(1) Consortium slide

(2) Seed Germination II Factors Affecting Seed Germination

Mark A. Bennett
Seed Biology Program
Dept. of Horticulture and Crop Science
Ohio State University
Columbus, OH 43210-1086
[email protected]

(3)Substantial progress has been made in our understanding of physiological mechanisms

and factors in seeds that confer the ability to germinate under a wide range of conditions.
The objective of this module is to describe various factors affecting seed germination
including a wide range of internal mechanisms, environmental factors and chemical
promoters. Germination and seedling establishment are the end result of a complex and
interactive process involving a number of physiological, morphological, environmental
and cultural factors. The discussion of these factors as individual topics needs to be put
in context of this interacting environment. Germination is completed at various times for
the individual seeds within a population which leads therefore to the distribution of
germination times and a characteristic cumulative germination curve for any given seed
lot. Aspects of internal seed physiology include the topics of seed vitality, the genetic
potential of a seed to germinate, seed maturation, and seed dormancy.
(4) In most seeds, a desiccation period which leads to embryo quiescence, is naturally
observed between development and germination. The sequence of events associated with
seed development, germination, and growth are illustrated using pea (Pisum sativum), as
a model. Various levels of development including histodifferentiation, maturation, or cell
expansion, desiccation and seed drydown are involved prior to the germination and
growth phases. During histodifferentiation, cell division, cell expansion and reserve
deposition occur, and the seed is typically desiccation intolerant. As the seed approaches

the end of the development period where drydown has proceeded adequately, the seed is
typically classified as desiccation tolerant. Upon exposure to germination conditions,
including appropriate temperature ranges, water, and atmospheric conditions, the seed
then again becomes desiccation intolerant as metabolism is renewed and reserve and
storage materials are broken down for seedling growth.
(5)Seeds typically do not germinate during development on the parent plant, but undergo
a process of maturation which includes the desiccation stage before being shed. Alfalfa
seeds typically will not germinate (filled circles Fig. A) until excised from the pod late
in development after maturation drying has occurred, shown here as stages 6, 7 and 8.
However, isolated embryos filled squares in the Fig. A - will germinate from an early
stage of development especially if placed on a nutrient medium. When embryos were
placed on water - open circles in Fig. A -, the response was closer to that of the whole
seed. For seeds which develop in a fully hydrated environment such as those of tomato ,
germination of isolated embryos- open circles in Fig B - can again occur at an earlier
stage of development if they are placed on nutrient medium. Intact tomato seeds black
circles in Fig B - typically take about 60 days after pollination (DAP) to reach full
maturity but will germinate at near 90 % levels by about 40 DAP.
(6) Endogenous hormones are often involved with seed development. Seed ABA content
commonly increases during development and then declines during later maturation or the
drying phase. Again, for seeds that develop within fleshy fruits (e.g. cucurbits, tomato)
ABA content plus low water potential of the fruit tissues will both prevent precocious
germination or vivipary. In this figure, wild-type Arabidopsis seeds are shown in the
open circles, an ABA-deficient mutant is shown in the triangles and the downward
triangles indicate an ABA-insensitive mutant response. Developing seeds of wild-type
Arabidopsis do not germinate on water upon being dissected out of the silique.

Seeds of

the mutant lacking ABA germinate precociously under identical conditions as do those of
the mutant that contains more ABA than the wild-type but which are insensitive to it.
Mutants of other species, including tomato show similar responses.
(7) Viviparous mutants have seeds that germinate precociously, that is, while still on the
mother plant. Mutants of this type have been reported for several species including
various cereals (e.g. maize, wheat, rye and barley) and those that produce seeds in fleshy

fruits such as lemon, orange, tomato and melon,plus Arabidopsis. The effect of fluridone
(an ABA biosynthesis inhibitor) on the germination of two lines of maize during the seed
development is shown here. Developing kernels were sprayed with fluridone at 9 to 14
days after planting for the Tx 5855 line or at 5-15 days after planting stage for the Va 35
line. These plants were then allowed to mature to 4550 days after planting before
germination was tested. As can be seen by the different responses of these two lines, the
effectiveness of fluridone varies with time of application during seed development.
(8) Seeds are not capable of withstanding desiccation or drydown at all stages during
their development, but acquisition of desiccation tolerance is usually considerably earlier
than the maturation drying itself. Germination results are shown of dried or fresh intact
seeds of developing castor bean which were harvested at various days after pollination
(DAP) and placed on water. Fresh seeds not subjected to drying do not germinate until
50 DAP, and even these seeds do not achieve normal growth (e.g. full germination) until
60 DAP. When excised seeds are first desiccated, germination is seen by 25 DAP, and
the growth of seeds dried prematurely from 35 days onward exhibit normal postgerminative seedling growth.

