Seed germination

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Seed germination (種子発芽)

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

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Def. germination; the development of a plant from a seed or spore after a period of dormancy e.g., seed germination

Def. emergence: the process of becoming visible after being concealed Ex. seedling emergence

seed → germination → seedling

seed dispersal →
seed dormancy → seedbank (seedbank estimation methods)

Germination signal

Two signals: dormancy-break signal + germination signal → germination
Germination: The initiation of embryo growth and seedling development.
Dormant seed: Viable seed in a non-germinable condition due to internal and/or external barriers, e.g., hard seed coat, water defficiency, and no light. imbitition

Seed imbibition

Measurement of seed imbibition rate - seed imbibition (rate) curve
5 lots of 10 seeds × 9 replications = 45 lots
Therefore, you need more than 450 seeds for 1 species
Measure seed weights at 3 hr intervals until no more seed weight increase is observed

Warm stratification

Any seeds that are indicated as needing a period of warm stratification followed by cold stratification should be subjected to the same measures, but the seeds should additionally be stratified in a warm area first, followed by the cold period in a refrigerator later. Warm stratification requires temperatures of 15-20°C. In many instances, warm stratification followed by cold stratification requirements can also be met by planting the seeds in summer in a mulched bed for expected germination the following spring. Some seeds may not germinate until the second spring.

Heat exposure (熱)

Dry heat:
In an oven at 75°C for 25 min, using 40 species (Tsuyuzaki & Miyoshi 2009)
In a water bath, ranging from 35°C to 95°C for 30 min, for three Asteraceous species (Yuan & Wen 2018)

Dry heat: without pouring water in the flask
Wet heat: with pouring a few drops of distilled water in the flask

Seed germination (種子発芽)

Germination indices

Germination percentage (germinability), g_p

g_p = N_g/N_t × 100 (%)

N_g = the number of germinated seeds
N_t = the total number of seeds used

Time for the first germination or germination time lag, t₀

the time for first germination observed (unit: time, e.g., day)

Time for the last germination, t_g

the time for last germination observed (time)

Time spread of germination, or germination distribution, T_s

T_s = T_g - T₀ (time)

Peak period or modal time of germination

the time showing the highest frequency of germinated seeds (time)

Median germination time, t₅₀

1. Coolbear et al. (1984), t₅₀ = T_i + [{(N + 1)/2}(T_j - T_i)]/(N_j - N_i) (time)

t₅₀: median germination time
N: the final number of germinated seeds
N_i and N_j: the total number of seeds germinated at T_i and T_j
condition: N_i < (N + 1)/2 < N_j

2. Farooq et al. (2005), t₅₀ = T_i + {(N/2 - N_i)(T_j - T_i)}/(N_j - N_i) (time)

Mean (T_a), variance (s²_T) and standard error (s_T) of germination time

mean germination time = mean length of incubation time, germination resistance or sprouting index
T_a = Σ_i=1^kN_iT_i/Σ_i=1^kN_i (time)

T_i: the time from the start of experiment
N_i: number of seeds germinated at ith time (not cumulative number)
k: last time of germination

s²_T = Σ_i=1^kN_i(T_i - T_a)²/(Σ_i=1^kN_i - 1) (time)
s_T = √(s²_T/Σ_i=1^kN_i) (time)

Mean germination rate (V_a), variance (s²_V) and standard error (s_{V_a})

V_a = Σ_i=1^kN_i/Σ_i=1^kN_iT_i = 1/T_a (time^-1)
s²_V = V_a⁴·s²_T (time^-2)
s_{V_a} = √(s²_v/Σ_i=1^kN_i) (time^-1)

Coefficient of germination velocity, C_gv

= Coefficient of germination rate (C_gr) or Kotowski's coefficient of velocity
C_gv = (Σ_i=1^kN_i/Σ_i=1^kN_iT_i)·100 = V_a·100 (% day^-1)

Germination rate as the reciprocal of the median time, v₅₀

v₅₀ = 1/t₅₀ (time^-1)

Speed of germination, S

= germination or emergence rate index, or index of germination velocity
S = N₁/T₁ + N₂/T₂ + N₃/T₃ + … + N_n/T_n = Σ_i=1ⁿ(N_i/T_i) (% time^-1)

Speed of accumulated germination, S_a

S_a = (N₁/T₁) + Σ_i=1²(N_i/T₂) + Σ_i=1³(N_i/T₃) + … + Σ_i=1ⁿ(N_i/T_n) (% time^-1)

Corrected germination rate index, S_c

S_c = S/g_p (time^-1)

