Experimental Plan - Phase 1

Objective: Evaluation of Cryopreservation Technology Transfer In International Genebanks

Variables

Variable 1: Institute, Country (4)

Lab 1 Research Institute of Horticulture and Viniculture, Poland

Lab 2 Kazakh Research Institute if Horticulture and Viniculture, Kazakhstan

Lab 3 Department of Forest Gene Conservation, Germany

Lab 4 University of Abertay Dundee, Scotland

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Variable 2: Cryopreservation Protocol (2)

PVS2 Vitrification Method

Encapsulation Method

Statistical analysis 2 stages are considered

.

1. Between method performance Ho: There are no differences between methods
2. Within method performance (to test performance at each step of the method, this will pinpoint which steps of the protocol are the most susceptible to variation between genebanks)
   1.  
   2. PVS2

      • Tissue culture performance
      • Dissection controls (critical point for operator skills acquisition)
      • Cryoprotectant control (critical point for expediency of methodology)
      • Frozen

Consider cold acclimatization (should this be included?)

Consider in vitro performance tests e.g. the status of the cultures before cryopreservation.

1.  
2. Encapsulation/dehydration

   • Tissue culture performance
   • Dissection controls (critical point for operator skills acquisition)
   • Encapsulation and 0.75 M sucrose pre-growth treatment
   • Encapsulation, 0.75 M sucrose pre-growth treatment, 4 hrs air drying
   • Frozen

Replication

20 shoot-tips per treatment (note that tissue culture performance will be evaluated on material before test treatments so the same plants can be used). Experiments will be replicated three times.

Total number required for each cryopreservation method

PVS2 = 20 x 3 = 60

Encapsulation/dehydration = 20 x 4 = 80

Total shoot tips required = 140

Additional Considerations

1. Status of tissue cultures will be assessed at the start of each experiment.
   • Color (chlorosis check, height)
   • Each experiment will be standardized in terms of the subculture procedure, taking shoots with single nodes at 3 weeks followed by 1 week of cold acclimatization.
2. Conditions in the lab will be assessed during the cryopreservation procedure temperature and humidity can be critical points for desiccation especially. Take temperature and if possible humidity readings (in the flow hood).
3. Assessments of recovery (weekly up to 6 weeks). Recovery as shoot development is the critical point performance indicator.

Critical Point Assessments:

Develop a critical point assessment of major cryopreservation techniques.

Rank by meeting or not meeting minimum requirements (+ or -) with a comment of why it does not meet and options for meeting the requirements.

1. Source plant status
   1. Origin
   2. Time in culture
   3. Transfer interval
   4. Genotype (test genotype ‘Ojebyn’)
2. Culture conditions
   1. Standard culture regime
      1. growth room parameters
      2. culture medium
      3. growth regulators
      4. transfer (subculture) interval
   2. Pregrowth
      1. acclimation
      2. pretreatment
   3. Recovery
      1. medium
      2. growth room parameters
      3. transfers
3. Step by step instructions for procedures and solution preparation aided protocol standardization.
   1. Solution preparation
   2. Meristem dissection
   3. Encapsulation
   4. Pregrowth
   5. Vitrification protocol
   6. Dehydration
   7. Cryopreservation/Rewarming
   8. Rehydration
   9. Plating
   10. Controls
4. Cryogenic facilities
   1. Type of dewar
   2. Vials
   3. Canes/Boxes
   4. Inventory system
   5. Labeling
   6. Nitrogen availability
   7. Ice
5. Facilities
   1. Growth room
   2. Laminar flow benches
   3. Water bath
   4. Cold hardening facilities
   5. General lab facilities
6. Personnel
   1. Background skills (medium preparation, solutions, calculations)
   2. Tissue culture expertise
   3. Dissecting skill
   4. Attention to detail
   5. Success in validation of protocol