การพัฒนาเป็นพืชต้นใหม่และการเพิ่มชุดโครโมโซมของกระชายดำในหลอดทดลอง (Kaempferia parviflora wall. ex Baker)

Abstract

A simple and cost effective protocol for large-scale propagation of black galingale, a medicinal plant, has been studied. Sterilized shoot explants were excised and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of Benzylaminopurine (BAP) (1, 2, 3, 4 and 5 mg/L). The results showed that MS medium supplemented with 3 mg/L BAP gave the highest shoot induction (95 percent), number of shoots (3.75 shoots/explant) and shoot length (4.76 cm) after culturing for 6 weeks. For shoot proliferation, shoots from a previous study were excised and cultured on different types of culture media (solid, semi-solid and liquid MS medium, supplemented with 3 mg/L BAP). The highest percentage of shoot proliferation (100%), number of shoots (6.30 shoots/explant) and shoot length (3.94 cm) were obtained from liquid MS medium after 6 weeks of culture. After that, the shoots were cultured on different strengths of MS medium (full MS, 1/2MS, 1/3MS and 1/4MS). The results showed that 1/4MS medium gave the highest root induction (100%), number of roots (7.35 roots/shoot) and root length (7.22 cm) after culturing for 3 weeks. For acclimatization, the in vitro plantlets and ex vitro sprouting buds were transplanted singly in polyethylene bags containing soil, peat moss, and compost, at the ratio of 1:1:1 and kept in a greenhouse for 9 weeks. The results showed that both sources of plants can produce tillers at 100 percent, but the regenerated plantlets produced more tillers (3.67 tillers/clump), more leaves (7 leaves/clump) and taller plants (28 cm), than the ex vitro spouting buds. For callus induction, shoots were cultured on MS medium supplemented with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) (0.2 0.5 1.0 and 2.0 mg/L) in combination with 1-Naphthaleneacetic acid (NAA) (0.2, 0.5 and 1.0 mg/L). The results showed that 0.5 mg/L 2,4-D in combination with 0.2 mg/L NAA gave the highest percentage of callus induction (45%) and the maximum size callus (1.32 cm). For proliferation, 150 mg calluses were cultured on MS medium supplemented with various concentrations of 2,4-D (0.1 and 0.2 mg/L) and NAA (0.1 and 0.5 mg/L). The results showed that 0.2 mg/L 2,4-D in combination with 0.1 mg/L NAA gave the best percentage of proliferation (100%) and callus fresh weight (1,127 mg). MS medium with reduced concentrations of nutrients could not induce plantlets from callus. For chromosome duplication, shoots of black galingale were immersed in colchicine solutions of 0.0, 0.1, 0.15, 0.20 and 0.25% and incubated for 24, 48 and 72 hours. The concentration of colchicine at 0.10% for 48 hours gave the highest survival rate (70%) and induced the highest percentage of tetraploid plants (37.5%) as analyzed by flow cytometry technique. And the result showed that LDso was obtained from 0.12% colchicine solution for 48 hours and 0.09 % colchicine solution for 72 hours as evaluated by regression equation. For physiological characteristics, the results showed that guard cell density at 2.8 cells/um2, guard cell width at 3.59 pm, guard cell length at 6.05 μm and chloroplast numbers at 51.90 chloroplasts/guard cell were obtained. Twelve weeks after transferring to ex vitro environments, morphological characteristics of the induced tetraploid plants were measured. The results showed that tetraploid plants gave the highest percentage of sprout induction (100%), number of tillers (2.80 tillers/clump), plant height (22.7 cm), number of leaves (6.20 leaves/clump), leaf width (5.84 cm), leaf length (13.89 cm) and leaf thickness (0.35 mm).