Alternative methods for storing the Collection Preliminary List of Options Points for and against

APPENDIX 1

• 
  Knowledge of biophysical properties of water within cells.
  
• 
  Physiological understanding of critical content for desiccation damage.
  
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  Hydraulic conductivity of water.
  
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  Thermal loads that effect cooling rates
  

To determine the effect of the physiology of starting material on the cryopreservation of meristems

Development work needed for each species to find optimum conditions for cryopreservation, thawing and recovery; (Slow freeze easier; large numbers can be processed; conditions less critical; toxicity lower and success rate usually higher. Vitrification marginally better if carried out experienced person.)
Understanding the stresses caused by drying, cooling, freezing and the effect of time on these processes

A better understanding of the physiology and biochemistry of species to be cryopreserved and of meristem biology

A better understanding of the effect of thawing on tissues

Examination of genetic erosion: what happens during cryo storage and what happens during regeneration?

Work on the nature of toxicity of cryoprotectants

The investigation of beneficial elevated sugar levels in pre-culture

The examination of the reason for vine buds being ‘very leaky’. The effect of electrolyte leakage on cryopreservability needs to be examined.

Evaluation of culture systems needed for recovery

Acclimation - Do cells of accessions which can be cryopreserved, produce their own cryoprotectants?

Do species which can be cryopreserved have the ‘right’ genes or do susceptible species fail to turn on their genes

How do buds acclimate in the fall and increase in winter hardiness?

What factors limit the hardiness of buds?

What features distinguish a hardy from a non-hardy bud?

What factors and conditions are required to maintain maximum bud hardiness?

What controls the de-acclimation of flower buds and the progressive loss of cold hardiness?

Studies needed on the epigenetic effects (Phenotypic expression due to variation in expression) and on plant and hormone physiology.

Assessment of hormonal and nutritional needs required in vitro.

Further studies in developmental biology to examine the induction of roots, multiple shoots and flowers

The control of endophytic bacteria

The rejuvenation of woody species

Episotic growth (eg in vitro Oak cultures)

How to get recalcitrant species in culture

Factors responsible for cold sensitivity of grape in vitro

C. Low temperature tissue culture
Pro As for tissue culture above Maintenance and labour costs may be lower than for standard tissue culture above Less frequent subculture required	Con As for tissue culture above, although maintenance and labour costs may be lower Need to go through multiplication and rooting media at 20oC (few months) before retuning to cold. Still labour intensive Loss of some accessions Needs system for producing vigorous shots free of contaminants/endophytes prior to cold storage

Development work:
At present, Ribes found to be too ‘dirty’ to be stored in cold storage tissue culture. Need to develop method of removing surface contaminants and endophytes Pers. Comm. Janine de Paz, (NCGR, Corvallis, Oregon, USA)

Factors effecting cold hardiness

Factors effecting bud break and survival at sub-optimum levels

The effect of reducing nutrient and sugar levels

Research is needed to develop health procedures during the collection and introduction of new accessions into field gene-banks. Also faster and more efficient ways of screening and disease indexing new accessions

Research is needed to improve management and maintenance of field collections

Development of low-input management strategies can help reduce costs of maintaining field collections.

General procedures for field collections include propagation methods, selection of planting sites, planting procedures, cultivation practices, disease and pest management and harvest and storage of propagules. Monitoring the genetic stability of the crop requires careful vigilance on the part of the curator and field staff, careful roguing, labelling and protection of the plants from biotic and abiotic dangers are important to the safety of the germplasm. (Reed et al, 2004)

E. Ultra Dwarfing Rootstock (e.g. M27 for apples)
	Pro Less land required Material readily available Morphological characteristics readily recorded Samples of propagating wood, leaves, fruit and pollen available Allows accessions to be characterised Allows users to select most appropriate material Position of accessions fixed in collection for many years with site maps giving location (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e. would reduce major cost) (User of material supplied responsible for import permits and quarantines etc)		Con Prone to pests and diseases Prone to vandalism Prone to climatic changes May not show all characteristics of full-sized plants Fewer flowers (for breeders) Less material available than standard trees May not produce enough fruit for crosses on trees Greater intensity of care necessary Smaller root balls M27 need wire frames to support scion Shorter life Doyenne du Comice interstock probably needed for pears More frequent re-propagation and parallel collections Effect of cherry and plum dwarfing rootstocks (Gisela 5 and Pixy) on many scion cultivars not known

Development work:
Need good genetic markers to identify accessions in field and those coming out of cryopreservation
Importance of secondary structure of proteins for dried or frozen material
Effect of cherry and plum dwarfing rootstocks (Gisela 5 and Pixy) on many scion cultivars is not known. Before using these very dwarfing rootstocks research will be needed to look at the long term effects of growing a wide range of scions on such rootstocks.

