X. Regeneration of Accessions

What is regeneration?

This is the renewal of an accession by taking a random sample of seeds, sowing and growing the resulting plants under conditions so that the seeds harvested will possess the same characteristics as the original population.

Why is regeneration necessary?

The most important part of conservation of material is that the seeds maintained in the genebank should always produce plants which show the representative characteristics of the accession. If seed viability begins to decrease, then genetic changes can occur in the seeds, thus changing the characteristics of the accession. Therefore renewal of the accession is necessary and must be carried out in the correct way to maintain the characteristics of the original population. Regeneration is also necessary when the number of seeds of the accession in store falls below a critical level.

When are accessions regenerated?

Regeneration is a time-consuming and expensive task which is potentially dangerous to accessions because it can allow genetic change and the accession is at the risk of pests and diseases during the growing cycle. Therefore regeneration should be kept to a minimum. It is necessary when the seeds of the accession begin to lose viability, when small seed samples are entered into the genebank or when seed stocks become low due to distribution from active collections. The IBPGR Advisory Committee on Seed Storage recommends that for most species an accession should be regenerated when the seed viability falls below 85%. Regeneration is also required when the number of seeds in an accession falls below the number required to complete 3 regeneration cycles for that species. If there is doubt about the condition of the seeds, their regeneration must take priority; otherwise the sample may be lost. Some very small seed samples may need several cycles of regeneration before sufficient seeds are harvested to reach the recommended sample sizes. FOR INFORMATION ON SAMPLE SIZE, SEE SECTION VII.

How should regeneration be carried out?

Regeneration is carried out by growing out and harvesting a representative sample of seeds from an accession. The techniques used must ensure that the seeds produced from regeneration are fully representative of the original population. Since species differ in agronomic requirements, breeding systems, compatibility and population structure, the techniques used are crop-specific and will not be discussed in detail in this manual. It is recommended that curators consult with plant breeders and experts on the various crops to identify appropriate methods for regeneration and that these be incorporated into the genebank's regeneration procedures.

Principle factors to consider when selecting a suitable regeneration method

* Suitability of the environment for each accession to ensure that natural selection is kept to a minimum and there will be good seed production.

* Special requirements needed to stimulate germination, to break dormancy and induce flowering (vernalization or photoperiod).

* Optimum number of plants to fully represent the population and ensure that all the characteristics of the accession are maintained.

* Optimum number of plants to ensure that enough seeds are produced to reach the recommended sample size.

* The correct spacing for the plants to ensure optimum seed set and production of good quality seeds.

* The breeding system of the plant (outbreeder or inbreeder) to determine whether isolation is needed.

* The need for an artificial pollination method, either by the introduction of insect pollinators or hand pollination, to ensure seed set.

* The presence of compatibility barriers to pollination which may have to be overcome artificially.

* The sexuality of the plants (dioecious, monoecious or hermaphrodite) to ensure that sufficient male and female plants are available for pollination and good seed production.

* The need to mix approximately equal proportions of seeds from different mother plants to avoid maternal effects from particular plants in the population.

STEP 1. DETERMINE HOW MANY ACCESSIONS CAN BE REGENERATED IN ONE CYCLE

1. Seek advice on the techniques and environmental conditions required for each crop from plant breeders and the curator.

2. Consider the area of the land available for regeneration.

3. Consider the number of plants per plot and their spacing to determine plot size.

4. Consider the area required for isolation and paths between plots.

5. Divide the amount of land available by the amount of land required for one accession including paths. The nearest low whole number is the number of accessions that can be grown in the space available.

6. The curator will consider the work involved and the staff available and decide how many accessions to handle.

Notes and Examples

Do not handle too much material at one time - it is better to handle fewer accessions well than many accessions badly. Generally speaking, seeds stored in the genebank can usually be kept for another year without greatly decreasing seed viability.

