Crop Genebank Knowledge Base

Regeneration

Regeneration guidelines for forage grass genetic resources

View regeneration guidelines in full (in PDF)
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Contributors to this page: ILRI, Addis Ababa, Ethiopia (Jean Hanson); CIAT, Cali, Colombia (Daniel Debouck, Rainer Schultze-Kraft); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Before reading the regeneration details for this crop, read the general introduction that gives general guidelines to follow by clicking here.

Introduction

Most forage grasses are maintained as seeds except for few shy seeders that rarely produce seeds or produce seeds without caryopses. These should be maintained in field genebanks using similar procedures to those described below for regeneration. Several forage grasses have seeds which are short-lived and back-up collections are also retained in field genebanks. Grasses belong to a wide range of genera and over 600 species are currently used for grazing and livestock feeds. With such a wide range of diversity, specific conditions and methods are required for each species. These general guidelines are only indicative and specific information should be sought in the literature whenever possible.

Choice of environment and planting season

Planting season

Regeneration should be undertaken in the rainy season or under irrigation for perennial species.
Planting is often done at the start of the rainy season to aid establishment.

Preparation for regeneration

When to regenerate

When seed stocks are less than 1000 seeds.
When percent germination is reduced to 65% (FAO/IPGRI, 1994).

Field selection and preparation

Select the environment and soil type best suited for the species. Information on adaptation of species can be found from the Tropical Forage ( http://www.tropicalforages.info/key/Forages/Media/Html/index.htm) and the FAO Grasslands Index (http://www.fao.org/ag/AGP/AGPC/doc/GBASE/mainmenu.htm) websites.

Select the field based on appropriate rotation and infection history to avoid mixtures and infection/infestation with different pests.
Soil should have good drainage and be well prepared for sowing.
Soil should be ploughed and disked, weeds and grasses removed. Take care to remove grass stolons that can grow and cuase mixtures in the plots. The soil should be prepared by tillage to obtain a well prepared and level seed bed prior to planting.
Deep plough to invert soil, followed by harrowing two or three times, to produce a fine and an even flat seedbed.
The majority of forage grass species are small seeded on size. The depth of planting should not exceed more than 3 cm.

Other precautions

For out-breeding grasses, a population size of at least 100 plants should ideally be used for regeneration in order to maintain genetic variation.
For those reproduced vegetatively, one plant is sufficient to maintain the genetic variation but more plants are needed for security and it is recommended that a minimum of five plants are maintained for each accession.

Method of Regeneration

Planting layout, density and distance

Aim for a final plant number of 100 in plots of approximately 25 m² for outbreeders and 25 plants in plots of 10 m² for vegetatively propagated grasses.
Plant in 11 rows of 5 m long, each row 50 cm apart with within row spacing of 50 cm giving a density of 100 plants per plot.
Forages vary in their breeding systems and species must be planted at different isolation distances (see table or Species Compendium).
Many forage grasses are out-breeding and it is recommended to use an isolation distance of at least 100 m between accessions.
Plant seeds of accessions of other species that do not hybridize or of other genera in between plots to increase the isolation distance.
Using tall plants or artificial barriers is very effective as wind breaks for wind pollinated species.

Planting method

Direct sowing

Weigh the amount of seeds to be planted per row and place in separate envelopes/bags.
Label the plot with the accession number and plot number as a minimum (taxonomic identification and origin of accession are also recommended).
Lay out the plots at the chosen row spacing.
Mark rows about 3 cm depth.
Check that the accession number is correct and place one envelope/bag on the end of each row.
Open the envelope and plant directly into a flat seedbed at a depth of 3 cm.
Scatter seeds along the row by hand sowing.
Cover with soil and lightly compact the soil in the row.