F. Cordons (for apples and pears)
Pro Less land required Material readily available Morphological characteristics readily recorded Samples of propagating wood, leaves, fruit and pollen available Allows accessions to be characterised Allows users to select most appropriate material Position of accessions fixed in collection for many years with site maps giving location (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e. would reduce major cost) (User of material supplied responsible for import permits and quarantines etc)	Con Prone to pests and diseases Prone to vandalism Prone to climatic changes More susceptible to Fire Blight May not show all characteristics of full-sized plants Fewer flowers (for breeders) Less material available than standard trees May not produce enough fruit for crosses on trees (but could use pollen instead) Greater intensity of care necessary Posts and wires needed Cherry and plums not well suited to cordon cultivation

Development work:
Need good genetic markers to identify accessions in field and those coming out of cryopreservation

G. Potted Plants
Pro Can be transported to new locations Less land required Morphological characteristics readily recorded Samples of propagating wood, leaves, fruit and pollen available Allows accessions to be characterised Allows users to select most appropriate material (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e. would reduce major cost) (User of material supplied responsible for import permits and quarantines etc) Can be kept in a screen house to protect from pests and extreme climatic conditions	Con Prone to drying out High regular commitment to watering Large mature plants not possible May not show all the characteristics of mature plant Periodic re-potting (Annual?) Labour costs of re-potting etc Loss of labels especially during re-potting (mislabelling causes masses of work) Restricted access to public because of danger of label theft Shorter life (more frequent re-propagation) Disease is more of a problem. Effected by extreme environmental conditions Risk of vandalism Climate changes may eventually effect fruiting due to lack of winter chill for flower initiation

Notes:
At NCGR, the problem of label loss is counteracted by pushing a second label, upside-down, completely into the ground at the edge of the pot (so that it is not obvious to the general public, and can also be retrieved if the visible label falls out by accident).

Genetic fingerprinting of the whole collection would also help to overcome label loss. Any doubts could be checked with the stored information.

Importance of secondary structure of proteins for dried or frozen or frozen seeds

Effect of lipid composition of storage of seeds

How changes in volatiles can indicate deterioration of seeds

ARS National Seed Storage Laboratory, Fort Collins, CO, showing acceptable viability levels in recovery grafting at the Plant Genetic Resource Unit, Geneva, NY.

From Forsline, P. L., J. R. McFerson, et al. (1999). Development of base and active collections of Malus germplasm with cryopreserved dormant buds. Acta Horticulturae; Proceedings of EUCARPIA Symposium on Fruit Breeding and Genetics. K. R. Tobutt, Alston, F.H. Oxford, England. 484: 75-77.

by grafting in the active collection at the USDA-ARS Plant Genetics Resources Unit, Geneva, NY.

From Forsline, P. L., J. R. McFerson, et al. (1999). Development of base and active collections of Malus germplasm with cryopreserved dormant buds. Acta Horticulturae; Proceedings of EUCARPIA Symposium on Fruit Breeding and Genetics. K. R. Tobutt, Alston, F.H. Oxford, England. 484: 75-77.

IDENTIFY

DETERMINE

PRIORITIZE

CHOOSE AN

SET UP DATABASE

SET UP TESTING

LOCATE A SAFE

PLAN

INITIATE

From

Facilitates development of micro-satellite markers (Varshney, Graner et al. 2005; Varshney, Sigmund et al. 2005)

Need to establish uniform nomenclature for Pyrus,

The application of LTR Retrotransposons as molecular markers in described in detail by Schulman, Flavell et al. (2003).