Calculator/graph paper
Packets
Harvest bags/packets
Labels
Stakes
Isolation bags or cages

1. Make a list of all accessions with a seed viability of less than 85%.

2. Make a list of new accessions which contain less than the recommended number of seeds for storage in the genebank (for details see Section VII).

3. Make a list of all accessions with the number of seeds remaining less than three times the number of seeds used for regeneration of that species.

4. The curator will decide which accessions have priority. As a general rule first priority should be given to the accessions with the lowest seed viability, second priority to any accessions which are required urgently from either list and lastly to accessions with high viability but few seeds.

5. Make a list of the accessions which the curator has decided to regenerate in this cycle and a second list of other accessions which are awaiting regeneration in the future.

6. Record the location of each accession in the genebank from the list of those to be regenerated.

STEP 3. DETERMINE WHERE TO GROW THE MATERIAL

1. Check the passport data to find which environmental conditions are needed for that accession.

2. Check the number of seeds that are available.

3. If few seeds are available and the accession is sensitive to the prevailing environment, grow under controlled conditions or in a glasshouse.

4. If enough seeds are available and the accession is well adapted to the outdoor climate or special conditions are not available, grow in the field.

Notes and Examples

Select the right environment for the accession. Take special note of day-length requirements in certain crops or they may not flower. In some cases where seed dormancy is present it may be necessary to treat the seeds first before sowing in the field.

1. Draw a scale map of the area to be used.

2. Mark this plan off into plots of the correct size for each species, allowing room for isolation blocks, inter-planting of species and paths for access.

3. Fill in the accession numbers from the list already prepared into the appropriate plots, taking account of any isolation distances, species and maturity differences necessary to prevent crossing between plots.

STEP 5. PREPARE THE SEEDS FOR PLANTING

1. Remove the container from the genebank.

2. Allow the container to warm to room temperature before opening. This may be several hours for large containers.

3. Write the accession number and genus and species of the seeds on the outside of a seed packet.

4. Open the container and quickly weigh or count the required number of seeds already decided as being appropriate for the regeneration of accessions of that species, but do not use more than half of the seeds which are available for that accession. Work in a dehumidified room, if possible and close the containers as soon as the seeds are removed to prevent the seeds absorbing moisture from the air.

5. Place the seeds in the labelled paper packet and seal.

6. Record the number or weight of seeds removed from each accession.

Notes and Examples

Always use random sampling techniques to remove seeds from the container so that the resulting sample will provide a good representation of the original population.

1. Follow the planting plan using techniques outlined by the breeders and curator to ensure that regeneration is done well and no crossing occurs between accessions.

STEP 7. HARVEST THE NEW SEEDS

1. Harvest each accession separately when the seeds are fully mature.

2. Mix the same weight of seeds from all the plants of one accession to minimize maternal effects and still keep as much variation as possible within the sample. Make sure that population samples are not divided into lines but keep the accession complete.

Notes and Examples

Make sure that the population is kept whole and not split into lines. If lines are required for plant breeding needs, it is possible to keep these separate in a different programme, but the population should also be maintained intact.

1. Carry out seed cleaning (Section II) and measure the weight or number of true seeds harvested.

2. Do not mix the seeds from the different regeneration cycles.

3. Use the seeds from earlier regeneration cycles to supply seed requests.

4. Regeneration may need to be carried out several times to reach the recommended sample size for genebanks (Section VII).

5. Use the other half of the original sample for the second regeneration and representative samples from these two for further regeneration.

Notes and Examples

It is better to go back to the original seeds to sow for regeneration, if these seeds still have a high viability, because each time that seeds are regenerated the characteristics of the population can be changed from recombination of genes and selection. Therefore it is better to have seeds from the least number of regeneration cycles in order that genetic integrity is maintained.

1. Enter all inventory data referring to the new seed into the appropriate data files.

2. Characterization can also be carried out during regeneration and this type of data should be entered into the appropriate data file.

Table of regeneration procedures used in your genebank

Fill in this table to use as your future personal guide:

Species	Number of Plants	Spacing	Breeding System	Isolation	Pollination Method