| Summary: | 碩士 === 國立高雄大學 === 生物科技研究所 === 95 === Bioreactors provide a rapid and efficient plant propagation system for plant cells, tissues, or somatic embryos. Previous studies showed that there were many defects in the marketing bioreactor, which including the input airflow was too fast to cause easily foam formation during immersion, the depressuring rate was also too fast to result in an uneven distribution of plant materials after immersion, and the air exchange only occurred in the growth compartment during immersion. Hence, this thesis was firstly to modify the marketing bioreactor, namely, to add a vent hole and insert a CO2 sensor in the growth compartment, then, to install a pneumatic server and connect a computer. It can accurately adjust the immersion frequency of medium-holding compartment and the airflow rate of growth compartment, separately via a program logical control . So, a programmable bioreactor was developed. This new type bioreactor not only solves the above-mentioned problems of marketing bioreactor but also can real-time monitor the CO2 concentration in the bioreactor. Secondly, the adventitious buds of Phalaenopsis Sogo Yukidiam ‘V3’ were cultured at different airflow rates and immersion frequencies using the programmable bioreactor, simultaneously compared with commercial bioreactor and traditional flask, to search an optimal growth condition for adventitious buds and to study the effects of different cultural conditions on the physiological and biochemical properties of Phalaenopsis plantlets.
The results clearly revealed that the increases in immersion frequency was conducive to utilize efficiently the medium components, such as sucrose, NH4+, K+, NO3-, phosphate, and malate, for adventitious buds, and leading to a higher growth value occurred. Concerning the circadian rhythm of CO2 concentration in the growth compartment, it exhibited a CAM pattern when adventitious buds were cultured in programmable bioreactor with 4 ml per minute airflow rate and 1 min immersion every 6 h after 2 months. Under same airflow condition, however, the decrease in immersion frequency into 24 h resulted in the CO2 pattern became a C3 type. Evidently, immersion frequency will affect the CO2 metabolism of Phalaenopsis plantlets. Once the immersion frequency was 36 h, the CO2 evolution was still similar to a C3 type, but the CO2 concentration of growth compartment always reached above 8000 ppm during the whole day, and the growth of adventitious buds was significantly inhibited. This result strongly implies that the relationship between the growth of plant materials and the CO2 level of environmental condition is not so straightforwardly akin to the CO2 enrichment study. Furthermore, increasing airflow rate and decreasing immersion frequency simultaneously not merely reduced growth value, but also enhanced the levels of H2O2 and ascorbate as well as the activities of antioxidative-related enzymes, such as superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11). Nonetheless, chlorophyll fluorescence analysis showed that the maximum quantum yield (Φp0) was no significant difference among the above conditions. This data demonstrates that the PSII of Phalaenopsis plantlets still work normally.
Although the conditions of airflow and immersion frequency adopted in the programmable bioreactor did not significantly improve the growth value and bud formation of Phalaenopsis adventitious buds, even the results were worse than that cultured in the marketing bioreactor with similar immersion frequency or the traditional flask containing agar-solided culture; however, this thesis clearly showed that bud formation was inhibited by a higher airflow rate and the residuary medium of the best growth condition in programmable bioreactor, still. rich amounts of glucose and fructose were not consumed. Therefore, it is convinced that the best growth condition for Phalaenopsis adventitious buds could be found in the future via increasing immersion frequency to hasten the utilization of medium nutrients and simultaneous decreasing the airflow rate in growth compartment.
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