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African Crop Science Conference Proceedings, Vol. 9. pp. 289 - 291

Printed in Uganda. All rights reserved

ISSN 1023-070X/2009 $ 4.00

2009, African Crop Science Society

Seed regeneration practices at the Southern African Development

Cooperation (SADC) Plant Genetic Resources Centre

L. LEROTHOLI QHOBELA & W.Y.F. MARANDU

SADC-Plant Genetic Resources Centre, Private/Bag CH6, ZA15302, Great East Road, Chalimbaba, Lusaka, Zambia

Bioversity International, Programme on Understanding and Managing Biodiversity. c/o AVRDC-RCA,

P.O. Box 10 Duluti, Arusha, Tanzania

Abstract Seed genebanks provide the most practical method for preserving large amounts of genetic material within a small space.Seeds in genebanks, eventually, deteriorate whatever the conservation conditions. Genebanks managers are, therefore, faced with the

challenge of ensuring that samples stored are genetically very similar to the original sample collected in the field. Low temperature

and optimizing seed moisture content delays seed deterioration, but, when seed viability decreases to below 85%, regeneration is

recommended. Regeneration process appears routine and necessary but it may lead to losses of valuable traits if sufficient attention

is not given to factors affecting genetic integrity of the accession. Genebank managers at the SADC Plant Genetic Resources Centre

(SPGRC) follow the recommendations published by Bioversity International (formerly IPGRI) and other Advanced ResearchCentres during a regeneration process. This paper gives an overview of practical considerations and procedures carried out at SPGRC

while regenerating germplasm of orthodox seed crops.

Key words: Genetic integrity, genetic resources, regeneration, seed viability, SPGRC

Introduction

The Southern Africa Development Community (SADC)

comprises 15 countries with a wide range of agro-climatic

conditions, rich in biodiversity and known for

domestication of several cultivated species. Before the

1980s, there were no formal genebanks for long-termconservation of genetic resources in the region. The

utilization of modern varieties and agricultural practices

were seen then to be eroding most of this wealth of agro-

biodiversity in the field. SPGRC was established in Lusaka

Zambia in 1989 to hold a base (long term) collection and

facilitate the establishment of National Plant Genetic

Resources Centres (NPGRCs) which hold the active (short

term) germplasm collections in their countries.

A critical objective of a seed conservation programme

is to preserve the genes with the same frequency as found

when they were collected in the field. Seed banks provide

the most practical method for preserving large amounts ofgenetic material within a small space. Nonetheless, seeds

in genebanks eventually deteriorate whatever the

conservation conditions. Low temperature and optimizing

seed moisture content can delay seed deterioration, but,

when seed viability decrease to below 85%, regeneration

is recommended. Although the process of regenerating

genetic resources in genebanks may appear routine and a

necessary operation to many scientists, it may lead to

losses of valuable material when conducted without

sufficient attention, to the genetic quality of the accession

thus leading to genetic erosion. Burton 1975 for example

made a comparison of the last generation with the first in

six different germplasm accessions of pearl millet andshowed that advancing germplasm of the crop narrowed

phenotypic variability, lost genes and obscured some

characters, with the population then appearing to be more

uniform. Therefore, along with the need for seed

regeneration, the process has to be managed in a manner

that the integrity of accessions is maintained as far as

possible. Genebank managers at the SPGRC closely follow

the recommendations published by Bioversity

International and other Advanced Research Centres inorder to maintain the genetic composition of an accession

during a regeneration process. This paper gives an

overview of practical considerations and procedures

carried out at SPGRC while regenerating germplasm of

some orthodox seed crops.

Regeneration procedures

Seed sources for regeneration. National genebanks hold

active collections in their genebanks while SPGRC holds

the regions base collection. The active collections are

used for evaluation, monitoring, utilization and exchangepurposes. Samples for regeneration are taken from active

collection when seed viability or the amount of seed in

the active collection is depleted. The base collection at

SPGRC is maintained under the most appropriate

conditions for long storage as a back-up in case the

accessions are lost in the active collection. However, no

matter how good the conditions of storage are, the seed

will undergo aging process. Therefore, if viability of seed

in base collection drops below the threshold recommended

by Bioversity, the seed accession will be taken from the

base collection for regeneration.