(9) In maturing seeds of several species (e.g. white mustard, soybean, brassica and maize
tested to date), the concentration of certain sugars and oligosaccharides increase in
association with the onset of desiccation tolerance and the early phases of water loss. In
data shown here from developing soybean embryos, there is an increase in some sugars
and oligosaccharides within embryos that were induced to become desiccation tolerant by
slow drying (filled circles), but not in embryos maintained in the intolerant state (open
circles).

(10) Different groups of transcripts and their protein translation products arise at discrete
times during seed development and maturation. One group, which was first identified in
cotton embryos was named LEA or late embryogenesis abundant proteins. LEA proteins
occur as two distinct classes with one class increasing at the mid-point of cotton seed
development, coincident with ABA peaks. The second LEA class increases at the
maturation drying stage, about 45-50 days after planting. LEA transcripts are now

known to occur in embryos of many species including pea, soybean, canola, carrot, castor
bean, Arabidopsis, and several cereals.

(11) Following the various steps of seed maturation, the seed now enters the situation of
dormant or non-dormant status. Seeds are often dispersed from the parent plant with
different levels of dormancy, linked to different conditions due to genotype effects and
the environment during seed development. Frequently, this variation is also observed in
seed color, seed size and coat thickness. Also note in this figure that for dormant seed
various light, temperature, and after-ripening treatments may release the seed from
dormancy and allow seedling development to occur. Secondary dormancy (such as
thermodormancy in lettuce) can also occur which will then have to be alleviated for
proper germination and seedling growth.

(12) Dormancy in many cases is observed only in the intact seed, due to a coat imposed
and/or embryo dormancy. Note the wide range of grains, woody plants and other species
in these two categories. Both types of dormancy can exist simultaneously, or
successively in some species. In apple seeds, embryo dormancy predominates but later
on a contribution is made by the covering tissues (endosperm and testa) and their removal
reduces the amount of dormancy-breaking treatment or chilling that is required.
(13) In many cases the expression of seed dormancy shows a strong temperature
dependence. For example, grains of several grasses and cereals such as wheat, barley and
oats are dormant only at temperatures above a particular value, a condition which is
known as relative dormancy. This critical temperature will frequently shift with seed
age or various treatments including cytokinins, nitrate, and gibberillic acids.
(14) Dormant seeds can slowly lose their dormancy by the process known as
afterripening, perhaps requiring as little time as a few weeks (e.g. barley), or as long as
60 months in the case of some weed species such as curly dock (Rumex crispus). Speed
of afterripening depends on the environmental conditions such as moisture, temperature
and oxygen. Temperature can be used to accelerate the loss of dormancy in agriculturally
important species such as barley, wheat and rice. For rice, note the rapid loss of
dormancy in terms of days needed as temperatures increase from 27-37 C and higher.

(15) Secondary dormancy typically develops in already dispersed mature seeds in

response to unfavorable germination conditions; some examples are given here. Several
categories of secondary dormancy are related to light requirements involving red light,
far-red light, and prolonged white light as well as temperature extremes, excessive or
below normal conditions and water stress. Secondary dormancy is of great importance in
germination ecophysiology, since it develops in many species when exposed to
unfavorable conditions.
(16) Environmental Factors and Seed Germination. The response of seeds to a given
combination of environmental factors depends on their (a) origin, (b) genotype, and (c)
any enhancements or cultural practices employed in crop seeding systems. Of all the
many environmental factors involved, water availability, temperature, and oxygen
availability are of paramount importance. For some species, light variables and smoke
are also important for germination.
(17) The uptake of H2O by seeds is an essential early step in germination. Water taken
up during imbibition is generally about 2 -3x the seed dry weight. The rate of water
uptake declines with time, and declines more quickly in sandy soils due to their low water
holding capacity. Water uptake by chickpea (Cicer arietinum) seeds in distilled H2O or
soils at various moisture contents is shown here. Soil water contents are expressed as
percentages.
(18) Other factors also play a role in the rate and extent of water uptake, regardless of the
difference in H2O potential between the seed and soil. Seed size, seed shape, seed coat
characteristics, and production of mucilage can all influence seed water uptake. Small
seeds, mucilaginous seeds, and smooth-coated seeds are more efficient in H2O uptake,
because their better contact with soil.

