Cryopreservation and recovery of temperate fruit germplasm using dormant bud technology
Date
2020
Authors
Tanner, Justin D., author
Minas, Ioannis S., advisor
Wallner, Stephen J., advisor
Volk, Gayle, committee member
Byrne, Patrick, committee member
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Abstract
Cryopreservation of dormant budwood allows for the efficient processing and long-term storage of some temperate deciduous genetic resources and holds promise for the secure storage of countless others. The method is of particular interest because it is more cost effective than preservation using in vitro methods. By investigating the process of dormant bud cryopreservation reported in literature in conjunction with novel studies of pretreatment effects on regrowth after liquid nitrogen exposure and bud freeze resistance, critical factors affecting survival have been identified. These factors can be broadly classified into two categories; factors related to the condition of budwood before storage and factors impacting recovery after storage. Preconditioning treatments such as air drying to ~30% moisture content and slow cooling of dormant budwood before liquid nitrogen storage are among the most critical pre-storage factors for survival. Preconditioning treatments can also have a significant effect on increasing freeze resistance. Differential thermal analysis was used to investigate the pretreatment effects and identify the best preconditioning method for the cryostorage of peach dormant buds. Apple, apricot, cherry, peach and pear were used for cryorecovery studies but not tested by differential thermal analysis. Treatments tested include air desiccation of budwood to 30% moisture content, exposure to sucrose solution using concentration and exposure treatment levels selected for greatest increase in freeze resistance in pretrial experiments, and a combination treatment of sucrose solution exposure before air desiccation. An additional group of twig segments was not treated but processed in the fresh state to compare treatment effects. Of the four pretreatment groups, desiccation alone had the greatest impact at increasing freeze resistance and cryosurvival in most species tested. A follow up experiment was conducted to investigate moisture content effect cryosurvival and freeze resistance in peach dormant buds. For all treatment levels tested, survival and shoot development were low. Best pre-storage moisture level in peach was higher than expected at 35% (fresh weight basis) suggesting desiccation sensitivity may be contributing to low cryosurvival. A linear relationship between dormant bud moisture content and LT50 was also observed in freeze resistance trials. Posttreatment factors affecting growth and establishment include warming rate and recovery method. Recovery of cryostored dormant budwood can be accomplished by either grafting, in vitro culture, or direct rooting. In order to increase efficiently in storage and recovery, a new approach to recovery was tested; antimicrobial forced bud development followed by in vitro culture initiation using shoot tips of sprouted buds. This approach aims to combine the efficiency of forced bud development with secure establishment of in vitro shoot cultures after cryopreservation. Successful establishment of shoot cultures from dormant buds recovered using antimicrobial forced bud development before culture initiation was correlated with the development of shoots with leaves. The earlier developmental stages were not significant for culture establishment. This method has successfully been applied to several fruit and nut species namely apple, apricot, cherry, peach, pear and little walnut and has the potential for use with many other species of deciduous species in need of clonal preservation. As a two-step process, antimicrobial forced bud development can be used as a standalone viability test, or when needed, used as a platform for shoot culture induction for the reestablishment of cryopreserved dormant bud germplasm.
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Subject
cryogenic preservation
freeze resistance
tissue culture establishment
forced bud development
clonal germplasm storage
temperate tree fruit