Seedling transfer

Germinate seeds in petri dishes in an incubator. The conditions vary with species (see table for use in germination testing).
As soon as the roots start to emerge, plant the young seedlings individually in seedling trays or pots using sterilized compost or forest soil.
Maintain the pots in a warm place and cover at night to retain moisture.
Keep the pots away from direct sun but with good light intensity or in a greenhouse.
Water carefully using a spray bottle so the pots remain moist but not wet.
Once seedlings reach about 15 cm in height and are strong and growing well, place the pots outside for the seedlings to harden off for one week, watering carefully.
Label the plot with the accession number, planting date and plot number.
Peg out the plots at the chosen row spacing and make holes at 50 cm along the row.
Transplant the seedlings to the field, one seedling per hole, taking care not to damage the roots when the seedlings are transferred from the pots, and water after transplanting.
Maintain few plants or weak seedlings in 8 inch pots for regeneration in the greenhouse.

Crop management

Thinning

If direct sown, thin to one plant per hole at 4-6 weeks after establishment when plants are growing well to keep plant density at about 100 plants per plot.
Avoid competition that will result in weak plants and low seed yields.
Avoid selecting only strong plants that would reduce genetic variation.
Thinning can be done simultaneously with the first weeding.

Weed management

Early growth can be slow and hand weeding 4 weeks after establishment is recommended.
Cultivate between rows twice during early stages of plant growth.
Eliminate plants growing off-row. Rogue plants that are genuine mixtures.

Irrigation

Irrigate the field after sowing and when needed subsequently.
Do not allow the leaves to wilt at any stage. Ensure enough moisture in soil at the time of flowering.

Fertilization

It is recommended to apply phosphorous at planting using a fertilizer such as diammonium phosphate or other phosphorus rich fertilizer at 100 kg per hectare of P.
An additional application of 50-60 kg N per ha as a top dressing at early flowering stage will ensure good seed quality.

Common pests and diseases

Forage grasses are susceptible to many fungal diseases and few viruses and phytoplasma (see table).

Pest and disease control

Coordinate periodic field inspections with pathologists and virologists during the growing season.
Spray with appropriate chemicals when necessary. Spray with fungicide to control mildew during the rainy season or when using irrigation and with insecticide at the first sign of insect damage.
Rogue out any infested material to eliminate all parasites before seed production and burn the residues.
Pay particular attention to army worm and spray at the first infestation.

Harvesting

Inspect the field daily once seeds start to ripen to determine the right date to harvest.
Seeds often ripen unevenly and may shatter once ripe. It is suggested to harvest once about 50% of the seeds ripen to obtain a reasonable seed yield.
Harvest by cutting the stems or heads when seeds are mature but before fully ripe seeds start to dehisce and shatter.
Wrap the heads in a bundle and stook the bundles to allow final ripening and air drying before threshing to separate the seeds.
Collect the seeds from each plant in labeled cloth or paper bags with an additional label inside each bag. Paper bags should only be used in dry climates.
Thresh the seeds on a tarpaulin by gently beating or in a threshing machine and return the seeds to their labeled bag.
For small quantities of seeds, a hand rubber thresher is recommended.
Ensure that seed mixing does not occur during threshing by thoroughly cleaning all equipment and implements between each sample, including washing cloth bags and not reusing paper bags.
Where resources are short and bags have to be reused, check carefully in folds for trapped seeds and use bags for different species so any mixed seeds can easily be spotted and removed.

Post-harvest management

Clean the seeds of debris by hand picking, hand winnowing or using a seed blower.
Hand pick over the seeds in trays to remove any shriveled, discolored, infected or damaged seeds from each plant. Incinerate the waste to avoid spread of seed borne diseases.
In order to have equal replications of all individuals in a population take equal quantities of seeds from each plant and mix in one paper bag labeled inside and outside. Once you are sure you have all the seeds needed, the plants can be removed from the field or screen house.
Where grasses grow in a sward and individual plants cannot be identified, the best practice is to ensure the seeds are harvested from all parts of the plot thoroughly mixed and kept as a bulk sample.
Retain the bags of each accession in temporary storage until seed drying.
Take a sample of the seeds for seed health testing. If the fresh seeds are infected with seed borne pathogens and more original seeds are available for a second regeneration, destroy the seeds by incineration. If no original seeds are available, schedule the seeds for a further regeneration in controlled environmental conditions using agrochemicals to obtain clean seeds.
If the seeds are free from pests and diseases, dry the seeds in low relative humidity at 15°C until they reach between 3-7% moisture content (see the seed drying page in this site for more details).
Remove the seeds from the drying room, weigh and pack directly into storage containers. Options for containers for medium term storage include using plastic containers or cans with sealed lids for storage in environments with humidity control or laminated aluminum foil packets for storage in environments without relative humidity control. Use of laminated aluminium foil packets is more suitable for long-term storage. Seal the containers or packets immediately.
Sample and test the viability of the seeds and record the results following standard germination methods (ISTA, 2008). If viability is high, proceed to storage, if viability is low, reschedule the accession for a further regeneration from the original seeds.
Store seeds in the genebank at 5-10°C in medium-term storage or at -18°C in long-term storage.

