Varietal identification of cucumber using seed protein analysis by ultrathin layer isoelectric focusing technique
2010
Onwimol, D., Kasetsart Univ., Nakhon 73140 (Thailand). Dept. of Horticulture | Chanprame, S., Kasetsart Univ., Nakhon Pathom 73140 (Thailand). Center for Agricultural Biotechnology | Thongket, T., Kasetsart Univ., Nakhon Pathom 73140 (Thailand). Dept. of Horticulture
Genetic purity test is crucial for quality control in seed production, especially for F-1 hybrid variety. The test should be reliable, rapid, simple and affordable. Unfortunately, the most reliable DNA fingerprint test is costly while the simple grow-out test is a very time-consuming method. The ultrathin-layer isoelectric focusing (UTLIEF) test was reported as a new, rapid and relatively cheap technique to differentiate plant varieties based on the polymorphism of feed protein in rice, corn and tomato (Yan et al., 2006; Leist and Knoblauch, 2003; Wang et al., 2000). Cucumber (Cucumis sativus L.) is the major crop for F-1 hybrid seed production of Thailand. The applicable UTLIEF technique as the genetic purity test for cucumber seed will greatly enhance its seed quality control efficiency. The objective of this experiment was to find a suitable extraction buffer and pH gradient to be used for running the UTLIEF on the protein stored within the cucumber seed in the IEF-SYS horizontal electrophoresis system. Four buffer solutions including water, 0.005 M NaCl, phosphate buffer and 0.005 M Na2 EDTA were tested in combination with two pH gradients of 2-11 and 4-5 plus 3-10 range. It was found that pH range of 2-11 on polyacrylamide gel and extracted protein by water yielded the most polymorphic and clearest bands. Positive result was found when the technique was used to identify 5 open pollinate commercial cucumber varieties. The efficiency of UTLIEF technique in hybridity test for F-1 hybrid cucumber seed needs to be tested to confirm the feasibility of using this UTLIEF technique in routine genetic purity test for cucumber seed production.
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