Construction Of Homozygous Strains And Cytological Studies On Growth And Development Of Porphyra Haitanensis Chang Et Zheng

Porphyra haitanensis Chang et Zheng was the unique economic seaweed in China. It has been cultivated widely along the coasts of South China with great economic and ecological significances. With the growing of the scale of the P. haitanensis cultivation, the phenomenon including degradation of germplasm, frequent occurrence of diseases and decline of yields and quality became increasingly serious, which affected sustainable and healthy development of Porphyra industry and put forword new demands on seedling cultivation techniques and germplasm. The growth and development of sporophytes (free-living conchocelis) and the development and differentiation of cells isolated from gametophytes (blades) in P. haitanensis were observed in detail. The alternation of nuclear phases of conchocelis developed from a single cell isolated from gametophytes and wild conchocelis were also studied, and on the basis of which, genetic homozygous strains of P. haitanensis were established, which are helpful for analysis of economic traits and molecular marker-assisted breeding in P. haitanensis.1. The growth and development of sporophytes:the sporophytes were cultured with aeration, and germinated into a large number of new buds under the conditions of right temperature, light and adequate nutrition. When cultured under heat-induced conditions, the conchosporangia grew directly from the majority of the filamentoas, there were, however, cases in which a few filamentoas gradually and evenly enlarged, became adventitious filaments, then continued to enlarge and finally transformed to the conchosporangia. After culturing for 30 days, the number of inflated cells gradually increased, the apical cells of conchosporangial branches became round with a large vacuole and lighter pigment in color, while the basal cells with a small vacuole and the deep pigment in color, then there were less typical sporangia with two conchospores in a few conchosporangia. By slow cooling and flow-water stimulation, the conchosporangia were fully mature with a large number of sporangia with two conchospores, then the wall of the apical cells or the middle cells of conchosporangia melted and formed a crack. A large number of conchospores moved forward along the conchosporangial tubes with deformed motion and escaped to outside through the cracks.2. Studies on the development of cells isolated from gametophytes of P. haitanensis:in order to investigate the developmental patterns of somatic cells isolated from male and female gametophytes in P. haitanensis and the developmental patterns of the single cells isolated from different parts of the gametophytic thallus (rhizoid section, bottom section, central section, top section, mature section), the single cells were enzymatically isolated from both male and female gametophytes of P. haitanensis and cultured, respectively. The results showed that although the developmental patterns of somatic cells of male and female gametophytes in P. haitanensis were nearly the same, some differences existed including that some somatic cells isolated from female gametophytes developed into carposporangia, a few somatic cells developed into conchosporangia-like cells; but some somatic cells isolated from male gametophytes developed into spermatangia from which spermatia were released, only few somatic cells developed into carposporangia-like cells. Researches on regeneration of the single cells isolated from different parts of a long gametophytic thallus showed that the developmental patterns of somatic cells isolated from gametophytes were affected by the part of the gametophytic thallus. The somatic cells isolated from different parts of the thallus regenerated into different types of plants because of the difference of the differentiation stages of the cells in the thallus. With the sites where the cells were isolated moving upward from rhizoid section, the percentages of both normal thalli and thalli regenerated from rhizoid cells decreased, and the percentages of the abnormal thalli also decreased or had no significant difference, but the percentages of the conchocelis increased.3. Construction of homozygous strains:ten gametophytic thalli with obvious traits were selected in farming population, from which single somatic cells were isolated enzymatically, cultured and developed into the strains of homozygous conchocelis. The homozygous conchocelis were prematurely cultivated to obtain mass filial generation gametophytes. The comparative results of parent gametophytes and filial generation gametophytes of every strain showed that the sex and shape of filial generation gametophytes were similar to that of the parent gametophytes. The filial generation gametophytes held the traits of the parent gametophytes, which demonstrated that the conchocelis strains were homozygous diploid.4. The alternation of nuclear phases of conchocelis developed from a single cell isolated from gametophytes:the alternation of nuclear phases of conchocelis from single somatic cell clone cultivation and wild conchocelis were abserved by cytofluorescence microscopy. The results showed that in early prophase of mitotic division loosely coiled chromatic threads appeared. In late prophase and early metaphase ten chromosomes were observed (2n=10), basically similar to the alternation of nuclear phases of wild conchocelis. It is showed that single somatic cells isolated from gametophytes occurred the natural chromosome doubling, resulting in developing into homozygous diploid sporophytes.