| Neoporphyra haitanensis is a traditional coastal aquaculture species in China; it is rich in essential amino acids, minerals, and vitamins. It is not only beneficial economically, but also adjusts the ecosystem. However, fine varieties of N. haitanensis are lacking even with the enlargement and extension of the N. haitanensis growing area. Since humans began breeding N. haitanensis, most produced strains have been domesticated from wild N. haitanensis collected from rocks. The germplasm collection and usage is considered the bottleneck issue that often causes N. haitanensis quality degradation. Therefore, it is important to breed new varieties. Selective breeding, cross-breeding, and mutation breeding are among the most common methods used to breed new N. haitanensis varieties. Mutation breeding is a quick, simple, and convenient genetic tools. For several decades, natural means to induce genetic diversity have been exploited to breed new varieties, as the naturally occurring mutations are insufficient. One of the most important genetic breakthroughs was the invention of artificial methods to induce mutations. Physical mutagenesis is a safer and more efficient procedure than chemical mutagenesis. To obtain artificial mutants, N. haitanensis (NSD35) gametophytes were treated with different γ-ray irradiation intensities (700 Gy, 900 Gy, 1100 Gy, 1300 Gy, and 1500 Gy). After culture recovery, the results showed that gametophytic cell death increased with the irradiation escalation, with more than 90% cell viability after low radiation dose treatment (700 Gy and 900 Gy). With doses more than 1100 Gy, the cell viability decreased, in which the NSD35 cellular space became larger and a few middle cells died, although no obvious change was observed in tip cells. After induction, the cell morphology changed with higher radiation doses (1300 Gy and 1500 Gy). Some cells exhibited microscopic features, such as enlarged size and alternation from irregular cell shape to circular for some cells. Notably, the cell death rate decreased systematically from the blade base to its tip and from that to its center. Microscopic examination revealed that the number of mutant cells increased and then decreased with the radiation dose rise. The pigmented mutant cells frequency was higher in low radiation doses, further increasing with the rise in irradiation from 700 Gy to 1300 Gy. Nonetheless, the pigmented mutant cells frequency decreased with the highest dose of 1500 Gy. The best mutagenic effect was observed in gametophytes treated with 1300 Gy as they had the largest number of pigmented mutant cells. Cells cultured in vitro were obtained by enzymatic hydrolysis and individual regeneration. Preliminary screening of 67 mutants was performed to construct the mutant library, and 45 pigmented mutant strains, 51 morphological mutants, and 11 other mutants were obtained. Most of the mutants showed two or more mutations of phenotypic character. Twenty-one F1 generation cell lines were screened, and 14 traits (length, width, fresh weight, daily length growth rate, daily width growth rate, daily weight growth rate, length-breadth ratio, base thickness, center thickness, tip thickness, color, base section form, leaf form, saw tooth size, twist, and maturity) were analyzed by correlation analysis and system analysis. The coefficients of variation results showed significant phenotypic differences between F1 and control cells. The F1 coefficients of variation ranged from 8.74% to 59.49%, revealing a moderate variability. Correlation analysis also showed that most of the traits had significant correlations. There was no significant correlation between the length and width or fresh weight, while a significant positive correlation was observed between the width and leaf fresh weight (P<0.01). Significant positive correlations were also found between quantitative traits, while there was no correlation between qualitative and quantitative traits. These allow indirect selection to improve breeding efficiency. Cluster analysis showed that at the Euclidean distance of 20, all 21 individuals analyzed were clustered into four groups. Overall, the phenotypic traits of different groups were significantly different. Mutants are critical materials for studying the genetic regulation of mechanisms involved in the control of economically important N. haitanensis traits. Pigmented mutants are a dominant-marker trait used for breeding that have high value for theoretical research and practical applications. Our study identified many pigmented mutants that can be used to study relevant biological mechanisms. Nevertheless, the variation coefficients of four quantitative characters (length, width, fresh weight, and thickness) of the F1 N. haitanensis generation were less than those of the control group and were biased towards negative variation. For example, more thin leaf mutants were obtained after the gamma rays´ treatment. It is interesting as a thin N. haitanensis is more suitable for automatic processing, and its primary products can be reprocessed with higher added value. In conclusion, after being treated with γ-ray, the N. haitanensis blades were guided to variation enabling the selection and cultivation of new strains, from which strains with improved traits strains were selected. This study provides interesting materials for accelerating the N. haitanensis breeding research and the selection of excellent varieties. |