Weighted germination %, g_w

g_w = Σ_i=1^t{(t - i + 1)N_i/(t·N)}·100

Mean germination % per unit time, g_u

g_u = g_p/T_n

Number of seeds germinated per unit time, g_N

g_N = N_g/T_n

Timson's index, T, or germination energy index

T = Σn = Σ_i=1^tG_i = Σ_i=1^tg_i(t - j), j = i - 1

G_i: the cumulative germination % in time interval i
t: the total number of time intervals
g_i: the germination (not cumulative)

Modified Timson's index, T_m

1. Labouriau, T_m = T/Σ_i=1^tg_i
2. Khan and Unger, T_m = T/t

George's index, G_r

G_r = Σ_i=1^tN_iK_i

K_i: the number of intervals until the end of the test

Peak value (P_V) or emergence energy (E_E)

the accumulated number of seeds germinated at the point on the germination curve at which the rate of germination starts to decrease

Germination value, g_v

1. Czabator, g_v = p_v·g_md

g_md: the mean daily germination % from the onset of germination (computed for other time intervals of successive germination counts, by replacing g_md with g_p)

2. Diavanshir & Pourbiek, g_v = (Σg_ds)/N·g_o·k

g_ds: the temoporal germination speed by dividing cumulative germination % by the number of days since the onset of germination
g_o: the germination % expressed over 100
k: constant

Coefficient of germination uniformity, g_uc

g_uc = (Σ_i=1^kN_i)/(Σ_i=1^k(T_a - T_i)²N_i)

Coefficient of germination time variation, g_tc

g_tc = √(s²_T/T)

Synchronization index, E_a

= uncertainty of the germination process, U, or informational entropy, H
E_a = -Σ_i=1^kf_ilog₂f_i

f_i: relative frequency of germination = N_i/Σ_i=1^kN_i

Germination synchrony (Z index)

Z = (Σ_i=1^kC_{N_i,2}/C_{ΣN_i,2})

C_{N_i,2} = (N_i(N_i - 1))/2
C_{ΣN_i,2}: the partial combination of the two germinated seeds from among the total number of seeds germinated at the last count

(Gummerson 1986)

Hydrothermal time model (HTT model)

Fig. 1. Diurnal temperature courses as applied in the 13 different temperature regimes in Experiment 2 for germination tests of Solanum sisymbriifolium seeds. Separate temperature regimes are indicated by numbers in the rest of the manuscript: solid lines represent regime 1, which was a constant 9°C treatment. Regimes 2, 3, 4, 5, 6 were treatments with fluctuating temperatures around 9°C with maximum amplitudes of 2, 4, 6, 8, and 10°C, respectively. Dotted lines represent regime 7, which was a constant 15°C treatment, and regimes 8, 9, 10, 11, 12, which were treatments that fluctuate around 15°C with maximum amplitudes of 2, 4, 6, 8 and 10°C, respectively. The broken line is regime 13, a constant 30°C treatment. Fluctuations were sine functions (of wavelength 1 d) with daily means of 9°C (treatments 2-6) and 15°C (treatments 8-12), respectively. The functions were truncated at 4°C, thus avoiding potential damage due to very low temperatures. (Timmermans et al. 1997)
____Rumex obtusifolius___Polygonum longisetum___Oenothera biennis

Fig. 1. Seed germination curves for three species buried at three soil depths (3, 10 and 30 cm). Mean germination percentage on six replicates was shown. All standard errors are less than 4 and are not shown in the figure, Closed symbols indicate temperature changes from low to high (L-H) and open symbols from high to low (H-L). Circles, squares and triangles are for seeds buried at depths of 3, 10 and 30 cm, respectively (Tsuyuzaki 2006)

Induction techniques on seed germination

Pre-treatment

Cold stratification: keeping seed in a cool, moist environment for a period of time to simulate overwintering
Gibberelin (GA): promote germination under specific concentration. The effect is similar with cold treatment
[1st step] Control = 0% GA, 27C in dark
```
  0% GA           0% GA        0% GA        0% GA
  x% GA           x% GA        x% GA        x% GA
  27C with light  27C in dark  27C in dark  27C in dark
```
[2nd step]
temperature range: 0, 10, 15, 20, 25, 30, 35, 40, 45, 50C (10 series)
→ temperature fluctuations between 5-40C
+ cold treatment
Kinetin (KA): Promote germination for some species. Cf. cytokinin
H₂SO₄ or sand: Promote seed germination by seed coat damage
Heat
KNO₃
Smoke (Tsuyuzaki & Miyoshi 2009)

Sterilization

To reduce the damages of seeds by fugui (and bacteria), sterlization may be required. We usually use benlate or 1% sodium hypochlorite (NaClO, bleach).

