Submitted to: CryoLetters
Publication Type: Abstract Only
Publication Acceptance Date: 9/20/2005
Publication Date: 9/15/2005
Citation: Reed, B.M. 2005. Cold acclimation as a cryopreservation pretreatment strategy. CryoLetters. p. 25. Interpretive Summary: Cryopreservation protocols for storing shoot tips in liquid nitrogen are often designed to take advantage of the natural processes of plants. Cold acclimation (CA) is the natural process that prepares a plant for the onset of cold weather. Artificial CA using the natural warm and cold cycle of an early winter day can produce increased cold tolerance in many plants. CA was used to induce cold hardiness in tissue-cultured shoots of several types of plants. In strawberry plants shoot tips of plants CA for 1 week could be cryopreserved with double the regrowth of non- acclimated shoots. Blackberry shoots CA for 2 weeks had double or triple regrowth compared to non-acclimated shoots. A large group of pears tested for cryopreservation displayed a wide range of responses to CA. The pear shoot cultures tested required one to 14 weeks CA for optimal recovery from cryopreservation. The length of CA required could be reduced by growing the plants on medium high in sugar or treated with other chemicals. Similar studies with grasses were also successful. Using these CA pretreatments, plants from subtropical to sub arctic regions could be successfully cryopreserved.
Technical Abstract: Pretreatment strategies are designed to take advantage of natural physiological processes. The efficacy of cold acclimation may vary with genotype due to the variations in natural mechanisms found in diverse plant groups. Studies in our laboratory show that many types of pretreatments improve cryopreservation success. ABA, cold acclimation duration and type, extended culture and sucrose pretreatments all influence meristem recovery from cryopreservation. Pretreatment interactions are also important to the success of a protocol. Cold acclimation (CA) was first shown to be effective in the cryopreservation of strawberries and one week of CA more than doubled regrowth compared to nonacclimated controls. For blackberries one week of CA doubled or tripled regrowth from shoot tips and with a 1 week 50'M ABA pretreatment greatly improved regrowth for one genotype. Pears are more complex with a wide range of responses to CA. Extending the duration of culture and CA of in vitro-grown Pyrus plants was very effective for improving regrowth following cryopreservation. Regrowth of moderate or difficult to cryopreserve genotypes was significantly improved by extending the CA to four or more weeks. Long culture duration improved regrowth and also reduced the length of CA required for high regrowth of pear meristems following cryopreservation. The type of CA was also crucial in improving plantlet cold hardiness. The LT50 of plantlets grown with 10 weeks alternating warm and cold-CA was –25'C; with constant cold-CA, -14.7'C; and no CA controls –10'C. The optimum length of CA varied with plant genotype from one to 14 weeks. The optimal shoot pretreatment for successful recovery of cryopreserved P. cordata shoot tips was 3-wk culture on either 50 'M ABA or 5-7% sucrose medium followed by 2-wk LT, and increased shoot tip regrowth from zero to greater than 70%. Using these techniques plants from sub tropical to sub arctic regions can be cryopreserved.