| Pyropia haitanensis as a typical warm-temperate cultivar uniqued to China,has been mainly cultivated in the coastal areas of Zhejiang,Fujian and Guangdong provinces.Its production occupying about 75%of the annual national total production of laver in China.So far,self-retained species are dominated in the large-scale artificial cultivation of P.haitanensis,which resulted inobvious germplasm degradation,yield and quality declinein production.Genetic breeding new varieties is still an important demand for nori farming and industrial development.In this study,the germinating conchospores of P.haitanensis were originally used as experimental materials for induction of pigmentation mutants via illumination of short-wavelength ultraviolet ray radiation（UV-C）.Moreover,the wild-type strain（WT）collected from the open sea area and stored strains（PUT,PR）preserved in laboratory were used as materials for mutagenesis of thin-blade strains and high-temperature resistant strains using UV-C irradiation,together with somatic cell cloning method and selective breeding,respectively.The growth indexes and physiological characteristics of the new strains,their parents and wild-type strains were comparatively analyzed.This study explored a new method for genetic breeding of P.haitanensis via mutagenesis of germinating conchospores and also provided a technical basis for more efficient and safe selection and breeding of high quality and environment resistant strains.The relevant researches and their results are as follows.1.A novel pathway to produce pigmentation mutants by short-wavelength ultraviolet ray irradiation on the germinating conchospore in P.haitanensisIn this study,short-wave ultraviolet（UV-C）irradiation with different doses（50,100,200,300,400,500 and 600 J/m2）was used to induce pigmentation mutants during the germination of conchospores in P.haitanensis.The results showed that the low-dose irradiation（50 J/m2）promoted the germination of conchospores,while irradiation doses above 100 J/m2inhibited the germination and growth of tetrad germlings.Therefore,low-dose of UV-C irradiation was used in the production to promote the germination of conchospores and improve the production efficiency of P.haitanensis.In the dose range of 50-400 J/m2,the frequency of color chimeras increased with the increase of irradiation intensity.When the dose was 300 J/m2and 400 J/m2,and the pigmentation mutation rate was 15.22%,17.18%.Among them,the regenerated color chimeras that appear were generally 2-color sectored chimera and 3-color sectored chimera,yet the proportion of 4-color sectored chimera was the least.The frequency of pigmentation mutants decreased with increasing irradiation dose above 400 J/m2,indicating that the mutagenesis effect was the best when the dose was 300 J/m2or 400 J/m2.At the same time,UV-C irradiation also led to the decrease in blade aspect ratios.Somatic cell germlings with single colored pigmentation were also isolated from color sectored chimeras by enzymatic hydrolysis.This study provides a novle pathway for preparation of artificial color mutants and mutation breeding in P.haitanensis.2.Selection and characterization of thin-blade strains of P.haitanensisMutation of pigmentation mutants and selection of thin-blade strains of P.haitanensis via irradiation of germinating conchospores using UV-C（300 J/m2）irradiation were researched.Two improved strains with obvious growth advantages were isolated and screened from the pigmentation mutants produced after the mutagenesis.These two improved strains with thin-blade trait（UR-2 and UR-6）were obtained using single cell cloning technique.Analysis of growth and biochemical indexes showed that the mean length of blade in the UR-2 strain at 60 days-old was61.67 cm,which was 2.08 and 3.52 times longer than that of the parental PR（29.67 cm）and WT（17.50 cm）strains,respectively;and the mean length of blade in the UR-6 strain was 72.33 cm,which was 2.44 and 4.13 times higher than that of the parental PR（29.67cm）and wild-type WT（17.50 cm）strains,respectively.The contents of Chl.a and phycobiliprotein of UR-2 strain were 12.13 mg/g and 162.09 mg/g respectively,which were 29.73%and 53.16%higher than those of PR strain,respectively,and 31.25%and60.37%higher than those of WT strain,respectively.The contents of Chl.a of UR-6strain were 12.69 mg/g which were 35.72%and 33.95%higher than those of PR and WT strains,respectively.Also,its