Cold stratification (低温湿層処理)

To increase the seed germination rate, cold stratification is required for many species that are distributed in cool and temperate regions.

Protocol
keep the seeds in 0-6°C for 1 month with moisture

moistened on three-layered filter papers in a petri dish
stored into a frig at 0-6oC for more than 1 month
check the condition at about 1 week interval

(proposed by Suzuki S)

Daily temperature fluctuation

Daily temperature fluctuations during seed germination test
A) Treatment under low temperature = simulation of fall condition

light: light/dark = 8 hr/16 hr with more than 1000 lux
temperature: high/low = 23C(8 hr)/10C(16 hr)
synchronize light and temperature fluctuations

B) Treatment under high temperature = simulation of spring condition

light: light/dark = 8 hr/16 hr
temperature: high/low = 33C(8 hr)/20C(16 hr)

Exp. Germination characteristics of Gaultheria miqueliana seeds

Preparation: 40 petri dishes, 120 filter papers (Whatman #1), dim green light, distilled water, twizzers, wrap
Sample: 50 seeds in each petri dish
Replication: 10 in each treatment
Treatment: Dark/Light germination (2) × Stratification/Non-stratificatoin (2)
1) L × N, 2) L × S, 3) D × N, 4) D × S
Caution: Observe the seed germination under dim green light when you handle the seeds with the treatment D.

H2SO4 soaking or sand scratch

Problems
1) concentration of sulfuric acids
2) treatment time
→ need preliminary survey

1) H2SO4 treatment (only): one sample = 50 seeds, time: 1 min, 2 min, 5 min, 10 min, 20 min, 40 min.
2) Cross-experiment with GA-pretreatment
It was sufficient for the seed coat damage of Trillium to treat H2SO4 for 2-4 min. These seeds were still viable 1 month after the seed germination test was conducted.

GA3 treatment

often using to promote the seed germination of buried seeds

concentration of GA3: 0 (control), 100, 200, 400 ppm
→ place on three-layered filter papers in petri dish → place into an incubator at 26°C in dark
GA3

GA treatment after H2SO4 pretreatment

↓ Soaking seeds into condensed H2SO4 solution within 1 minute
↓ Soak into GA-solution for 48 hrs (concentration: 0, 10, 25, 50, 100, 200, 400 ppm)
↓ Place them on filter papers in a petri dish
↓ Place in an incubator with fluctuated temperatures
↓ Observation (= germination test)
Materials: 50 × 6 × 2 seeds = 600 seeds

Seed germinatin experiment (発芽実験)

Greenhouse (温室)

A house that allows
plant growth even in
fields

Seed germination exeriment conducted in a greenhouse. The samples were collected from the former topsoil on Mount Usu 20 [left] and 30 [right] years after the 1977-78 eruptions. The most common species was Rumex obtusifolius (Left: Goto M. Right: ST).

A few photos on seeds

Juncus effusus var. decipiens
Polygonum weyrichii

Phytotron (ファイトトロン)

A closed system that regulates various environments used for investigating plant growth
LED light PRO180 SODATEC (out of order)

PAR (μmol): 1469.2
LED wavelengths (nm): 450-475, 620-630, 650-660 (white, IR)

[ dromancy ]

Seed dormancy (種子休眠)

A period in a life cycle when seed development is temporarily suspended
When mature seeds are placed under favorable conditions and fail to germinate, the seeds are dormant.

Innate dormancy (Primary dormancy)

seed is dormant during seed maturation (true dormancy)

After-riping: seeds requried a period to be mature after the dispersal

Induced or spontaneous dormancy (seconcary dormancy, 自発休眠)

seed does not germinate spontaneously until environmental conditions improve. The seed needs favorable environmental stimuli to germinate

Enforced or imposed dormancy (quiescence, 強制休眠)

seed germinates readily on removal of the environmental limitation

Conditions

water
light
temperature
gasses (O₂, CO₂, etc.)
smoke
mechanical restriction
seed coat
hormone

(Baskin & Baskin 2003)

Exogenous dormancy

caused by conditions outside the embryo

Physical dormancy: an impermeable seed coat - water-gap
Mechanical dormancy: seed coats or other coverings are too hard to allow the embryo to expand
Chemical dormancy: due to growth regulators

Endogenous dormancy

caused by conditions within the embryo itself

Physiological dormancy: chemical or hormonal changes
Drying
Photodormancy
Thermodormancy
Morphological dormancy: embryo is underdeveloped or undifferentiated
Morpho-physiological or morphophysiological dormancy: having both morphological and physiological dormancy - underdeveloped embryos having physiological components to dormancy

Stage

pre-dormancy (前休眠) or summer dormacny (夏休眠) →

true or winter dormancy (冬休眠) at innate dormancy stage →
post-dormancy (後休眠) at enforced dormancy stage

Water stress on seed germination

Water stress

When water demand exceeds the available amount, the various changes are observed with various sclales from molecule to ecosystem.

