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Seedbank (埋土種子)






Mount Usu / Sarobetsu post-mined peatland
From left: Crater basin in 1986 and 2006. Cottongrass / Daylily

[ seedbank estimation | centrifuged flotation method ]
[ seed | seed dispersal | seed germination ]

Seedbank, seed-bank, or seed bank
  1. A store of viable seed (種子) buried and dormant (休眠) in the soil or underwater sediments.
  2. The place where seeds of rare plants or obsolete varieties are sotred in carefully controlled conditions to preserve their genetic material for research and possible future use.
I use the meaning #1 in my lectures.
Seeder vs sprouter
Obligate seeder: Regeneration of the rare Morro manzanita (Arctostaphylos morroensis) is dependent on fire. This plant is an obligate seeder; that is, individuals do not resprout after fire, and the species maintains itself solely by reproducing from seed.
Obligate sprouter

Seedbank in the former topsoil on Mount Usu


索引

Fig. 1
Fig. 1. Spatial distribution of total number of seeds in a 50 × 50 × 10 cm quadrat of which density was the highest in the four quadrats surveyed. The sizes of circles indicate that number of seeds (flotation method) or seedlings (germination method) in each 10 x 10 cm subquadrat. The maximum circle indicates 52 seeds. Numeral within each box represents number of species. (Tsuyuzaki & Goto 2001)

Carex oxyandraHypericum erectumLuzula capitataRumex obtusifolius

Seedbank1 Dig2
[1] seeds of dominant species (Rumex obtusifolius, and others) in the former topsoil 20 years after the 1977-78 eruptions on Mount Usu (Goto M). The seeds were extracted by a centrifuged floatation method (Tsuyuzaki 1994). [2] how to dig! (April 29 1988)

Methods of seedbank estimation (埋土種子集団推定法)


Table 1. Types of seedbank estimation (After Tsuyuzaki 1990)
  1. Germination: sowing soil on pot
    easy way
    waste long time (occasionally more than 6 months), low accuracy (all seeds do not always germinate)
    low seed recovery rate in general
  2. Hand sorting: power play under a binocular stereomicroscope
    easy way
    restricted sample volume, Boring job
    high seed recovery rate
  3. Sieving: seiving with various mesh sizes
    easy way, quick treatment
    difficult to extract all sedds, in particular, small seeds
    seed recovery rate depending on the soil sample
  4. Flotation: seeds floated by a high-concentration solution
    quick treatment, high seed recovery
    complicated manipulation, seeds damaged by flotation solution
    very-high seed recovery rate

(Feast & Robert 1973)

Germination test

for the estimation of the density of buried seeds
Weigh soil and place into earthenware dishes (10 cm in diameter)
↓ soil thickness = 0.5 cm (ca. 40 cm³) and 1.0 cm (ca. 80 cm³)
↓ three replications = 40 × 3 + 80 × 3 = 360 cm³
Place into an incubator
↓ Add water as necessary
↓ Stirs sample soils every 2 months (not necessarily)
Count number of seeds germinated

(Ter Heerdt et al. 1996)

Improved germination test

Washing soil samples by coarse and fine sieves

Coarse = 4.00 mm mesh width
Fine = 0.212 mm

= concentrated samples [and greenhouse space]
[Sample soils = clay, peaty, and sandy soils]

|

Spreading in a 3-5 mm thick layer on sterilized potting compost
  1. Seedling emerged more than by unconcentrate samples
  2. Hand-sorting afterwards shows that the germination rates vary between 81 and 100% of the viable seeds present
  3. 95% seedlings emerge within 6 weeks

(Traba, et al. 1998)

Concentrating samples can lead to seed losses in soil bank estimations

Concentration, C

Traba et al. (1998) = 0.100 mm-diameter sterile nylon mesh

[ter Heerdt et al. (1996) = 0.212 mm in mesh width]

vs

Non-concentration, NC
Seedling emergence (/432 cm³)
C__199.7 ± 79
NC_161.5 ± 11.8
P < 0.001 (paired t-test, n = 10)
No significant difference in the number of species per sample (P = 0.153, n = 10)
Seven species (Crassula tillaea, Hernaria hirsuta, Sagina apetala, Teesdalia coronopifolia, Trifolium arvense, Tuberaria gutata and Vulpia spp.)

