การเพาะเลี้ยงเซลล์เอนโดสเปอร์มจากข้าวไร่แบบแขวนลอยและการใช้ประโยชน์เพื่อศึกษาเอนไซม์ในวิถีการสังเคราะห์แป้ง

Abstract

Fungus contamination is a major problem encountered in the induction of callus from endosperms of immature spikelets of rice grown in natural environments. Sterilization requires stirring of immature spikelets in 2.6 % sodium hypochlorite in vacuo for 1 hour and 40 minutes, after washing in detergent and 70% ethanol. Using MS agar medium containing 3 % sucrose and 1 mg/l 2,4-dichloro- phenoxyacetic acid (2,4-D), friable calluses were induced from immature endosperms of upland rice cultivars, Dok Payom which is a fragrance rice, Khu Mueng Luong and Sew Mae Jun, as well as the recommended lowland cultivars, Hom Mali 105 (Jasmine rice) and Phatalung 60. These calluses could not proliferate in suspension culture with the widely used standard growth media viz. MS, LS, White's and No. By doubling the contents of vitamins and glycine in N, medium (N,2) as well as adding proline at 0.02 M in the presence of 4 % sucrose and 1 mg/l 2,4-D, callus formation could be induced directly in a suspension culture of immature endosperm from Dok Payom cultivar. The calluses in suspension culture were curved-plate in shape which grew sideways. Inclusion of 0.1 % Macerozyme in the culture medium helped prevent increase in size of the callusos but could not generate single-cell suspension. The endosperm suspension culture obtained from Dok Payom cultivar could increase by 60% in fresh weight after 15 days of subculture and could be used as a source of enzymes for the study of starch synthesis. Detection of the activity of starch synthase in the callus was done by measuring the rate of transfer of "Clglucose from ADP-[C]glucose to the primer starch molecules in the crude extract, which was found at 1.28 nmol/min per milligram of protein. Studies of other enzymes in the pathway will be carried out in the near future.