Monitoring accession identity

Comparisons with previous passport or morphological data

Distinctive traits are specific for each species. In general tillers, flower or spikelet parts, pubescence and stem traits may be useful for comparison.
Digital images are useful for identification and comparison.
Herbarium specimens are important to compare specimens to verify acession identity.

Documentation of information during regeneration

The following information should be collected during regeneration:

Regeneration site name and map/GPS reference.
Name of data collector.
Field/plot/nursery/glasshouse reference.
Accession number; population identification.
Source of seed.
Generation or previous multiplication or regeneration (if generation is not known).
Preparation of planting materials (pre-treatments).
Sowing date.
Field layout and density used.
Field management details (watering, fertilizer, weeding, pest and disease control, stresses recorded, others).
Environmental conditions (altitude, precipitation, soil type, others).
Emergence in the field or green house (number of plants germinated).
Days from sowing to flowering.
Number of plants established and harvested.
Pollination control method used.
Harvest date and method.
Quantity of seeds harvested/plant.
Comparisons with reference materials (record any identification numbers or references of any samples or herbarium specimens taken from this regeneration plot).
Agronomic evaluation; agro-morphological traits recorded.
Taxonomic identification.
Post harvest procedures.

References and further reading

Click here for a list of selected references on breeding systems in forage grass species.

Bray RA. 1983. Strategies for gene maintenance. In: McIvor JG, Bray RA, editors. Genetic Resources of Forage Plants. CSIRO, Melbourne, Australia. p157-168.

Dafni A, Firmage D. 2000. Pollen viability and longevity: practical, ecological and evolutionary implications. Plant Systematics and Evolution 222, 113–132.

Fairey DT, Hampton JG, editors. 1997. Forage Seed Production volume I:Temperate Species. CABI International, Cambridge, UK.

FAO/IPGRI. 1994. Genebank standards. Food and Agriculture Organization of the United Nations, Rome and International Plant Genetic Resources Institute, Rome. Available in English, Spanish, French and Arabic.

Friedman J, Barrett SCH, 2009. Wind of change: new insights on the ecology and evolution of pollination and mating in wind-pollinated plants. Annals of Botany 103, 1515-1527.

Hacker B, Hanson J. 1999. Crop growth and development: reproduction. In: Loch DS, Ferguson J, editors. Forage Seed Production vol II Tropical and Subtropical Species. CABI International, Cambridge, UK. pp. 93-111.

Humphreys LR, Riveros F. 1986. Tropical Pasture Seed Production. FAO Plant Production and Protection Paper 8, FAO, Rome, Italy. 203pp.

ISTA. 2008. International Rules for Seed Testing. International Seed Testing Association. ISTA secretariat, CH-Switzerland. Available from: www.seedtest.org/.

Loch DS, Ferguson J, editors. 1999. Forage Seed Production vol 2. Tropical and Subtropical Species. CABI Publishing, UK.

Neal PR, Anderson GJ. 2004. Does the `Old Bag' make a good `Wind Bag'?: Comparison of four fabrics commonly used as exclusion bags in studies of pollination and reproductive biology. Annals of Botany 93, 603-607.

Thormann I, Metz T, Engels JMM. 2004. The Species Compendium (release 1.0; December 2004). [online] Available from: http://www.bioversityinternational.org/databases/species_compendium_database/index.html. Date accessed 17 Jan 2011.

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