Elementary Studies On Sargassum Thunbergii (Phaephyta) Cultured In Vitro

Sargassum thunbergii (Mert.) O. Kuntze (Sargassum Phaeophyta) is a common sargassum species distributed in tidal and subtidal area along Chinese coast. It is an important economic resource for industrial extraction and bait for sea cucumber and abalone aquaculture. S. thunbergii is also the spawning and feeding refuge for crabs, mollusks, and clam worm in tidal and subtidal area. In recently years, the nature resource has been over-exploited as more and more aquaculture fields are built, and as a result, the demand for seaweed is increasing. It would be a great help to apply seaweed cultivation instead of exploiting wild resource for seaside ecology preservation.Tissue culture is an efficient approach of micro propagation for S. thunbergii which would help to promote S. thunbergii cultivation. Only a few available studies have described the process of tissue culture for micro propagation in Sargassum species. Polne-Fuller (1987) successfully got pigmented callus by holdfast explants of Sargassum muticum. Shinji (1996) established axenic tissue culture of Sargassum confusum and obtained brown callus cells and primary lateral branches. Sections of Sargassum heterophyllum (Mooney and van Staden, 1985) and Sargassum ringgoldianum (Notoya, 1992) directly produced shoots from tissues in liquid medium. Reddy (2006) carried out the Sargassum tenerrimum callus induction in solidified PESI medium, but no further study was performed. As to S. thunbergii, few studies that focus on its axenic tissue culture involving callus induction, plantlets regeneration and protoplast preparation are available. In the present study, a series of experiments are carried out in hopes of developing an efficient callus inducement method, from which the clonal micro propagation and new plantlet regeneration of S. thunbergii could be achieved: (1) plant growth regulators (PGR) are applied and its effect on explant morphogenesis responses is estimated; (2) different portions of S. thunbergii are selected and their morphogenesis responses in different origins of explants are compared. (3) Both solid medium and liquid medium are applied and the differences between the two culture systems are analyzed. The results are exhibited below:1. The effect of PGR on morphogenesis responsesThe presence of three PGR (NAA, 6-BA, 2,4-D) strongly promotes the induction of transparent callus, pigmented callus, bottom callus and filaments. Whatever the concentration of PGR is, one or more of these morphogenesis responses could be observed in PESI-PGR liquid media. PESI media without PGR only have transparent callus growing, and in seawater media, callus or filament were never found. However, the appearance of any morphogenesis response is not determined by a certain concentration of PGR.2. The regeneration of adventive shootsIn the present study, PGR had little simulative effect or had negative effect on adventive shoots regeneration because the highest regeneration rate was found in seawater media. Adventive shoots were only observed on holdfast explants. It was autoclaved seawater medium that got a 50% adventive shoots regeneration rate compared with any nutrition enriched medium (PESI mediums and PESI-PGR mediums). The highest regeneration rate in PESI-PGR media series was 6.6%, and no adventive shoots was found in PESI media.3. The difference of morphogenesis responses among four kinds of explants Holdfast of S. thunbergii is the best choice for tissue culture. The explants of holdfast exhibited the overwhelming quality of all kinds of morphogenesis (transparent callus, pigmented callus, filaments, bottom callus and adventive shoots regeneration) than explants of stem, branch and leaf in liquid mediums. Stem explants are the second choice, which only developed transparent callus in early stage of culture, and little pigmented callus five months later. Explants of branches and leaves had not represented obvious morphogenesis.4. Comparison of liquid medium and solid mediumIn present study, liquid medium is superior to solid medium in tissue culture of S. thunbergii. Most of the explants, no matter the origins of explants, have not presented morphogenesis responses. Only a little of pigmented callus and adventive shoots appeared on four explants in solid media. In contrast with solid medium, morphogenesis responses of liquid medium are multiform and common. The difference may result from the unique quality of S. thunbergii, or because the culture environment is suitable for liquid medium culture but not optimal for solid medium culture.5. High adventive shoots regeneration rate were observed in seawater medium accompany with diatom contamination. On the contrary, seawater medium with germanium dioxide and PESI medium with germanium dioxide have not exhibited any adventive shoots regeneration. It is suggested that, the presence of germanium dioxide or diotom may affect the adventive shoots regeneration of S. thunbergii.