Water stress on seed germination

When water is deficient, seed germination percentage often decreases to avoid drought stress on seedlings.

Protocol

to detect the effects of water stress on seed germination

Method 1 (classical method)

Changes in the thickness of seed bed by adjustijng nunmber of filter peaprs, and keeping constant water table by adjusting water supply

Method 2

Preparation
Making an osmotic pressure gradient by the concentration of polyethylene glycol (PEG)

Equation on making osmotic perssure using PEG-6000

ψs = -(1.18 × 10^-2)C - (1.18 × 10^-4)C² + (2.67 × 10^-4)CT

+ (8.39 × 10^-7)C²T

ψs: osmotic pressure (bar)
C: concentration (g/kg)
T: temperature (°C)

(Michel & Kaufmann 1973)

Polyethylene glycol (PEG)

≈ polyethylene oxide (PEO), and polyoxyethylene (POE)
PEG: tended to refer to oligomers and polymers with a molecular mass < 20000 g/mol
PEO: tended to polymers with a molecular mass > 20000 g/mol
POE: tended to a polymer of any molecular mass.

Seed germination (種子発芽)

Germination signal

Seed imbibition

Warm stratification

Heat exposure (熱)

Seed germination (種子発芽)

Germination indices

Germination percentage (germinability), gp

Time for the first germination or germination time lag, t0

Time for the last germination, tg

Time spread of germination, or germination distribution, Ts

Peak period or modal time of germination

Median germination time, t50

Mean (Ta), variance (s2T) and standard error (sT) of germination time

Mean germination rate (Va), variance (s2V) and standard error (sVa)

Coefficient of germination velocity, Cgv

Germination rate as the reciprocal of the median time, v50

Speed of germination, S

Speed of accumulated germination, Sa

Corrected germination rate index, Sc

Weighted germination %, gw

Mean germination % per unit time, gu

Number of seeds germinated per unit time, gN

Timson's index, T, or germination energy index

Modified Timson's index, Tm

George's index, Gr

Peak value (PV) or emergence energy (EE)

Germination value, gv

Coefficient of germination uniformity, guc

Coefficient of germination time variation, gtc

Synchronization index, Ea

Germination synchrony (Z index)

Hydrothermal time model (HTT model)

Induction techniques on seed germination

Pre-treatment

Sterilization

Cold stratification (低温湿層処理)

Daily temperature fluctuation

H2SO4 soaking or sand scratch

GA3 treatment

Seed germinatin experiment (発芽実験)

Greenhouse (温室)

A few photos on seeds

Phytotron (ファイトトロン)

Seed dormancy (種子休眠)

Innate dormancy (Primary dormancy)

Induced or spontaneous dormancy (seconcary dormancy, 自発休眠)

Enforced or imposed dormancy (quiescence, 強制休眠)

Conditions

Exogenous dormancy

Endogenous dormancy

Stage

Water stress on seed germination

Water stress

Water stress on seed germination

Protocol

Method 1 (classical method)

Method 2

Equation on making osmotic perssure using PEG-6000

Polyethylene glycol (PEG)

Germination percentage (germinability), g_p

Time for the first germination or germination time lag, t₀

Time for the last germination, t_g

Time spread of germination, or germination distribution, T_s

Median germination time, t₅₀

Mean (T_a), variance (s²_T) and standard error (s_T) of germination time

Mean germination rate (V_a), variance (s²_V) and standard error (s_{V_a})

Coefficient of germination velocity, C_gv

Germination rate as the reciprocal of the median time, v₅₀

Speed of accumulated germination, S_a

Corrected germination rate index, S_c

Weighted germination %, g_w

Mean germination % per unit time, g_u

Number of seeds germinated per unit time, g_N

Modified Timson's index, T_m

George's index, G_r

Peak value (P_V) or emergence energy (E_E)

Germination value, g_v

Coefficient of germination uniformity, g_uc

Coefficient of germination time variation, g_tc

Synchronization index, E_a