Reliable encapsulation-based cryopreservation protocol for safe storage and recovery of grapevine embryogenic cell cultures

- Cryopreservation of embryogenic cell lines.
- Dehydration-encapsulation method.
- Storage of grapevine embryogenic germplasm.
- Enhancement of embryogenic competence and plant regeneration.

An efficient and reliable cryopreservation protocol by dehydration-encapsulation technique was developed for several grapevine cell suspensions. Three embryogenic lines of grapevine (Vitis sp. rootstock 110 Richter as well as Vitis vinifera cv. Tempranillo and cv. Riesling) have been successfully cryopreserved with direct immersion in liquid nitrogen using one-step freezing procedure. Cryoprotection consisted of pretreatment into liquid culture with increasing sucrose concentration followed by loading with sucrose/glycerol solution. Alginate-encapsulated beads were rewarmed and placed on recovery medium which led to regrowth of frozen embryogenic tissues within 6-8 weeks culture. Factors affecting survival of cryopreserved tissues were investigated. Recovery rates varied among genotypes (from 43% to 78%). Following regrowth, beads transferred to liquid medium generated new proliferating cell suspensions showing active multiplication and higher morphogenetic competence as revealed by RNA yield and quality. The cryogenic procedure adopted here allowed high embryo survival after exposure to liquid nitrogen with successful plantlet regeneration. This high throughput protocol is useful for the conservation of a large collection of embryogenic tissues from endangered Vitis species.