.)

A simple and efficient protocol was developed for culturing Cu-tolerant and Cu-accumulating plants via pre-adaptation to Cu during plant tissue culture. We induced multiple shoots from begonia (Begonia evansiana Andr.) leaf explants on MS medium supplemented with naphtaieneacetic acid and benzyladenine. After 3 months, small plantlets were transferred to MS medium supplemented with $100{\mu}M\;CuCl_2$ for pre-adaptation to Cu and cultured for 5 months. Then, these plantlets were individually planted in pots containing artificial soil. An additional 500 mg of Cu dissolved in 1/4 strength MS solution was applied to each pot during irrigation over the course of 2 months. We planted pre-adapted and control begonias in soil from the II-Kwang Mine, an abandoned Cu mine in Pusan, Korea, to examine their ability to tolerate and accumulate Cu for phytore-mediation. Pre-adapted begonias accumulated $1,200{\mu}g$ Cu/g dry root tissue over the course of 45 days. On the other hand, non-Cu-adapted controls accumulated only $85{\mu}g$ Cu/g dry root tissue. To enhance Cu extraction, chelating agents, ethylenediamine tetraacetic acid (EDTA)-dipotassiun and pyridine-2,6-dicarboxylic acid (PDA), were applied. While the chelating agents did not enhance accumulation of Cu in the roots of control begonias, EDTA application increased the level of Cu in the roots of pre-adapted begonias twofold (to $2,500{\mu}g$ Cu/g dry root tissue). Because pre-adapted begonias accumulated a large amount of Cu, mainly in their roots, they could be used for phytostabilization of Cu-contaminated soils. In addition, as a flowering plant, begonias can be used to create aesthetically pleasing remediation sites.