Cryopreservation Of Two Diatoms Used As Food In Mariculture By Encapsulation-Vitrification Method

Content: This paper takes Nitzschia closterium minutissima Allen et Nelson and Chaetoceros muelleri as experiment materials, and studies the crypreservation of the two under ultra-low temperature (-196℃) with encapsulation-vitrification method. Encapsulation-vitrification is a new technique for the cryopreservation of plant germ-plasm, which is developed on the basis of vitrification and encapsulation-dehydration. It has integrated the advantages of both encapsulation-dehydration and vitrification: the ability to treat a large amount of materials at the same time, rapid recovery growth after treatment, low toxic action on the material. It has gained success in the germ-plasm preservation of more than twenty kinds of advanced plants. However, there are few reports of its application in algae. This experiment filled this gap and proved the feasibility of this technique in the cryopreservation of algae.In encapsulation-vitrification experiment,discuss the influence of such factors as the composition of the PVS, the concentration of loading solution and the loading time, the dehydration time and the washing method on the viability of the algae was studied.1. The influence of the PVS composition on viability in cryopreservation:Among the seven selected vitrification formulas in this experiment, PVS2 formula(PVS2:30%(w/v)glycerol + 15%(w/v)ethylene glycol + 15%(w/v)dimethyl sulfoxide in f/2 medium containing 0.4 mol·L-1 SUC ) is suitable for the cryopreservation of the two algae.2. The influence of PVS dehydration time on viability in cryopreservation: Set different treatment time of the rubber balls in 100%PVS solution: 0,20,40,60,80,100 min. Results show that the most suitable time for Nitzschia closterium minutissima Allen et Nelson and Chaetoceros muelleri are both 60 min.3. The influence of loading time and loading solution concentration on viability in cryopreservation:Choose five points in loading time: 0,20,40,60,80,100 min. Experiment results show: when loaded with 25%PVS, the two algae get the highest viability at the point of 60 min; when loaded with 50%PVS, Nitzschia closterium minutissima Allen et Nelson gets the highest viability at the point of 60 min and Chaetoceros muelleri 40 min. As for the loading solution concentration, it's best to use 50%PVS for the cryopreservation of the two algae. 4. The influence of sucrose concentration in washing solution, washing method and washing time on viability in cryopreservation:After rewarming, choose five types of washing solution based on different sucrose concentration. Experiment shows: gradual washing in sucrose solution is beneficial for the viability of Nitzschia closterium minutissima Allen et Nelson cells (original concentration of the sucrose solution being 1mol·L-1); washing in 1.2 mol·L-1 sucrose solution is beneficial for the viability of Chaetoceros muelleri cells. As for washing time, among the selected five points, 0,10,20,30,60 min,30 min is the best time for the cryopreservation of the two algae.In conclusion, chodse the best,the most suitable conditions for the cryopreservation of the two algae are as follows:The highest viability of Nitzschia closterium minutissima Allen et Nelson, 74.1 %, is obtained when the beads containing the algal cells are loaded with 50%PVS2 for 60 min under 0℃,then dehydrated with 100% PVS2 for 60 min under 0℃and washed gradually by sucrose for 30 min at 20℃.The highest viability of Chaetoceros muelleri, 48.2%, is obtained when the beads containing the algal cells are loaded with 50%PVS2 for 40 min under 0℃,then dehydrated with 100% PVS2 for 60 min under 0℃and washed by 1.2 mol·L-1 sucrose for 30 min at 20℃.