| The genetic engineering of Laminaria japonica is being perfected and put into practical use. The high density culture of transgenic kelp gametophytes for effective expression of foreign gene is one of the key technologies for its application. To avoid problems of contamination of culturing transgenic marine plants in open systems, the biochemical engineering technology including photobioreactors utilized in the cultivation of transgenic gametophyte clone is an effective alternative to the commercial aquiculture of transgenic kelp. Up to now, few papers have been published on the high density culture of transgenic seed for effective expression of foreign gene. In this thesis, the female gametophytes of transgenic Laminaria japonica expressing rt-PA were as the materials. The research had been divided into three parts. Firstly, transgenic kelp gametophyte cells were cultured in the shake flask to find the growth characteristic of gametophytes. Secondly, a bubble-column photo-bioreactor with a working column of 300 ml had been designed and manufactured. The result showed that the bioreactor is very favorable to culture the gametophyte of kelp and it can be used easily. Through the experiment of optimizing the process conditions, A maximum final dry cell weight of 1,120 mg l-1 was obtained in batch culture with an initial dry cell weight of 126 mg l-1, light intensity of 30 μE m-2 s-1, aeration rate of 1.2 l air min-1l-1 culture (vvm), nitrate of 1.5 mM and phosphate of 0.17 mM during 20-day cultivation period. At last, the culture system can be scaled up to 2500 ml and effect of different light regimes on culture growth of gametophytes was investigated. The fluctuating light increasing from 10 μE m-2 s-1 to 105 μE m-2 s-1 with a photoperiod of 14h light /10 h dark will significantly improve the biomass. The research results would serve as the theory basis and technology support for the commercial aquiculture of transgenic Laminaria japonica gametophytes. |
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