Criteria for scheduling regeneration of accessions frombase collection. The initial seed germination percentages

for all accessions held in the base collection are known

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L. LEROTHOLI QHOBELA & W.Y.F. MARANDU290

from a register of the internally developed SPGRC

Documentation and Information System (SDIS) database

that is updated each time a sample of any accession is

entered into the collection. Seed accessions in the

genebank are monitored at intervals of five years for

germination percentage and quantity of seed. Accessions

that have low viability and those with insufficient seed

quantities are marked for regeneration and the list is passedto Regeneration and Multiplication section which will

determine the number of accessions to be regenerated

based on the availability of resources (financial and

personnel) and the type of accessions (wild, weedy,

cultivated) before regeneration is undertaken. First priority

is given to accessions with low seed germination

percentage and insufficient seed quantity; followed by

accessions with low seed germination percentage with

sufficient seed quantity and least prioritized are seed

accessions with high germination percentage with

insufficient seed quantity.

Field practices. Regeneration practices aim to produce

sufficient seed with genetic composition as close to the

original sample as possible; but experience shows that it

is not always possible to produce sufficient seed in one

regeneration cycle from small seed sample sizes commonly

received from collection missions. When the seed sample

size is sufficient the appropriate size of a plot for production

of adequate quantities is chosen. For most field crops the

normal size of a plot is five meters long with 0.80 meters

between rows. The number of rows will vary with the sample

size. On the whole, seed sowing follows the agronomic

requirements of the crop. Proper land preparation, plot

markings in the field and sowing also depend on the crop.Sowing is done in December and early January and seed

is sown in ridges to encourage drainage. Both basal and

top dressing fertilizers are recommended and

supplementary irrigation is applied during dry spells.

Weeding is done manually and chemicals are used as a

last resort to control pests and diseases. Field rotations

are considered on routine basis to minimize accumulation

and spread of pests and diseases. Strict isolation

procedures to avoid adulteration by foreign pollen (Rao,

et al., 2006) are followed for all cross pollinating

germplasm. Isolation is mainly achieved by controlled

pollination using waxy pollination bags used for collectionof pollen and isolating individual plants or flowers.

Appropriate cost effective mating system such as selfing

as many plants as possible or sibbing is normally used. In

a few cases individual accessions are isolated by distance

or by time. In a number of cases multiple cycles of

regenerations are required to achieve the target amount

of seed for long term storage. Since each multiplication

cycle may affect the genetic composition of the original

accession by altering the frequencies from one generation

to another or by the loss of alleles a target minimum number

of plants to be grown for regeneration is normally set. For

example for pearl millet, at least a minimum of 200 plants

are required from each regeneration cycle while 150 plantsare grown for sorghum accessions 150 plants are grown

out for maize accessions to maintain genetic integrity and

get sufficient seed quantities.

Seed storage facilities and seed longevity. The growing

conditions, post harvesting handling, drying, packaging

and storage conditions determine seed longevity. To

achieve high initial seed viability, infestation of seed by

pest and diseases is minimized. However, some germplasmaccessions are more vulnerable than others and problems

such as diseases and pest attacks are sometimes

encountered. If the problem is experienced for the first

time and appears to have a substantial effect on the plants,

where possible chemicals are applied to control the pests

or diseases otherwise application of chemicals is not

encouraged during seed regeneration activities in the field.

Attention is also paid to harvesting each accession at the

right time, giving priority to shattering types. Post-

harvesting handling activities such as threshing are carried

out with great care before the pre-storage activities begin.

The genebank uses the dehumidification method of drying

germplasm with drying temperature of 150C and relative

humidity of 15% to dry seed to between 5-3% of moisture

content achieved over a period of 2 to 4 weeks for many

crop species in a walk-in drier. The method minimizes

damage to the seed even to seed with high initial moisture

content. When seed has dried to required low moisture

content, it is placed in airtight bottle for conservation and

in five aluminum foil packets each used to monitor viability

status of the accession on intervals of five years or more.

The sealing operation is carried out with care and as fast

as possible to ensure seed does not re-absorb moisture

after drying and before it is stored at -20oC in the genebank.

