Induction of in vitro tetraploids in Kaempferia parviflora Wall. ex Baker and its characterization

Kaempferia parviflora Wall. ex Baker is a valuable crop with high medicinal potential that, unfortunately, suffers from low rhizome and phytochemical yield. Among the breeding techniques that could improve crop vitality, induction of polyploidy was proven to be one of the most successful approach. In this study, stable tetraploids of K. parviflora were developed and characterized for the first time. Pre-established multiple shoots (6-wk-old) were separated into single shoots and sliced at the meristematic region to obtain ample transverse thin cell layers (tTCLs), which were then immersed in colchicine solution of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% for 6, 12, and 24 h in each concentration. The treated tTCLs were regenerated on Murashige and Skoog (MS) medium (added with 4.0 mg L−1 6-benzyladenine and 0.8 mg L−1 indole-3-acetic acid) for 12 wk. tTCLs treated with 0.4% colchicine for 12 and 24 h displayed larger shoot thickness (64%), leaf length (17%), and leaf width (27%), which were indications of potential polyploidization. The ploidy level of regenerants with altered morphologies was confirmed via flow cytometry analysis as compared to controls (without soaking). The highest tetraploid frequency (38.8%) was obtained in 0.4% colchicine treatment for 12 h. Tetraploid regenerants displayed contrasting stomatal characteristics where the frequency was significantly lower, and the size was larger than diploid counterparts. Leaves of tetraploids showed elevated chlorophyll content, whereas roots harbored enhanced flavonoid and phenolic concentration (flavonoid, 90% increase; phenolic, 63% increase). High-performance liquid chromatography analysis of root extracts revealed tetraploid plantlets produced a significantly higher (~ two-fold) 5,7-dimethoxyflavone and 3,5,7,3′,4′-pentamethoxyflavone content in comparison to the diploids. Thus, it is evident from the present study that in vitro tetraploid induction offers a promising approach for production of improved K. parviflora planting material with higher rhizome yield and secondary metabolite content.

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