(19) The range of germination responses among species to water stress is also wide, from
very sensitive (e.g. soybean) to more resistant (e.g. pearl millet). Seeds of more resistant
species may have an ecological advantage, establishing seedlings in areas where waterstress sensitive species will be unable to germinate.
(20) Seed permeability to H2O is influenced by morphology, structure, composition,
initial water content and temperature. Soybean embryos with higher initial moisture

contents imbibed faster than those with a lower (8%) initial moisture level. All embryos
imbibed more water as temperatures increased. Rates of water uptake are critical to
successful germination; if uptake is too slow, germ/emergence is reduced because seed
may deteriorate. If uptake is too rapid, seeds may exhibit imbibitional damage.
(21) As seeds begin to imbibe, a rapid leakage of solutes into the surrounding medium
occurs. These solutes may stimulate the growth of soil microbes. Fig A shows Initial
solute leakage from pea embryos (Fig. B) lasts for ~ 30 min, and occurs only from the
outermost cell layers.
(22) Some seeds (e.g. soybeans) leak proteinase inhibitors and lectins, compounds which
may serve as protective agents against soil microbes and insects. Also, seeds of many
legumes germinate poorly when hydrated without their testas; damage to the seed coat
during harvest or planting can seriously reduce emergence and eventual yield.
(23) Temperature affects both the capacity for germination, and the rate of germination.
Temperatures ranges for germination can vary widely by species; Gypsophila (solid line),
leek (open circles), and Lychnis germination patterns are shown here.
(24) Species can exhibit widely different temperature minima and maxima. For
experimental or descriptive purposes it may be useful to also define the temperatures at
which 50% germination (GT50) occurs. The GT50 of several species can be affected by
application of growth regulators. Kinetin raises the upper GT50 value for lettuce seeds
(in light) from 31 to 40 C, for example.
(25) When considering temp effects on germination, dormancy issues may arise. The
unchilled Delphinium seeds in this figure are dormant at 20 C and above, while the
chilled seeds have no dormancy and germinate at 30+ C.
(26) Although all seeds of a species (or cultivar, or seedlot) may germinate over a fairly
wide range of temps, the time needed for maximum germination potential to be achieved
varies with temperature. The maximum germination rate usually occurs over a narrow
range, or at about 33 C for the Dolichos (horse gram) species shown here.
(27) In this example, Brussels sprouts seeds were set to germinate over the temperature
range of 4 to 42 C, and germination percentages were recorded after 2 and 14 days. The
highest germination rates were at temperatures ~ 25 C (per 2 days counts), but all seeds
were capable of germination at temperatures from ~ 10 to 35 C.

(28) The consumption of O2 by many seeds follows the basic pattern shown here, where
embryo tissue respiration (fig. A) differs from that by storage tissues (fig. B).
Respiration in phase I increases linearly with tissue hydration. O2 uptake in phase II
increases slowly. Between phases II and III, radicle emergence occurs. A second
respiratory burst is seen with the onset of phase III. Phase IV occurs only in storage
tissues, coincident with reserve depletion and senescence.
(29) Oxygen levels in soil and greenhouse mixes are generally quite adequate for
germination. Fine-textured (clay) soil particles may encase seeds under wet conditions,
and lead to poor germination. Soil crusting (O2 levels below 10%), deep planting, and
flooding may also limit oxygen availability in extreme situations. Flooding-tolerant
genotypes of maize, soybean and other crops have been identified for use in crop
breeding programs as a partial remedy for short-term flooding situations.
(30) In flooded soils, O2 concentrations can decrease to 1% or less (and CO2 can
increase to 5-8% in clay or muddy soils with decomposing organic matter). Flooding
with warm water is also more detrimental to germination since O2 solubility increases
with decreasing temperature.
(31) Many seeds experience conditions of temporary hypoxia (or anoxia) during the lag
phase (phase II) of germination. Ethanol and lactic acid can accumulate within the seed.
Alcohol dehydrogenase and lactate dehydrogenase are responsible for both the synthesis
and removal of ethanol and lactate, respectively. After germination has been completed
and when conditions are more aerobic, Alcohol dehydrogenase shown in this figure and lactate dehydrogenase levels become negligible.
(32) The layers of tissue surrounding the embryo might limit gaseous exchange by the
embryo in two ways: (1) O2 entry is impeded, and (2) CO2 escape may be hindered. The
effects of tissue removal (e.g. testa, hull, pericarp, endosperm), seed coat puncturing, and
oxygen-enriched atmospheres have different effects on the germination of species with
coat-imposed dormancy.
(33) In some species, seed coats have a powerful chemical inhibitory effect, rather than a
physical restriction. The inhibitor ABA is present in many seed coat tissues; phenolic
acids, tannins, coumarin, and other inhibitors are also listed here. In cases where repeated
washing or leaching of the seed relieves dormancy, inhibitors are known to be removed.

