Evidence Project Final Report

** Note: some accessions have more than one image of fruit on the tree.

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  An initial set of 20 candidate accessions from the apple collection was selected for cryopreservation trials in 2009 (Milestone 1) based on previous work carried out by Imperial College Wye (ICW, Defra project GC0138NTF). Dormant graftwood was collected from these accessions and taken through the standard cryopreservation process (as used previously by ICW, and described by Forsline et al., 1998 J. Amer. Soc. Hort. Sci. 123:365-379; and Forsline et al., 2004 Cryoletters 25:323-334). Samples were recovered from storage after a period of approximately 1-month for viability testing (Milestone 9) alongside material which had been stored over 12-months previously as part of a pilot study by ICW. Cryopreserved material was tested for survival by recovery and chip budding (to assess both graft survival [indicating the viability of cambial tissue to form a successful graft] and bud survival [indicating the viability of the axillary bud to allow scion growth]). A minimum of 50% survival was found for the material stored for 1-month, although lower survival was found for the longer stored material (see 4.1). It was noted that this older material had been transferred from a pilot study at ICW and the constancy of storage conditions through the handover was not absolutely clear (further material stored at the University of Reading for 12-months was therefore also tested at a later date);
  
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  Attempts were made to establish material from the vine, cobnut and currant collections on tissue culture with a view to testing in-vitro approaches to cryopreservation (Milestone 14). However, difficulties were encountered with establishing sterile in vitro cultures due to high levels of persistent contamination. In these circumstances, the inefficiency of establishing material in sterile culture was deemed to make these approaches impractical. In vitro methods including encapsulation-dehydration and vitrification-based cryopreservation were also considered for the apple collection but, given these difficulties, and because it was generally felt that the dormant bud system would be applicable across the large majority of the apple collection, efforts were focussed entirely on dormant bud cryopreservation (Milestone 21).
  
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  Further testing of material placed in cryopreservation during 2009 and stored for 12-months was carried out and survival was generally found to be greater than 40% (see 4.2) with an exception of a sample of Malus sylvestris. It was noted that no testing was carried out for this specific sample in the previous 1-month storage trial and so it could not be determined if this was an effect of storage, or a low baseline survival rate for this specific sample. Short term storage trialling was also repeated to test both the repeatability of the process (Milestone 13) and to assess the potential to extend the dormant season (Milestone 15) by holding collected material at 4^oC for an extended period after collection. Survival was scored as primary (in instances where the axillary bud grew out from the grafted material) and secondary (where the primary bud may have failed but secondary growth was seen from the axillary meristem in the bud axil). Since both of these were deemed to allow the recovery and growth of the scion they were scored together as bud survival;
  
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  In collaboration with the University of Copenhagen, trials were conducted to determine the importance of each stage of the cryopreservation protocol. ‘Holsteiner Cox’, ‘Maglemer’ and ‘Prima’, which had been found to exhibit high, low and intermediate recovery, were sampled from both Brogdale and the Nordic Fruit Collection in order to conduct large, replicated trials. We established that: the -4^oC dessication treatment was not sufficient on its own to allow post-thaw survival but was vital in combination with the slow cooling to -30^oC; the vast majority of the post thaw rehydration took place in the first 24 hours so this 14 day step could be shortened; ice-derived tissue disruption could be identified within inadequately dehydrated buds using differential scanning calorimetry and cryo-scanning electron microscopy and these techniques represented useful tools to further optimise the protocol (findings of these studies were published in the scientific literature and publications are listed in section 6.1);
  
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  On the basis of these findings, the routine cryopreservation of the apple collection for safety back-up was begun. Over a period of 4 years (2010-13), approximately 1,500 accessions were placed into cryopreservation [Milestone 28]. The final round of including material in the safety back-up was scheduled for 2014 but was delayed, in part due to the relatively warm conditions experienced during the winter 2013/14. The final round of inclusion of material into the cryopreservation back-up is expected to be carried out in February-March 2015 (resources were held back at UoR to complete the delivery of this work). Completion of this final round will result in a total of over 1,750 accessions to held in cryopreservation going forwards (Milestone 34);
  
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  Our original plan had included an expansion of the dormant bud system to the sour cherry cultivars in the NFC (Milestone 19). This was however, abandoned in light of the plant health embargo placed across the Prunus collection. In effect, the embargo meant that any Prunus placed in cryopreservation could only have been done so under license as research material which would require its disposal at the end of the process and therefore would not be effective in forming a safety back-up. Since techniques for sour cherry were already established (Towill and Forsline, 1999 Cryoletters 20:4 215-222) and this process would offer little additional security to the field collections, efforts were focussed on the apple collection.