Rosaceae is also considered the model family (and Prunus the model genus) for fruit/nut trees,

or more samples could be extracted per person per day (even a hundred or more, if leaf material has been previously prepared - freeze-dried then ground to a fine powder in a ball mill or some such). Given such a massive collection, a quick method is recommended as long as equipment exists to easily powder the leaves.

The number of accessions divided by the number of samples that can be extracted per day, multiplied by 150%,

equals the number of man-days required. This can be done by a technician-level scientist.
Running the DNA tests depends on the technology available. An automated system that can run many hundreds of

samples per day given the size of the collection, is recommended.

LiCor (cheaper, as most can be prepared in the lab): approx. $0.50 per sample.

ABI/16 (must buy gel and other necessities from the ABI company): approx. $1.25 per sample. Plus $1000 per "array", where one array will last for 400-500 runs (so I calculate from this that with about 570 samples per day [= per run], you'd need a new array every 250,000 samples, which is less than a cent a sample. Basically negligible. One array would last for something like 50 to 70 separate DNA tests per sample, i.e. 25 to 35 DNA tests per accession when everything is done in duplicate).

ABI/96: $1.25 per sample, $4000 per array

LiCor: 95 samples, or can multiplex 2 tests at a time (i.e. 190 samples per day)

ABI (16 capillary): 190 samples, or can multiplex up to 4 tests at a time (i.e. 760 samples per day for x4 multiplex)

ABI (96 capillary): 96 samples with 30-36 multiplexing (i.e. 3000-3600 samples per day)

* No. samples = 8000 DNA extracts x 30 DNA tests = 240,000

* No. samples per day = 500 (with multiplexing, and allowing for reruns where

necessary)

* No. 1-person days required = 240,000 / 500 = 480

* Labour costs = 480 days x $100-$150 per day (£50-70.) = $48,000 to

$72,000.

* Running costs = 240,000 x $1.25 = $320,000

* Total cost = $368,000 to $392,000 plus the yearly maintenance cost.

480 days of continuously running the ABI probably difficult especially if more than 30 DNA tests are desired, so the 96 capillary system would probably be the best way to handle so many samples.

* No. samples = 240,000

* No. samples per day = 150 (with multiplexing, and allowing for reruns where

necessary)

* No. 1-person days required = 240,000 / 150 = 1600

* Labour costs = 1600 days x $100-$150 per day = $160,000 to $240,000.

* Running costs = 240,000 x $0.50 = $120,000

* Total cost = $280,000 to $360,000 plus the yearly maintenance cost. A little

cheaper, but takes a lot longer.

Same calculations for ABI/96:

* No. samples = 240,000

* No. samples per day = 2500 (with multiplexing, and allowing for reruns

where necessary)

* No. 1-person days required = 240,000 / 2500 = 96

* Labour costs = 96 days x $100-$150 per day = $10,000 to $15,000.

* Running costs = 240,000 x $1.25 = $320,000

* Total cost = $330,000 to $335,000 plus the yearly maintenance cost. Similar

price but so much quicker, and most desirable when many different DNA

tests are to be conducted over the years.
Same calculations for simple polyacrylamide gels (for simplicity assuming silver staining rather than radioactivity):

* No. samples = 240,000

* No. samples per day = 75 (with multiplexing, and allowing for reruns where

necessary)

* No. 1-person days required = 240,000 / 75 = 3200

* Labour costs = 3200 days x $100-$150 per day = $320,000 to $480,000.

* Running costs = 240,000 x $0.75 = $160,000

* Total cost = $480,000 to $640,000

240,000 samples, it would take perhaps 480 person-days, costing $48,000-$72,000, just to prepare the samples for the machines.

APPENDIX 6

The following have expressed a wish to be involved in any future work which may arise as a result of recommendations made in this report on ‘Safeguarding the national fruit collection’.

Professor Phil Forsline, PGRU, Cornell University, Geneva, NY 14456, USA

Radovan Boskovic, East Malling Research, Maidstone, Kent

Cameron Peace, Kearney agricultural Center, 9240 S. Riverbend Av., Parlier, CA 93648, USA

Biological Sciences, School of Education, Health and Science, University of Derby, Kedleston Road, Derby, DE22 1GB

Conservation & Environmental Chemistry Centre, School of Contemporary Sciences, Kydd Building, University of Abertay, Dundee, Bell Street, Dundee, Scotland,DD1 1HG,