(34) Only 7 species are known to germinate under anoxia including 4 species of
Echinochloa (barnyard grass), and rice while several aquatic plant species germinate
better under reduced O2 concentrations than in air (see Table 1). O2 requirements for
germination may depend on other environmental factors (e.g. temperature, osmotic
pressure of the germination medium, light) as well.
(35) Germination of fatty seeds (e.g. lettuce, sunflower, cabbage, soybean, and tomato) is
completely inhibited when O2 concentrations approach 2%. This figure shows that
tomato seed germination at 25 C steadily decreased as atmospheres went from ambient
(21%) O2 in curve 5, to 15% (curve 4).10% (curve 3).5% (curve 2).and 3% in
curve 1. Mainly starchy seeds (e.g. rice, wheat, maize, sorghum, pea) are able to
germinate in atmospheres containing less than 1% oxygen.
(36) Scarification or coat removal promotes the germination of many nondormant seeds
in hypoxia, since isolated embryos require less oxygen than intact seeds. In most cases,
isolated embryos of apple, oat, sunflower and melon germinated well in 1 to 3% oxygen.
However, as for whole seeds, their O2 requirement increases when temperature increases.
In this figure curve 1 represents apple embryos, and curve 2 represents oat embryos.
(37) Light influences germination in two ways; it can promote or inhibit. Seeds of many
species are stimulated to germinate by light, but there is some debate as to whether this is
a direct effect on germination itself, or a dormancy-breaking effect. Light responses are
important to seed bank dynamics and other seed ecology responses where optimal timing
for seedling establishment is linked to this environmental trigger. The photoreceptor
responsible for most types of light-responses in seeds is phytochrome. Phytochrome is a
family of chromoproteins where the protein part (phy A-E, and possibly F in tomato) of
the molecules are encoded by different genes. (Black, 2006)
38) Examples of species whose seeds are sensitive to light are shown in this table. Light
requirements also depend on associated factors such as temperature, depth of dormancy,
seed coat integrity, seed development/maturation environment, water status, and the
presence of chemicals (e.g. nitrate).
39) Levels of photodormancy in seeds vary among genotypes, and seed lots of a given
genotype or cultivar. Photoconversions of the red-absorbing phytochrome, Pr (inactive
form) to the far-red-absorbing Pfr (active form) usually take place when seeds are

hydrated above 8% seed moisture content. Critical periods for light environment effects
on lettuce seed germination and storability can occur after physiological maturity (PM)
has been achieved.
40) Lettuce (cv. Tango) germination data of seeds produced from plants grown under
various daylength conditions are shown here. L= LD conditions throughout seed
development, and S=SD conditions throughout; S6=6 d of seed development in SD, then
LD, S12 = 12 d of seed development in SD, then LD, and so on. Even though
physiological maturity (PM) is seen for this lettuce cv. by 11 days after anthesis, note
how seeds from S6 and S12 treatments show a similar germination response to seeds
from the full L (LD) treatment.
41) Prolonged exposure to relatively high fluence-rate white light (sunlight or lamps)
also inhibits germination of many species This effect is attributed to the blue region of
the visible light spectrum, and is likely mediated thru cryptochrome. Two cryptochromes
(cry1, cry 2) exist in higher plants; cry 1 is light-stable, while cry 2 is light labile, being
rapidly degraded in blue light. White light photoinhibition is magnified for seeds under
water stress (e.g. very negative water potentials). Evidence from tomato seed studies
suggest that ABA biosynthesis is promoted at low H2O potentials, and that blue light
increases tissue sensitivity to ABA. (Black, 2006)
42) In addition to water, temperature, oxygen and light, a fifth environmental factor
influencing germination is smoke. First reported in 1990, the stimulatory effects of
smoke on more than 100 species of heathland (fynbos) and prairie plants from South
Africa, Australia and North America have been noted. Butenolide compounds are found
in smoke from burning plant materials, and these compounds have the needed attributes
for germination effects; they are (1) stable at high temperatures, (2) water soluble, (3)
biologically active across a broad range of concentrations, and (4) capable of promoting
the germination of a wide range of species from fire-prone environments.
43) Chemical Promotion of Seed Germination. Changes in endogenous hormones are
often occurring while the developing seed is accumulating its major storage reserves.
Auxin, gibberellins, cytokinins and ABA can all play important roles in the regulation of
seed growth, development and eventual germination. Ethylene is also involved in the
germination response of seeds from some higher plants.

