Chlamys Farreri Triploid Breeding Research

Cytology engeneering breeding technology was used in this study. The physiological factor, cytologic factor which influenced the triploid inducing, the suitable inducing parameters in the production and the biological characters of triploid scallops were studied combined with the method of cytobiology, histology, experimental ecology and biochemistry. All this study will settle foundation to the taking shape of the triploid scallop industry. The long time save of the germ cells of scallops is an important aspect in breeding. Spermatozoa cryopreservation of scallops Patinopecten yessoesis and Chlamys farreri were studied in 1997. Different diluents, different cryoprotectants, different freezing speeds, and different melting times were conducted to develop a suitable technique for the cryopreservation of the spermatozoa of scallops. To the two species of the scallops, the results showed that: Under the conditions that the natural sea water acted as the diluent and DMSO acted as cryoprotectant, and in the freezing process, spermatozoa were maitained from 25cm, 20cm to 10cm above LN2 surface for 8-10min respectively, then immersed in the LN2, and in the thawing process, the spermatozoa were maintained from the position of 10cm,15cm and 20cm for 6min respectively, and then immersed in tap water (7-11℃) till melting, the survival rate of the cryopreservative spermatozoa was 80-90%, the fertilization rate and hatchery rate were 85-95% and 70-85% respectively. Using DAPI stain, the developing process of the firtilized eggs of Chlamys (Azumapecten) farreri was obseved under fluorescence microscopic in this study. The chromosome of the matured eggs stayed in the metaphase of the meiotic I. After insemination, the eggs finished the first meiotic division and the second meiotic division, and the first polar body and the second polar body were extruded. The chromosome in the oocyte decondensated and enlarged, and the female pronucleus formed. The incorporated sperm nuclei experienced two enlarged stages: I. When the sperm incorporated the eggs, sperm nuleus decondensated and enlarged. II. After the second meiotic division, there was a dramatic enlargement and the male pronucleus formed. Female pronucleus and male pronucleus reached their maximal sizes at the same time, then the two pronuclei associated in the centre of fertilized eggs, and the first division began. Eggs became polyspermic when fertilized with a concentrated sperm sample, and the sperms incorporated an egg can developed into male pronulei. There wasn't a stable site where the sperm incoporated the eggs. The first polar body of some inseminated eggs can divided in two cells. Chlamys farreri eggs were treated 25 min after fertilization with 60mg/L 6-dimethylaminopurine(6-DMAP). The chromosome behavior and the nuclear stages of the insemination eggs were observed with fluorescence microscope. And the effects of 6-DMAP treatment at various meiotic stages on chromosome ploidy were investigated by assessing the percentage of triploids and embyos. It was concluded that three kinds of nuclear stages were observed after 5 to 15 min tratment. One nuclear stage showed the first polar body, one female proncleus and one male pronuleus; another showed the first polar body, two female pronuclei and one male pronucleus; and the third stage displayed one male pronucleus and two female pronuclei taking shape of bulliform structure with treatment duration. The triploid scallops induced by 6-DMAP to inhibit the second polar body originated mainly from the fertilized scallop eggs at metaphase of second meiosis, when a diploid female pronucleus combined with a male pronucleus forming triploid. Treated at anaphase of second meiosis, two female pronuclei and one male pronucleus of the fertilized eggs became abnormal during their fusion and separation procedure. Our experiment proved that 6-DMAP can induce triploids in fertilized scallop eggs by the ability to block extrusion of polar bodies when inhibiting chromosome separation and pronucleus motion. To produce triploids, fertilized eggs of Chlamys farreri were treated with 6-DMAP to inhibit the extrusion of the second polar body and the suitable induction parameters were studied. Using dihydrochloride (DAPI) stain and fluorescence microscopic observation, the relation between triploid induction rate, larval survival percentage and the nuclear stages before and after induction, the effects of egg maturation and spawning temperature on the synchronous development of fertilized eggs were analyzed. It was concluded that the appropriate induction parameters for farming industry are as following: completely mature eggs, initial treatment time when the first polar body occupied 40%, 60mg/L of drug concentration, 15 min of treatment...