Cryopreservation Of Female And Male Gametophytes Of Undaria Pinnatifida By Encapsulation-Vitrification

Content: Cryopreservation has long been considered an important method for the long-term storage of plant germplasm. This paper outlines new developments in the last 10 years of plant. As the development of natural resources of the marine life in recent years, algae play an important part in our production and daily life. Their potential has been growing in biotechnology. So the research of algae germplasm cryopreservation received increasing attention.Cryopreservation methods on algae germplasm have considerable research at home and abroad. While encapsulation-vitrification is a method developed from encapsulation-dehydration and vitrification in the 1990s. Compared to two step freezing and encapsulation-dehydration,encapsulation-vitrification have lots of advantages, such as simple operation, good recurrence and dealing with a lot of material at the same time. So encapsulation-vitrification has broad prospects in algae germplasm cryopreservation.Female and male gametophytes of Undaria pinnatifid were cryopreserved in liquid nitrogen using encapsulation-vitrification. Algal cells in early stationary phase were encapsulated in 3% Ca-alginate beads with 30‰NaCl, then cultured 1d in the dark and 3d in the light. Beads were loded with loding solution at 25℃, then dehydrated with 100% PVS (plant vitrification solution) at 0℃prior to a direct plunge into liquid nitrogen and warmed in 40℃water bath for 2min. The thawed matieral were rinsed in a washing solution, and then were determined the survival rate. The main factors influencing the gametophytes viability, such as the composition of loading solution and loading time, the composition of PVS and time of dehydration, the composition of washing solution and washing time were studied. The results are as follows:1. Female gametophytes of Undaria pinnatifid: The result showed that the high viability of 31% was obtained when the beads containing the gametophytes were loaded with a mixture of 2M glycerol and 0.6M sucrose for 120min at 25℃, then dehydrated with PVS2 for 50 min at 0℃prior to a direct plunge into liquid nitrogen and washed by 1.2mol/L sucrose two times(10min a time) after warming in 40℃water bath.2. Male gametophytes of Undaria pinnatifid: The result showed that the high viability of 26% was obtained when the beads containing the gametophytes were loaded with a mixture of 2M glycerol and 0.6M sucrose for 90min at 25℃, then dehydrated with PVS2 for 40 min at 0℃prior to a direct plunge into liquid nitrogen and washed by 1.2mol/L sucrose two times(10min a time) after warming in 40℃water bath.This experiment separated male and female gametophytes of Undaria pinnatifida, purificated and cryopreserved individually, which is conducive to the needs of breeding. The reserch on female and male gametophytes of Undaria pinnatifid cryopreservation has a complete regeneration and the ablity to developing into sporophytes.This experiment adopted the method of encapsulation–vitrification to cryopreserve male and female gametophytes of Undaria pinnatifida for the first time, which simplified the operation procedure of cryopreservation, so it was a new method for long-term storage of Undaria pinnatifida.