ALTERNATIVE METHODS FOR STORING THE COLLECTION PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST

Pyrus Expensive development workVolk and Walters2003a Material not immediately available to growers and other users Long wait of years for production of flowers andpollen for breeding pu

APPENDIX 1

ALTERNATIVE METHODS FOR STORING THE COLLECTION

PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST

A CryopreservationPro

Long term storage (Forsline, Towill et al

Desiccated dormant buds easily shipped

(Forsline, Towill et al 1998)

Grafted trees can be forced into early

flowering (Forsline, Towill et al 1998)

Clonal integrity maintained (Forsline, Towill et

Variable recovery rates (Forsline, Towill et

al 1998) May lose some accessions

Desiccation damage (Volk and Walters 2003a)Material needs to be decontaminated (Volk andWalters 2003a)

Some species need to go through culture stage prior to cryopreservationPrecultural conditions of both vegetative parentand explant material impact on success of cryopreservation (Helliot and deBoucaud 1997;

tissue-Wu, Engelmann et al 1999; tissue-Wu, Zhao et al 2001; Volk and Walters 2003a)

Cryopreservation diffusion rates in larger propagules may not be sufficient for survival after cryopreservation (Volk and Walters 2003a)

Smaller explants susceptible to physical damage of extraction and exposure to toxic cryoprotectants (Volk and Walters 2003a)Physiological condition important for health andsurvival after cryopreservation (Wu,

Engelmann et al 1999; Volk and Walters 2003a)

Mature specimens not available for characterisation

Back up systems needed in case of power/equipment failure

Meristem cryopreservation necessary for less

cold hardy species e.g Pyrus

Expensive development work(Volk and Walters2003a)

Material not immediately available to growers and other users

Long wait of years for production of flowers andpollen for breeding purposes (especially for

Loss of staff to maintain field collection results

in loss of technical backup and expert knowledge

Cryopreservation of different types of material (Reed 2002)

Dormant buds

Readily available form field genebanks

In vitro shoot tips

Available at any time of the year

Easy to manipulate physically and

physiologically

Embryogenic cultures

Callus is generally easy to cryopreserve

Degree of cold hardiness varies with reason and genotype

Buds only available for a few moths during the winter

Requires more storage space than other techniques

Requires grafting and budding expertise for recovery

Techniques are not developed for all plantsRequires a laboratory and skilled workers

Not all plants produce somatic embryos

Embryonic axes

Easy to remove and process

Techniques are not broadly applicable across species/accessions

In vitro systems needed to recover whole

plants

Cryopreservation; Future studies (Volk and Walters 2003a)

 Knowledge of biophysical properties of water within cells

 Physiological understanding of critical content for desiccation damage

 Hydraulic conductivity of water

 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

Other questions: Ashworth, 1986 #74}

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?

Notes

US varieties may be more tolerant of cryopreservation because of genetic make up

or exposure to harder winters producing hardening to a greater extent

B Tissue Culture Pro

Rapid multiplication possible if large numbers

required

No requirement for large land area

Not affected adverse weather

Cultures easily transported

International transport possible

Pest and disease-free stocks maintained

Propagation of recalcitrant species

Ease of rooting (especially for woody

materials)

Rapid regeneration documented for many of

NFC crops

Ease of germplasm exchange and shipment

both within and between countries

Con

Development work expensive and time consuming(Volk and Walters 2003a)High maintenance costs

Labour intensiveAccurate labelling at each subculture necessary (real danger of errors due to mislabelling)

Danger of microbial contamination (Volk and Walters 2003b)

Promotes endophytic bacteria (Reed, Buckley

et al 1995) (Reed, Mentzer et al 1998)Stresses of media additives to control endophytic bacteria may decrease vigour and survival of cultures (Volk and Walters 2003a)Mite/thrip infection possible

Physiological changes with time/subculturesSomaclonal variation(Swartz, Galletta et al 1981)

