Molecular Cytogenetic Methods for Studying Curcuma Plants from Thailand

Species in the genus Curcuma L. (Zingiberacace) that are cultivated widely in Thailand for phytoestrogen-producing rhizomes are called Wan chak motluk. Enzymatic-based methods were modified in order to obtain metaphase spreads with high quality of chromosomes from root tips of these Curcuma species. The results revealed that protoplast dropping with modification in the hypotonic and the post drying steps was an efficient method for chromosome preparation of C. comosa (2n=42, SB5403) using the combination of 8% Cellulase and 3% Pectinase (K8) enzyme mixture. In addition, a new enzymatic drop-squash method was developed in the present study produced well-spread metaphases with very little cytoplasmic background in Curcuma sp.2 (NK5501). Moreover, the enzymatic squash method using the combination of 10% Cellulase and 12% Pectinase (K12) enzyme mixture gave satisfactory results for C. latifolia (KB5451), C. elata (CB5316) and Curcuma sp.1 (PR5319), but moderate quality with C. comosa (2n=42, NP5411), C. comosa (2n=63, SB5403) and Curcuma sp.3 (TK5502). Conditions of the chromosome pretreatment and fluorescent in situ hybridization (FISH) were optimized in order to obtain suitable protocols for rDNA-FISH mapping of Curcuma species. The results showed that Proteinase K treatment could be omitted for chromosomes prepared from protoplast dropping and enzymatic drop-squash methods, whereas 3-10 µg/ml of Proteinase K was needed to treat chromosomes prepared from the conventional enzymatic squash method. Then, combined denaturation at 89°C for 10 min for re-probing was successful to obtain strong fluorescent signals of 18S-25S rDNA in most Curcuma species. However, the optimal denaturation temperature for C. comosa (2n=63, SB5403) was 92°C for 10 min followed by 89°C for 10 min in the second denaturation. The separate denaturation protocol whereby chromosomes were denatured in a Formamide and 1xSSC solution at temperature up to 80°C was also experimented. This work will be continued until the most efficient protocol for mapping of the ribosomal genes on chromosomes can be obtained for all Wan chak motluk species and cultivars from different regions in Thailand and then it will be possible to establish the genomic and genetic relationships among these taxa.

Scholarship from the Institute for the Promotion of Teaching Science and Technology under the Development and Promotion of Science and Technology Talents Project (DPST) of the Ministry of Higher Education, Thailand