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Research Project: Increasing Efficiency in Cryopreservation of Clonally Maintained High Priority Germplasm Collections Using Dormant Winter Bud Technology


Project Number: 3012-21000-015-15-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: May 1, 2015
End Date: Apr 30, 2020

The overall objective is to develop and optimize cryopreservation methods that increase both the 1) post cryopreservation viability, and 2) throughput efficiency of freeze/thaw protocols for dormant winter buds from important woody food crops. The initial focus will be on high priority germplasm collections of Juglans sp, including J. hindsii, J. major, and J. regia. As time and resources permit, other high priority collections (Pistacia, Prunus dulcis, Punica granatum) will be included in the study. Cryopreservation protocols will be developed from the results of experiments designed to achieve the following sub-objectives: 1. Develop innovative methods to assess post cryopreservation viability. 2. Determine the optimal time to harvest dormant buds. 3. Develop and optimize bud pretreatment methods. 4. Characterize intraspecific and interspecific variation for response to dormant bud cryopreservation protocols.

The approach will be comprised of numerous coordinated lines of research inquiry. Primary among these will be to critically review and assess the state of relevant research-based knowledge of the 1) cold hardiness whereby woody plant buds acquire the capacity to survive freezing in regions of adaptation, and 2) factors underlying successful cryopreservation of dormant woody buds in other collections maintained in the NPGS and elsewhere. Such information will guide the design of experiments to determine how best to apply the knowledge and mechanistic understanding gained from more fundamental research. Because of the multitude of potential variables, and complex interaction between those variables, the number of experimental treatments will be high, requiring sophisticated statistical design and analysis to assure useful results. To develop viability testing methods that support development of high throughput alternatives to existing, relatively labor– and time– consuming tests, we will explore a variety of non-destructive methods based on various contemporary image analysis methods. Efficacy will be assessed by comparison to methods that are well accepted, but which are much more time-consuming. Starting first with Juglans species and the repository at Davis, CA, we will develop collaborations that will facilitate the harvesting of dormant buds at intervals of their acclimation/deacclimation for time course experiments. Project personnel will travel to Davis to assure appropriate, consistent, and reproducible bud collection and handling procedures. In addition to assessing the influence of on tree temporal variation in physiological state, a wide arrange of targeted pretreatments will be evaluated for their cryoprotective effects. Much of this experimentation will be guided by the large body of published work on cryoprotection. It may also be that possible new pretreatments will be identified, in which cases preliminary experiments would determine possible new approaches to test for larger scale high throughput application. Once appropriate protocols are developed, we will select accessions of available Juglans and perhaps other species for the purpose of characterizing intraspecific and interspecific variation for response to the best available combination of cryopreservation treatments. Selection of experimental treatment variables and their combination, and perhaps especially the selection of accessions from large collections (446 accessions for J. regia alone) will require significant, well-coordinated assistance from statistical design and analysis as well as IT experts.