Establishment Of Tissue Culture System And Plantlet Regeneration Of Kappaphycus Alvarezii

Kappaphycus alvarezii was a large economic alga, which contained a lot of carrageenan, was an important raw material used in the extraction of carrageenan. Carrageenan in the algae was not only widely used in food, chemical, biochemistry, medicine, etc., having a significant economic value, but also an important raw material for preparation of oligosaccharides, therefore it had great prospects for development. Kappaphycus alvarezii became an emerging aquaculture industry in the coastal harbour when it was introduced into Hainan Province for decades, but due to frequent breeding diseases, germplasm resource degradation and the deterioration of the ecological conditions of the aquaculture water, its farming in Hainan Province was declining. In view of this, we studied the explants detoxification, medium screening, propagules and callus induction, organic carbon source and so on, to establish a suitable cell and tissue culture technology system for Kappaphycus alvarezii, which was conducive to its germplasm conservation, artificial breeding of detoxified seed, rapid propagation of breeding seed, and laid a foundation for future development of it on cell engineering and genetic engineering researches. Through a series of system studies, the researches were as follows:1. Establish a set of suitable explants detoxification methods for Kappaphycus alvarezii. Firstly, pretreated explants were sonicated for10minutes, and then soaked in a0.05%HClO solution for5seconds, washed away disinfectant adsorbed in the surface of explants, finally inoculated into the seawater containing30mg/L of ampicillin sodium salt and30mg/L carbenicillin sodium penicillin at the same time for72hours. It greatly reduced the damage to explants caused by the detoxification, significantly increased the survival rate, and obtained desired results in the further induction process.2. Successfully acquire an improved PES liquid medium formulation and an improved f/2solid medium formulation which were suitable for Kappaphycus alvarezii explants culture by screening and improvement. The explant survival rate was67%in the improved PES liquid medium, and the budding rate of propagules has also been significantly improved; meanwhile, both ends of the explants were induced shrubby propagules and white filamentous callus in the improved f/2solid medium.3. Through the test that the plant hormone induced propagules and callus on explants, we found:the induction of propagules was not affected significantly when IAA, GA and6-BA were added alone in the liquid medium, respectively; when the medium was added6-BA and2,4-D at the same time, the induction process of propagules appeared synergistic effect and the induction of propagules was promoted. In addition, a certain concentration of carbon source1was added to the medium containing plant hormones was significantly improve the average number of propagules, the average budding number was6.0+0.82, which was quite different from the other treatments(p<0.01). The study also found that the average budding number of propagules on the explants increased with the extension of the carbon source1towards explants processing time within a certain processing time.4. The study found that explants cultured in the improved PES liquid medium and the improved f/2solid medium were differentiated. Explants in liquid medium can only be induced propagules, and most of them only occurred from the incision of the physiological upper end of explants, having obvious physiological polarity; explants cultured in the improvement of f/2solid medium, whose incisions which were in contact with the solid medium could induce a large number of shrubby propagules, and callus could be induced in the other end of the incision, but the biological polarity of explants themselves did not affect propagules and callus induction.5. There were significant differences between a new type of callus induced in PES liquid medium containing2,4-D and the carbon source2as well as a new type of white filamentous callus we has gained and reported research results. Cell structure of callus was dense, and there were a large number of projection cells similar to buds formed in the surface of the callus. The callus inoculated in the PES liquid medium containing carbon source1,6-BA and IAA, can induce a large number of propagules, its average budding number was17.9±2.02.6. Callus inoculated in the f/2solid medium containing6-BA and2,4-D induced the regenerated plantlets successfully, and the cultured period of regenerated plantlets was shortened greatly, furthermore, new seedlings growth rate was higher than the previously reported results, their daily growth rate was4.49%; callus inoculated in the liquid medium also could induce a large number of regenerated propagules, their average budding number was21±6.77. and their daily growth rate was4.33%.