STUDIES ON GENOME MANIPULATION IN FISH 鈪? TETRAPLOIDY INDUCED BY HYDROSTATIC PRESSURE TREATMENT AND A COMBINATION OF HYDROSTATIC PRESSURE AND COLD TREATMENTS IN TRANSPARENT COLORED CRUCIAN CARP

The studies on the induction of tetraploidy, using either hydrostatic pressure treatment alone or a combination of hydrostatic pressure and cold treatment for blocking first cleavage, were attempted in transparent colored crucian carp (Carassius auratus transparent colored variety). The shock treatments involving hydrostatic pressure of 650 kg/cm2 lasting 3 minutes were applied at 35, 38, 40, 42, 45, 48, 50, 54, 55 or 60 min after fertilization. Tetraploidy embryos that include tetraploids, hypotetraploids and tetraploid/diploid mosaics were found in the pressure treatment groups at 50, 54, 55 and 60 min after fertilization, whereas the tetraploidy embryos were not found in the pressure treatment groups at 48, 45, 42, 40, 38 and 35 min after fertilization. The results suggest that hydrostatic pressure treatment at 50-60 min post-fertilization arrest the first cleavage and induce tetraploidy. The shocks involving a combination of hydrostatic pressure and cold treatment were applied at 40, 48, 50, 52, 55, 56 or 60 min post-fertilization. The tetraploidy embryos of tetraploids, hypotetraploids and tetraploid/diploid mosaics were observed in the combined treatment groups at 48, 50, 52, 55, 56 and 60 min post-fertilization. The tetraploidy rates in the combined treatment groups were higher than those in the pressure treatment groups. Both methods of treatments, especially the dombined treatment, strongly affected the development of fertilized eggs and decreased the survival rates of embryos. The fertilized eggs subjected to pressure shocks and the combined shocks not only produced tetraploids, hypotetraploids and tetraploid/diploid mosaics, but also developed diploids and hypodiploids. Some chromosome fragments caused by the suboptimal treatments were obviously observed in the metaphases of some hypotetraploids and hypodiploids. We also found a short stage at which fertilized eggs have stronger tolerance towards the shock treatments during the effective period for the inhibition of first mitosis and induction of tetraploidy. Before and after this short stage, the fertilized eggs were more sensitive to the shock treatments. Artificial tetraploid transparent colored crucian carp were identified and selected from the individuals developed from some shock groups through chromosome observation of tail-fin tissue cells of fingerlings and blood cultured cells of adult fish. The problems about the techniques of inducing tetraploidy, the effective period of blocking first mitosis for the doubling of chromosome set and the survival rates and viability of retraploidy embryos in fish were analysed and discussed based on the results obtained from this study and those reported by other investigators.