短波紫外线辐照坛紫菜壳孢子制备色彩突变体的新途径

A novel pathway to produce color mutants by short-wavelength ultraviolet ray irradiation on the germinating conchospore in Pyropia haitanensis

中文关键词: 坛紫菜  壳孢子  短波紫外线  色彩突变体  分离

英文关键词: Pyropia haitanensis  Conchospores  Short-wavelength ultraviolet  Color mutants  Isolation

中文摘要:

      壳孢子萌发时期是坛紫菜(Pyropia haitanensis)减数分裂发生的时期，壳孢子萌发形成的最初4个细胞呈线性排列，形成减数分裂四分体，且发生遗传重组的四分体细胞可以决定叶状体的发育模式和性状分离。因此，诱变产生色彩突变的嵌合叶状体相比诱变叶状体产生的点状色块，将更易于获得突变细胞。本研究为获得坛紫菜人工色彩嵌合突变体，使用不同剂量(50、100、200、300、400、500和600 J/m2)的短波紫外线(UV-C，λ=254 nm)辐照坛紫菜壳孢子，培养数天后，在壳孢子苗中出现了色彩突变的嵌合叶状体。结果显示，低剂量(50 J/m2)的辐射促进壳孢子萌发，而辐照剂量高于100 J/m2则会抑制壳孢子萌发和分裂。辐照剂量在50~400 J/m2范围内，色彩突变体出现的频率随辐照剂量的增加而增加，辐照剂量分别为300和400 J/m2时，突变率分别达到15.22%和17.18%。其中，出现色彩突变的嵌合叶状体以2色块嵌合体和3色块嵌合体居多，4色块嵌合体最少，但辐照剂量增加至400 J/m2以上时，随着辐照剂量的增加，色彩突变体出现的频率反而下降，表明最适宜的诱变剂量为300或400 J/m2。同时，短波紫外线辐照也使色彩突变嵌合体的长宽比下降，采用生物酶解法从色彩嵌合体中分离出了单色的体细胞萌发体。本研究为坛紫菜人工色彩突变体的制备和诱变育种提供了新途径。

英文摘要:

      Ultraviolet mutagenesis is a safe and efficient method to induce mutations in laver. It has the advantages of non-pollution, high efficiency, easy operation, and low cost. It has been primarily used for mutagenesis of the filament, protoplast, or blade of cultivated Pyropia species, but is rarely used to induce genetic mutants from germinating conchospore. The germination period of conchospores is the period of meiosis in Pyropia haitanensis. The four progeny cells of germinating conchospores are linearly arranged forming a meiotic tetrad. The tetrad cells that undergo genetic recombination can determine the developmental pattern and segregation of traits in the thallus. In 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 color mutants during the germination of conchospores in P. haitanensis. The results showed that low-dose irradiation (50 J/m2) promoted the germination of conchospores, while irradiation doses above 100 J/m2 inhibited the germination and growth of tetrad germlings. Therefore, low-doses of UV-C irradiation were used in the production to promote the germination of conchospores and improve the production efficiency of laver. In the dose range of 50–400 J/m2, the frequency of color chimeras increased with increasing irradiation intensity. When the dose was 300 J/m2 and 400 J/m2, and the pigmentation mutation rate was 15.22% and 17.18%, respectively, and the death rate of conchospores was 51.70% and 61.00%, respectively. In the dose range of 50–500 J/m2, with the increase in UV-C irradiation dose, the proportion of 2-color sectored chimera showed a trend of first decreasing and then increasing, and the proportion of 3- and 4-color sectored chimera showed a trend of first increasing and then decreasing. Among them, the regenerated color chimeras that appear were generally 2- and 3-color sectored chimera, yet the proportion of 4-color sectored chimera was the least. When the irradiation intensity reached 500 J/m2, the majority of the conchospores died, and the death rate was 83.98%, and the frequency of color mutants was significantly lower than that of 300 J/m2 and 400 J/m2 dose groups. These results indicate that the mutagenesis effect was the best when the dose was 300 or 400 J/m2, which was convenient to obtain abundant genetic recombination and genetic variation in progeny cells. In addition, UV-C irradiation also had a significant effect on the early development of conchospores and phenotypes of pigmentation mutant arranged in tetrad germlings, which was mainly manifested in the large number of color-sectored blades developed from the irradiated conchospores. Simultaneously, UV-C irradiation retarded the development of cells at the top of conchospore germlings, inhibited the development of cells at the middle base toward the poles, and increased the lateral development, resulting in the decrease in blade aspect ratios. Somatic cell germlings with single colored pigmentation were also isolated from color sectored chimeras by enzymatic hydrolysis. The color species of the five monochromatic mutants obtained were basically consistent with the pigment variant sectors observed on color-sectored thallus, indicating that the obtained color mutants were derived from a single mutant cell on the maternal color-sectored thallus. In conclusion, UV-C irradiations can effectively mutate the conchospores of P. haitanensis, and appropriate irradiation doses (300 or 400 J/m2) can obtain a certain number of color-sectored thallus. This study provides a novel pathway for the preparation of artificial color mutants and mutation breeding in P. haitanensis.