To store adequate seed in a bottle and ensure sufficientseed is available for monitoring viability over a period of

30 years, at least 2500 seeds from self pollinated species

and 4000 from cross pollinated species are required. The

genebank uses chest freezers to conserve the base

materials as appropriate technology for the region.

Discussion

Constraints. Regeneration is a costly difficult operation

that requires special skills. Two main factors are considered

at SPGRC during regeneration of germplasm: the quantity

of seed to be produced and preserving the genetic integrityof the accession. To preserve the genetic integrity of the

accession during seed regeneration, crop specific or

sometimes accession specific procedures are applied.

Theoretical knowledge on the factors that affect genetic

integrity of germplasm under regeneration is very

important. Regeneration guidelines have been developed

for a number of crops. However, the majority of accessions

handled by SPGRC are landraces consisting of mixtures

of populations, therefore, harvesting time and procedures

may not be the best for all the plants which may have an

effect on the longevity of the seeds in storage and

probably unwanted natural selection. This is a major

challenge that must be kept in the minds of staff all the

time to avoid accidental systematic selection. In addition,

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291Seed regeneration practices

wild species are particularly difficult to regenerate

optimally. Brown (1997) emphasizes that optimum

regeneration of wild species requires a clear definition of

objectives and priorities among accessions, monitoring

of mating system and genetic structure, maintenance of

accession purity and associated passport data, and

biologically realistic and flexible guidelines for sample size.

Most wild, weedy, and some cultivated species have notyet been regenerated at SPGRC partly because of this

problem. If an accession cannot be restored from active

collection, the national genebank is normally asked to make

arrangements for regeneration closest to a site where the

sample was adapted and collected, and usually, specialist

scientists who are familiar with the biology of wild, weedy

and other difficult species in the country of origin are

consulted. A site at SPGRC is used to regenerate accessions

with majority being from major cereals crop species

including some maize, sorghum and pearl millets which

grow in a wide range of ecological conditions. Data from

the SPGRC Documentation and Information System (SDIS)

database is used to determine on a case by case basis,

whether an accession can be regenerated at SPGRC fields.

Other constraints include incidences of wild animals

selecting out specific material during growing season

necessitating hiring people to scare them; power shortages

resulting in heavy dependence of the standby generator.

These constraints increase the cost of regeneration

considerably.

Research needs. Considering the number of crops and

the type of materials maintained by the SPGRC, there is

need for studies in the regeneration of mixed populations,

wild relatives of crops and some of lesser known crops.Paying attention to methods and tools such as use of

controlled pollinations, planting an appropriate number

and making balanced samples from the individuals

contributing seed to the next generation is important for

efficiency and quality of samples put in storage. Dormancy

breaking protocols for some wild, weedy and difficult

species including less known crop species need to be

developed. These kinds of studies can be coordinated in

the region as well as outside the region so as to get the

best input by different experts.

Conclusions

Regeneration of genebank accessions by SPGRC heavilydepends on the network linkage with NPGRCs which

provide appropriate sites for materials collected from their

countries and also expertise. By carefully sifting through

the documentation system it has been possible to narrow

down to crops and accessions that can be regenerated at

the SPGRC site allowing NPGRCs to delegate some of the

regeneration load whenever appropriate. A large number

of the materials under conservation are heterogeneous

landraces or wild crop relatives which are not well studied.

These materials therefore pose a major challenge to

scientists since they will soon need regeneration. It is,

therefore, necessary to initiate coordinated studies in the

region as well as outside the region so as to get the best

input by different experts.

Acknowledgement

This paper was prepared with permission of the Head of

SPGRC which is greatly appreciated.

References

Brown, A. H. D., Brubaker, C.L. & Grace, J.P. 1997.

Regeneration of Germplasm Samples: Wild versus

Cultivated Plant Species. Crop Science 37, 7-13Burton, G..W. 1975. Gene loss in Pearl millet germplasm

pools. Crop Science 16, 251-255.

Rao, N.K., Hanson, J., Dullo, M.E., Ghosh, K., Nowell, D.

& Larinde, M. 2006. Manual of Seed Handling in

Genebanks.Handbook for Genebanks No.8. Bioversity

International Rome, Italy.