total phycobiliprotein contents were 165.43 mg/g,which were 60.23%,and 63.68%higher than those of PR and WT strains,respectively.The mean thickness of blade was 22.92μm in UR-2 strain.Compared with PR（28.30μm）and WT（36.20μm）,the mean thickness of blade in the UR-2 strain was decreased by 19.01%and 36.68%,respectively.The mean thickness of blade was 17.95μm in UR-6 strain.Compared with PR and WT strains,the mean thickness of blade in UR-6 strain was decreased by 36.57%and 50.41%,respectively.The above results indicated that the UR-2 and UR-6 strains have the characteristics of fast growth,high quality and thin-blade,which can be served as germplasms for breeding thin-blade strain of P.haitanensis in the near future.3.Selection and characterization of high-temperature resistant strains of P.haitanensisMutation of pigmentation mutants and selection of high-temperature resistant strains of P.haitanensis using UV-C（300 J/m2）irradiation of germinating conchospores were researched.After two weeks culture at 24°C,the mutants from germinating conchospores were incubated at 28℃for another 2 weeks,and then restored to 24°C after majority of the conchospore germlings were died.We selected the regenerated thallus with fast growth and improved characteristics,and then obtained three excellent strains through somatic cell enzymatic hydrolysis and parthenogenesis.The 35-day-old blades from F1of WT,PUT,UT-6,UT-11 and UT-18 strains were used for preparing the blade discs.After cultured at 24℃,28℃and 30℃for heat temperature resistance,UT-6 and UT-18 strains were selected as high-temperature tolerant strains by measuring the growth rates of the blade discs in each strain and their capacities tolerant to the high temperature.When the conchospores of the UT-6 strain was cultured at 28℃for 15days,the survival rate of conchospores was 8.63 and 7.39 times than that of WT and PUT strains,respectively.The survival rate of conchospores of UT-18 strain was 10.48and 8.97 times than that of WT and PUT strains,respectively.When the conchospores of the UT-6 strain cultured at 30℃for 15 days,the survival rate of conchospores was10.34 and 9.07 times than that of WT and PUT strains,respectively.The survival rate of conchospores in UT-18 strain was 14.14 and 12.40 times than that of WT and PUT strains,respectively.When the conchospore germlings of UT-6 strain which have been cultured at 24℃for 35 days and then were cultured at 24℃,28℃and 30℃for another 25 days,the mean length of blade in UT-6 strain was 2.75,6.51 and 4.57 times than that of WT strain,and 1.22,2.02 and 1.47 times than that of PUT strain,respectively.The mean length of blade in UT-18 strain was 3.32,8.71 and 9.30 times than that of WT strain,and 1.47,2.70 and 3.00 times than that of PUT strain,respectively.In addition,the chlorophyll fluorescence activities（Fv/Fmand Yield）of UT-6 and UT-18 strains blades were significantly higher than those of WT and PUT strains.Compared to the in vivo absorption spectrum of the blades,the peak values of all curves of UT-6 and UT-18 strains were higher than those of WT and PUT strains between wavelength of 350 nm and 750 nm.When the blades of UT-6 strain cultured at24℃,28℃and 30℃for another 25 days,the total phycobiliprotein content of UT-6strain was increased by 23.06%,91.12%and 197.06%compared with those of WT strain,respectively,and 7.86%,2.72%and 16.83%compared with those of PUT strain,respectively.Also,the total phycobiliprotein content of UT-18 strain was increased by26.91%,169.80%and 206.13%compared with those of WT strain,respectively,and11.24%,45.01%and 20.39%compared with those of PUT strain,respectively.The contents of Chl.a of UT-6 and UT-18 strains blades were significantly higher than those of WT and PUT strains.These results showed that UT-6 and UT-18 strains were resistant to high-temperature,and the capacity of UT-18 strain was better than that of UT-6 strain.These above results in this study indicated that the method of using UV-C irradiation mutagenesis on Pyropia germinating conchospores was feasible.The artificially selected UR-2 and UR-6 strains had the characteristics of fast growth,high quality and thin-blade,which could be expected to be bred into thin-blade cultivars of P.haitanensis.The UT-6 and UT-18 strains had the traits of fast growth,good color in visible and high-temperature resistance,and had more heat-tolerant capacities than those of their parental strain（PUT）. |