NC > C (Wilcoxon range test, P < 0.05)
Those species produce small seeds.

Discriminant criteria on seed survival (種子生死判別)

TTC staining on seed embryo for viability check
Procedure: 0.6% TTC solution (pH 6-7 adjusted by 20 mM K2HPO4 or K2HPO4) → Cut seed vertically → Place them for 24 hr in the dark → Inspect the embryo under a binocular stereomicroscope

Preparation of artificial buried-seed populations

cup
Seeds
↓ mixed with sea sand (not necessarily)
↓ Packed into a holed plastic tube
Seeds burial into soil at any soil depths

Small plastic capsules, each of which contains 50 seeds and sea sand, are buried into soil. Four pin holes were drilled through each capsule so that the contents of air and humidity might equilibrate with those outside.

Flotation method to extract buried seeds


Seed extraction from soils by a flotation method (Tsuyuzaki 1994)
Abstract. In this study a flotation method is described which enables the rapid extraction of seeds from soil. Essentially, soil is mixed with 5.5 M K2CO3, and thoroughly dispersed by mechanical stirring for 3-6 min. The dispersed soil samples were delivered to plastic tubes, then centrifuged at more than 4,000 g for about 5 min. Buried seeds and lighter organic materials floated in the supernatant and could be removed by filtration, washed and subsequently identified. This method performed reliably using soil samples artificially burdened with white clover and a range of cultivated grass species. In addition, a wide range of weed seeds which differed in their size and densities were also effectively recovered from a range of soil types loam to gravel.

Principle

Seeds are floated by a high-concentration solution. We use 50% K2CO3 solution with cnetrifugation described below. This method is improved gradually.

Flotation method with centrifugation

Soil samples 1

↓ - add 50% K2CO3 (specific gravity = 1.54)
↓ - agitation by a high-power mechanical stirrer for a few min.²
↓ - centrifugation at > 3000 g for 3-5 min

Supernatant

↓ - filtration using an aspirator ³
Seeds and organic debris are collected on 2-3 layered miracloth
↓ - inspection uder a binocular stereomicroscope

Seed samples
[Remarks]
  1. The volume of soil sample is dependent on the volume and number of centrifugation tubes. I usually use four 200-ml centrifugation tubes at a time. Do not use glass tube.
  2. The power os Mini-stir is not enough to agitate soil sample.
  3. Wash the organic debris well using distilled water to reduce seed damage by salt solution. The side-wall of centrifugation tubes should also be washed wall to remove organic depris on the wall.

protocol (プロトコル)

Preparation

For flotation
Portable centrifugator
Centrifugation tubes (> 200-ml is nice)
Floatation solution (see below)
Distilled water
Beakers (size is dependent on the volume of solution)
Bottle with nozzle (to wash organic debris)
Pipette
Balance (for centrifugation)
Miracloth
Tweezers
Stirrer (shaft is better)
Aspirator
Buchner funnel
Suction bottle
Rag (floorcloth), and/or Kim-towel
Rubber gloves (if your skin is sensitive)
Flotation solution
50% K2CO3 (Tsuyuzaki 1993)
Add 770 g K2CO3 by bits to 1 l H2O in a beaker with stirring by using a mechanical stirrer
→ Appplication: (Tsuyuzaki 1989, Tsuyuzaki 2010, Ishikawa-Goto & Tsuyuzaki 2004)
Calgon mixture (Malone 1967)
10 g sodium hexametaphosphate (calgon, ヘキサメタリン酸ナトリウム), (NaPO3)nP2O5
5 g sodium bicarbonate (baking soda, 炭酸水素ナトリウム) NaHCO3
25 g magnesium sulphate (epsom salts, 瀉痢塩) MgSO4
200 mg tap water

For sorting

Binocular stereomicroscope
Tweezzers
Needle
Petri dishes
Of course, notebook and writing tools
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