| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 99 === Papaya (Carica papaya L.) is one of the important economical fruit crops in Taiwan, and it can be propagated by seeds, grafting, cutting and tissue culture. However, papaya is propagated mainly by seeds. Nevertheless, freshly harvested papaya seeds germinate poorly and unevenly. Even though numerous researchers had reported many kinds of treatments to improve the germination, results were different due to cultivars and environmental factors. Gibberellins and heat treatments have been reported to promote the germination of papaya seeds. However, the mechanism of how heat treatment breaks the dormancy of papaya seeds is not clear. Therefore, it is an important topic to understand how heat treatment induces the germination of papaya seeds.
In this research, I use the method of proteomic analysis to investigate the possible mechanism of heat treatment on the dormancy breakage of papaya seeds. Unfortunately, there are no related protocols have been reported for protein preparation from papaya seed for proteomics analysis. Therefore, in this study I established the optimized protocol of protein extraction for sample preparation of two-dimensional electrophoresis. The protein extraction mixture for 5 day-imbibed papaya seeds was a Tris-HCl base buffer. The extracted solution was then precipitated by 10% TCA/Acetone (TCA-A) or extracted by phenol followed by 0.1M ammonium acetate-methanol precipitation (P-AA-M). The results showed that the method of P-AA-M could increase at least 5 bands than TCA-A in SDS-PAGE analysis. Two of the bands were assessed via LC-MS/MS and the proteins identified included “unknown proteins”, with the highest ratio (34%), “energy metabolic proteins”, the minor category (20%), and “cell cycle proteins”( 14%). P-AA-M could reduce the problems of streaking and smearing in 2D gel analysis, enhance the efficiency of protein separation, and therefore, increase the number of protein spots identified.
Application of 36℃ hot water immersion for 5 hours to 5 day-imbibed papaya seeds would induce the expression of proteins related to calcium signal transduction (CBL and CDPK), small heat shock protein (17.5 sHSP), energy metabolism (G3PDH and ATP synthase epsilon chain), and transcription (GR-RBP). It is assumed that after the heat treatment, the papaya seeds would be released from dormancy by a series of physiological events including signal transduction and energy metabolism, increase of the embryo growth potential, overcome the physical constraint of testa, and then germination.
According to the results, the possible mechanism of heat treatment on dormancy breakage of papaya seeds would be through GA, but not brassinoids, biosynthesis, since brassinzole, a brassinoids synthesis inhibitor, was unable to inhibit the germination of the seeds treated with the hot heat immersion
Even though heat treatment could induce the germination of papaya seeds, the effects would be various depending on degree of the dormancy. Hot water immersion treatment with 34, 36, or 38℃ could enhance the germination of after-ripened seeds. The longer the after-ripening time applied, the better the efficacy of dormancy releasing. Moreover, the germination capability of papaya seeds acquired from heat treatment could be preserved up to 80% if the seeds were prepared by fast drying method (36℃ forced air for 6 hours), but could not be preserved by slow drying method.
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