The Ultraviolet Mutagenesis Breeding And Selection Of Stress Tolerance And Fast Growth Strains Of Gracilariopsis Lemaneiformis

Gracilariopsis lemaneiformis is a macroscopic red alga well known as the majoragarophyte in China. It becomes the third largest cultivated seaweed followingLaminaria and Porphyra in China. Gp. lemaneiformis distribute naturally along thecoast of Shandong Peninsula in China. The suitable temperature for growth of thewild population is between12-23°C. With the development of Gp. lemaneiformis, it isurgently needed to improve it’s heat tolerance, growth rate and agar content in orderto extend the cultivation period, enlarge the cultivation area and increase theproductivity and quality. Therefore it is essential to improve it genetically and toselect more worthy strains.In the present study, tetraspores and carpospores were firstly used as the materialfor Ultraviolet (UV) mutagenesis. Stress tolerance mutants were screened by hightemperature tolerance and growth rate mainly. The influence of high temperature onphysiological and molecular character of the mutants, cultivar981and wild strainswas analyzed. The aim of our research was to set up an appropriate means for geneticbreeding, to develop a suitable screening procedure for high temperature tolerantstains and to lay the foundation of breeding and application for stress tolerance andfast growth strains of Gp. lemaneiformis.Mutagenesis breeding is one of the most important methods for the selection ofnew strains of Gp. lemaneiformis. Newly attached tetraspores and carpospores weremutated by UV irradiation. The UV mutagenesis condition was set as:20W UV light,30cm distance，45s and48s irradiation respectively. The method of tetraspores andcarpospores UV mutagenesis was set up. The differences between haploid tetrasporesand diploid carpospores after UV mutagenesis were discovered. After UVmutagenesis, the pigment aggregation was discovered in tetraspores cell while it wasnot found in carpospores. Also, six pigment mutants were isolated after heat-stresswhile there was no in carpospores. It was indicated that the newly released tetrasporesand carpospores which were single cells were the nice materials for UV mutagenesis.A set of method for screening for high temperature tolerance mutants was set upsuccessfully. Mutants at different development stages were screened under heat shockin order to obtain mutants with constant heat-resistance. The screening ofthormo-tolerate gametophytes was divided into three phases. Heat shock of35°C inincubator for72h was imposed to tetraspores when they developed into gametophyticdiscs (2weeks after mutagenesis), germlings of gametophyte (6weeks aftermutagenesis) and mature algae (after cultivation in the sea) respectively. The survivalsafter the three screening were preliminary considered as thermo-tolerate strains. Thethormo-tolerate screening method of tetrasporophyte was optimized to two phases.When carpospores developed into5cm germlings of tetrasporophyte (8weeks aftermutagenesis), heat shock of41°C water bath for30min was carried on. When carpospores developed into mature algae (after cultivation in the sea), heat shock of35°C for72h in incubator was carried on. The survivals after the two screening phaseswere preliminary considered as thermo-tolerate strains.Two strains of female gametophytes and tetrasporophytes each withthermo-tolerance and high growth were isolated through high temperature screeningand analyzed with cultivar981and wild strains under heat stress. The specific growthrate (SGR) were compared and malondialdehyde (MDA) contents, SuperoxideDismutase (SOD) activities, proline contents and the expressions of heat shockproteins70(hsp70) under heat stress were also compared among the mutants, cultivar981and the wild strains. The agar contents were measured.Results demonstrated that the SGR of two female gametophytes growed and inlab and sea were similar with that of the cultivar981and higher significantly than thewild strains. While the SGR of two tetrasporophytes were higher than cultivar981and wild strains. It was inferred that tetrasporophytes may have the growth superioritybecause of diploid. The contents of MDA of gametophytes and tetrasporophytesmutants were lower than the wild, and SOD activities and hsp70expression werehigher than wild, which indicated their stress tolerance. All these may indicated thatthe mutants were more tolerant to high temperature and growed faster than the wild.The analysis process on the basis of these criteria may help to develop heat-toleranceGp. lemaneiformis cultivars in the future.In our study, the physiological and molecular responses of different strains to heatstress were coordinate. Research revealed, MDA contents that are the ultimate productof membrane peroxidation increased during the heat stress, which indicated the cellmembrane was harmed increasingly. SOD activity in all strains increased within thefirst day and decreased thereafter gradually within the heat stress period. Increasedactivity of SOD during short-term heat stress may provide protection from oxidativestress. We deduced that with prolonged periods of heat stress, the activities of SODwere inhibited and the degree of cell damage increased, which lead to MDA contentsaccumulated significantly. Proline plays an important role in the nonenzymaticantioxidant system and it’s contents increased firstly and then decreased under theheat stress. It showed proline can improve the stress tolerance of the strains at thebeginning. The expression of hsp70increased significantly after4h’s heat shock. Theexcessive expression of hsp70rapidly can relieve the harm of heat stress and producehigher heat tolerance. The physiological and molecular indicators provided data forunderstanding the heat stress mechanisms of Gp. lemaneiformis, and for breeding forhigh temperature resistant strains.