Ploidy problemsDifferences in growth and fruiting after regeneration

Need light and temperature controlNeed sterile working area

Delayed flowering

Mature specimens not available for characterisation

Future studies in tissue culture:

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

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 belower

Need to go through multiplication and rooting media at 20oC (few months) before retuning to cold

Still labour intensiveLoss of some accessionsNeeds 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

D DEFRA National Field Collection at Brogdale Pro

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

Con

Labour intensiveHigh cost

Large acreageProne to attack from pathogens & pestsEffected by extreme environmental conditionsRisk of vandalism

Climate changes may eventually effect fruiting due to lack of winter chill for flower initiation

import permits and quarantines etc)

Position of accessions fixed in collection for

many years with site maps giving location

Easier for public to see and appreciate

collection which may result in more monetary

support from foundations or the public-ie PR

exercise

Need for re-propagation and parallel collectionsM9 need stakes to support scions

Note:

At NCGR, accessions in pear collection cut back annually; either 25% off the top, or

left or right hand side of rows cut back with hedge trimmer on consecutive years to

reduce cost of maintaining trees at a manageable size, and keep trees rejuvenated

Development work:

Need good genetic markers to identify accessions in field and those coming out of

cryopreservation Research on genetic diversity can help define core collections for

field collections, so reducing collection size and costs More reliable identification

(fingerprinting) of accessions which are relatively easy and cheap to run, are needed

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)

Fewer flowers (for breeders)Less material available than standard treesMay not produce enough fruit for crosses on trees

Greater intensity of care necessarySmaller root balls

M27 need wire frames to support scionShorter 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 notknown

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)

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)

Fewer flowers (for breeders)Less material available than standard treesMay not produce enough fruit for crosses on trees (but could use pollen instead)

Greater intensity of care necessaryPosts 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

Periodic re-potting (Annual?)Labour costs of re-potting etc

Loss of labels especially during re-potting (mislabelling causes masses of work)

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

Restricted access to public because of danger

of label theftShorter life (more frequent re-propagation)Disease is more of a problem

Effected by extreme environmental conditionsRisk 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

H Seed Storage Pro

Long term storage

Simple and space efficient

Low maintenance requirements

Easy to use for appropriate seed types

(orthodox) i.e small seeded desiccation

tolerant , cold tolerant, not clonally propagated

accessions (Reed 2002)

Con May lose some accessions Mature specimens not available for characterisation

Not useful for large, cold sensitive or desiccation intolerant seeds (recalcitrant) or clonally propagated plants

(Reed 2002)Back up systems needed in case of power/equipment failure

Development work on content of collection not possible

Loss of staff to maintain field collection results

in loss of technical backup and expert knowledge

Seed increase process necessary when number of seeds in accession falls below 100

or viability falls below acceptable level (eg testsindicate that only probably 30 viable seeds left)-Peters ,J., NCGR

Seed increase process time consuming –Peters, J., NCGR

Note:

A seed collection would be complementary to, but not a replacement for a field

collection

Development work:

Developing a way of storing recalcitrant seeds

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

I Pollen Storage Pro

Long term storage (Towill and Walters 2000)

Simple and space efficient (Towill and Walters

2000)

Low maintenance requirements

Desiccated dormant buds easily shipped

(Forsline, Towill et al 1998)

Easy for many plant genera (Reed 2002)

Useful for breeding (Reed 2002)

Con May lose some accessions Mature specimens not available for characterisation

Back up systems needed in case of power/equipment failure

Development work on content of collection not possible

Loss of staff to maintain field collection results

in loss of technical backup and expert knowledge

J Leaf Storage Pro

Long term storage

Simple and space efficient

Low maintenance requirements

Material readily available for DNA extraction

Con Mature specimens not available for characterisation

Back up systems needed in case of power/equipment failure

Development work on content of collection not possible

Loss of staff to maintain field collection results

in loss of technical backup and expert knowledge

APPENDIX 2

Table A2.1 – Number of Malus accessions cryopreserved at the

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

4522115231542408142425121151120511100575

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.