Study On The Optimization Of Growth And Development Conditions And The Mechanism Of Development Process Of Gametophyte Clones In Saccharina Japonica

The clonal gametophyte of Saccharina japonica has the characteristics of developmental totipotency and genetic homozygosity.It is the object of kelp germplasm conservation and can provide abundant genetic material for kelp germplasm improvement and seed breeding.This study evaluated the impact of key environmental factors on the vegetative growth,development and reproduction of kelp gametophytes,and further optimized the environmental conditions regulating the growth,development and reproduction of kelp gametophytes.From the aspects of appearance,subcellular structure,physiological and biochemical and transcriptome,etc.,the characteristics and differences of gametophytes in the growth,development and reproduction stages are compared and analyzed.The main findings are as follows:（1）The effect of Fe³⁺on the vegetative growth and reproductive development of the kelp gametophyte clonal line.The results showed that 3.60μmol/L of Fe³⁺was the most effective at promoting vegetative growth in kelp gametophytes in seawater with N and P concentrations of 0.825 mmol/L and 0.0336 mmol/L,respectively.Under different N and P concentration conditions,the maximum fluorescence yield of kelp gametophyte could be increased when the Fe³⁺concentration was 3.60～17.80μmol/L,and there was no significant difference between each concentration group（P>0.05）.Under different nitrogen and phosphorus concentrations,the oogonium formation rate,ovulation rate,and seedling formation rate were significantly improved at Fe³⁺concentrations of 3.60μmoll/L,8.90μmol/L,and 17.80μmol/L.So the Fe³⁺could increase the developmental process.When the nutrient concentration of N and P reached 0.275 mmol/L and 0.0112 mmol/L,0.36～17.80μmol/L Fe³⁺could promote the transition from vegetative growth to reproductive growth.At Fe³⁺concentrations of 3.60～17.80μmol/L,the gametophyte development indicators did not differ（P>0.05）among different nitrogen and phosphorus groups.Even if the N and P concentrations reached 0.825 mmol/L and 0.0336 mmol/L,all of the gametophytes remained in vegetative growth without development under the iron-free conditions.If Fe³⁺was sufficiently available,most of the gametophytes（65%）remained in vegetative growth under low nitrogen and phosphorus conditions.A small proportion of gametophytes（35%）entered the developmental state,but the development lagged behind the other N and P concentration groups.Iron and N,P nutrients have a synergistic effect on the vegetative growth and reproductive growth of kelp gametophytes.Iron is a key factor in the process of vegetative growth,gametognesis and sexual reproduction of kelp gametophytes.Iron deficiency will seriously inhibit the reproductive development of gametophytes.（2）Study on optimal light conditions for vegetative growth and reproductive development of S.japonica gametophyte clones.The optimal light conditions for vegetative growth of female and male gametophytes of kelp are basically the same.The relative growth rate（RGR）kept increasing under the light intensity of 40～80μmol photons/（m²﹒s）.Under the same intensity of blue,red and white light,the RGR decreased in turn but the differences were not significant（P>0.05）.Long photoperiod of 16L:8D but not 24h continuous light was more conducive to vegetative growth compared to short photoperiod.It was found that light intensity of 60μmol photons/（m²﹒s）was the best for the gametangium development and reproduction.Compared to white light,blue light was more conductive to the reproductive development of gametophytes,while red light has inhibition effect.Short photoperiod（8L:16D）was more beneficial to the reproductive development of gametophytes compared to the long photoperiod（16L:8D and 24L:8D）.Summarily,the bule light of80μmol photons/（m²﹒s）with a photoperiod of 16L:8D was the best for the vegetative growth,while the blue light of 60μmol photons/（m²﹒s）with short photoperiod of8L:16D was the best for the gametangium development and reproduction of kelp gametophyte.The results can be applied in the S.japonica seedling breeding industry based on gametophyte clone.（3）Research on the cell structure and physiological and biochemical characteristics of S.japonica gametophyte in different growth and development stages.The dormant gametophytes were crushed and transferred into the appropriate conditions,then they enter into rapid vegetative stage of the best in the following 6d,during which the filament gametophyte branched and cells began to appear nuclear proteins.The soluble protein content is the highest,and the number and volume of mitochondria increase.The maximum fluorescence yield（Fv/Fm）,actual photosynthetic rate（Y（II））,initial slope of photosynthetic curveα,and apparent photosynthesis rate of photosystem II are all the maximum.At this stage,the stress resistance is strong,and the specific activities of superoxide dismutase,peroxidase and ascorbate peroxidase are all significantly higher.The gametophyte grows to the9th day,and it is in the transition from vegetative growth to reproductive development and the formation of oocysts.The formation of oocysts may be related to the golgi apparatus.This stage is in the early stage of gametocyte development,and its resistance to stress is poor,especially to light.Fv/Fm,Y（Ⅱ）,initial slope of photosynthesis curveα,respiratory oxygen consumption rate,apparent synthesis rate and Rubis CO,MDH,and soluble protein content all decreased.After the gametophyte was broken and cultured for 12 days,it is already in a state of normal development.Its stress resistance is enhanced,and the physiological metabolism of the gametophyte cell is improved,which accumulates nutrients for better development.Taken together,kelp gametophytes show different morphological structures and physiological and biochemical states during growth,development and reproduction.（4）Transcriptome analysis of different growth and development stages of S.japonica gametophyte.Compared with the"dormant"state,the number of differentially expressed genes（DEGs）in fast vegetatively growing gametophytes was6891,of which the number of genes up-regulated was 3024 and the number of genes down-regulated was 3869;Compared with the rapid vegetative growth stage,the number of DEGs was 12,304 when it entered the developmental stage,of which the number of genes with up-regulation was 7,135,and the number of genes with down-regulation was 5169.GO analysis of 9710 DEGs showed that 2023 DEGswere annotated to molecular function,and 1975 DEGs were classified as catalytic activity.There were 2 DEGs enriched in GO:0004784 superoxide dismutase activity,28 DEGs are enriched in GO:0004601 peroxidase activity.These genes enriched in catalytic activity may be involved in the catalysis of antioxidant enzymes during the growth and development of gametophytes.1093 DEGs were annotated to Cellular Component,of which 1933 DEGs were annotated as organelle,and 295 DEGs were enriched to GO:0005794 golgi apparatus,which may regulate the increase of Golgi apparatus during the growth and development of gametophytes.121 DEGs were enriched in GO:0044429 mitochondrial part,which was involved in mitochondrial enlargement and respiration during gametophyte growth.7323 DEGs were annotated to the biological process classification,2233 DEGs were significantly annotated to the function of biological regulation.9 DEGs were enriched into GO:0015996chlorophyll catabolic process.The 4 DEGs were enriched to GO:0010380 regulation of chlorophyll biosynthetic process.These genes may be involved in the regulation of gametophyte photosynthesis and changes in chlorophyll content.The results of KEGG enrichment analysis showed that genes in the ubiquitin-mediated proteolytic pathway were down-regulated during the transition from vegetative growth to development,and may be involved in the regulation of gametophyte development.The genes on the oxidative phosphorylation pathway were significantly up-regulated during the transition from vegetative growth to development,indicating that the genes on this pathway may be involved in the regulation of gametophytes from vegetative growth to development,maintaining the material and energy metabolism of the transition from gametophyte cells to gametes,and the normal operation of respiration.Genes enriched on the peroxidase pathway were significantly down-regulated from dormancy to vegetative growth,and may be involved in the regulation of reactive oxygen species during the growth and development of gametophytes.