| Artificial induction of gynogenesis refers to the development of eggs intoembryos without the participation of male pronuclei by physical, chemical orbiological methods. It shows great potential in rapid production of inbred lines whichare valuable resources for the genetic breeding. Furthermore, the genetic material ofgynogenetic offspring only comes from the female parent, so it’s perfect material forstudying the mechanism of sex determination, gene-centromere recombination, genemapping and cloning. In this study, gynogenesis induction by electrical stimulationwas performed for the first time in Chlamys farreri, a commercial shellfish species inthe north of China. To gain the optimal induction condition, several parameters weretested. In addition, Hoechest staining was applied for examining the activity of eggsfollowing electrical stimulation. Flow cytometry analysis and chromosome countingwere conducted for assessing the ploidy of gynogenetic embryos.1、In order to acquire the optimal condition for gynogenesis induction by electricalpulses in C. farreri, we used orthogonal design to investigate the induction effects ofelectric field strength, electric capacity, density of eggs, electro-activation solutionand pulse number.The results as follows:(1) Selection of the electro-activation solution. Three kinds of solutions (mannitol,sorbitol and Zimmerman) containing three concentration gradient of theircorresponding non-electrolyte (0.25mol/L,0.30mol/L and0.35mol/L) was testedunder the following electric parameters:0.98kv/cm,3μF and two pulses. Resultsshowed that for each of the three solutions, the highest cleavage rate could beacquired when the concentrations of mannitol, sorbitol and sucrose were0.30mol/L，0.25mol//L and0.30mol/L, respectively. Electro-activation solution, electrical fieldstrength and electric capacity were designed in an orthogonal way. Comparison of theeffects of different electro-activation solutions revealed that electro-activationsolution with mannitol had better effect on gynogenesis induction than that with sorbitol or Zimmerman in spite of the insignificant differences in cleavage ratesamong electro-activation solutions (p>0.05). Therefore, electro-activation solutionwith0.30mol/L mannitol was applied in the subsequent experiments.(2) The optimization of egg density. Egg density, electrical field strength andelectric capacity were designed in an orthogonal way of L9(34). When the electricalfield strength was1.30kv/cm or1.62kv/cm, the cleavage rate rose with the increase ofelectrical field strength. The optimal egg density was11.25×104eggs per ml, and thecleavage rate reached27.46%when the electrical field strength was1.30kv/cm, andthe electric capacity was10μF.(3) The effect of electrical field strength on gynogenesis induction. Single-factordesign method was applied, and the results showed that with the increase of fieldstrength from0.75to1.13kv/cm, the cleavage rate constantly rose. When theelectrical field strength was1.13kv/cm, the cleavage rate reached the maximum andthen declined significantly (p<0.05) with the increase of the electrical field strength.So the optimum electrical field strength ranged from1.00to1.75kv/cm.(4) The optimization of electric capacity and the screening of the optimalparameters combination. By using an orthogonal design of electrical voltage, electriccapacity and pulse number, we found there were significant differences between threeelectric capacity gradients,with the best induction effect of3μF and the worst effect of25μF.Concurrently, the optimal parameters combination was achieved: egg density of11.25×10~4eggs per ml, electro-activation solution containing0.30mol/L mannitol,electrical field strength of1.20kv/cm, electric capacity of3μF, and two pulses. Whenthe optimal parameters were applied in C. farreri, the cleavage rate was28.16%andthe survival rate of gynogenetic embryos reached98.37%.2、Cytological analysis of the gynogenetic embryos:(1) Detection of embryos development. Gynogenesis can be induced by electricalpulses. We observed that some eggs can be activated and proceed to cleavage, but thedevelopment was delayed. In addition, the membranes of eggs treated by electricalpulses came off easily and the eggs became abnormal.(2) The cytological analysis was also conducted for the gynogenetic embryos. By Hoechst staining, we found that the development of the embryos was later than thenormal group and most of the activated eggs entered the cleavage process, with only afew producing1or0polar body.(3) Ploidy investigation of the gynogenetic embryos. Two different methods wereapplied. Chromosome counting showed that the ratio of the haploid, non-aneuploidy,and diploid was48.98%,45.92%and5.10%, respectively. Flow cytometry analysisrevealed that the DNA content of the main peak of the electrically stimulated groupwas approximately half of that of control group, i.e. the gynogenetic embryos werehaploid. The two ploidy analysis methods proved that the electrical stimulation caninduce gynogenesis in C. farreri effectively. |
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