44) The major auxin in developing seeds is indoleacetic acid (IAA); IAA is formed from
tryptophan and is not derived from the parent plant. IAA is most abundant in developing
plant tissues (e.g. immature seeds, fruits). In pea (Pisum sativum) -shown in this figureand seed of most plants studied to date, auxin is first found in the endosperm solid line and is later detectable in the embryo dotted line - after the endosperm has been
reabsorbed. The final decline in free IAA is the result of metabolic conversions to bound
forms and other products. IAA is commonly conjugated to sugars, sugar alcohols and
amino acids (asp, glu, ala).
45) The gibberellins comprise a large family of compounds that can regulate seed
germination, reserve mobilization in cereal grains, and stem growth. The first GA
identified was isolated from a rice plant infected with the fungus Gibberella fujikuroi,
which causes foolish seedling disease. The first plant gibberellin (GA1) was isolated in
1957; since then over 125 naturally occurring GA-like compounds have been isolated
from higher and lower plants (mosses, algae). The biosynthetic pathway of GAs is
complex, beginning with conversion from geranylgeranyldisphosphate (GGPP) to GA12aldehyde.
46) Studies with Arabidopsis and tomato gibberellin-deficient mutants provide the basis
for the hormone balance theory, the concept that dormancy and germination are
regulated by the internal actions of hormones, predominantly gibberellin (promotive) and
abscisic acid (inhibitory). Some types of seed dormancy are also based on changes in GA
+ ABA content or sensitivity.
47) Abscisic acid (ABA) is multi-functional (e.g. promotes dormancy and maturation,
inhibits germination, promotes synthesis of certain storage protein) and is found in the
seeds of many species, as well as in leaves, roots, tubers, ripening fruit and dormant buds.
Like other growth regulators in immature seeds, ABA generally rises in concentration
during seed development, reaches 1 or 2 peaks as seen in this picture for developing
wheat grains- then (usually) declines rapidly at seed dry down.
48) Cytokinins also have diverse effects on plants; most notably in promoting cell
division. Cytokinins also occur in developing embryo (e.g. zeatin from maize kernels).
Cytokinins are synthesized primarily in root tips and transported (in xylem) to aerial plant
parts. Cytokinins can also be synthesized in shoot tips. The amount of cytokinins

increases markedly during seed development, - seen in this figure for developing lupine
seeds- then declines with seed maturation.
49) The plant hormone ethylene (C2H4) is made in most plant tissues, and is also induced
by a range of stresses and wounding. Ethylene dynamics can interact with other
hormones (e.g. Cytokinins, ABA), or it can self-regulate its own synthesis. Ethylene is
biosynthesized from ACC (1-aminocyclopropane-1-carboxylic acid), which is in turn
synthesized from SAM (s-adenosyl-L-methionine). Exogenous ethylene-generating
compounds (such as ethephon) can be used in seed enhancement treatments for improved
germination.
50) Exogenous ethylene stimulates the germination of various dormant and non-dormant
seeds (see table). This compilation includes cultivated plants (e.g. lettuce, peanut,
sunflower), trees (apple, beech), weeds, and parasitic plants (e.g. witch weed,
Orobanche). This gas can break coat-imposed dormancy (cocklebur, curled dock),
embryo dormancy (apple, sunflower), and can overcome thermodormancy (lettuce).
Ethylene can also inhibit germination of some species, or have no significant effect.
Ethylene enhances the action of light, and can interact with other factors such as
temperature, oxygen, osmotic agents and ABA.
51) Hydrogen peroxide (H2O2) was noted as a germination stimulant in addition to its
early role as a fungicidal seed treatment. H2O2 may accelerate early respiratory phases of
germination, but exact mechanisms are still unknown. Seed analysts do employ an
H2O2 test which results in more rapid radicle protrusion from the seed (SCST Training
Manual).
52) Potassium nitrate (KNO3) is also used in seed/germination testing. KNO3 is linked
to relieving light dormancy (by increasing sensitivity to light) in many species. Various
other chemicals (e.g. metabolic inhibitors, anaesthetics) also promote germination by
acting on dormancy or germination physiology (or both).
53) Germination stimulators are numerous, and of great interest because their use may
contribute to a fuller understanding of the biochemical and physiological mechanisms
involved in germination and/or dormancy. Ethanol, for example, may modify membrane
characteristics (ala anaesthetics) or may be involved metabolically as a respiratory
substrate since it increases the O2 uptake in oats (and wild oats).

54) A great variety of compounds are also known to inhibit germination (see table).
These include gases, aldehydes, organic acids, aromatic acids, coumarin, alkaloids,
tannins, proline, and many others. While a diverse set of inhibitors exist, their biological
action is less clear. More studies as to physiological roles, and mechanisms of action by
inhibitors will also contribute to our understanding of seed germination and dormancy.