Seed Germination Testing

Concept
of
Seed Germination TESTING
Prof. Kumari Rajani

Department of Seed Science & Technology
Bihar Agricultural University
Sabour, Bhagalpur-813210
Structure of Monocot Seed
Seed coat : Provides protection

Endosperm/Cotyledon : Store food
Aleurone cells : Store abundant proteins and enzymes
Prof. Kumari Rajani, DSST, BAU, Sabour
 The triploid endosperm is composed of two tissues:
 Starchy endosperm
 Aleurone layer
 The nonliving starchy endosperm consists of thin walled cells filled

with starch grains and it is centrally located
 Living cells of the aleurone layer, which surrounds the endosperm,

synthesize and release hydrolytic enzymes into the endosperm during
germination
 As a consequence, the stored food reserves of the endosperm are

broken down, and the solubilized sugars, amino acids, and other products
are transported to the growing embryo via the scutellum
 The isolated aleurone layer, consisting of a homogeneous population of

cells responsive to gibberellin, has been widely used to study the
gibberellin signal transduction pathway in the absence of non responding
cell types Prof. Kumari Rajani, DSST, BAU, Sabour
Structure of Dicot Seed
Radicle  Root
Plumule  Shoot and leaves

Seed Germination
“Germination begins with water uptake by the seed (imbibition) and
ends with the start of elongation by the embryonic axis, usually the
radicle”
Physiologist
“the emergence of radicle from a seed”
Seed Testing
“development of a normal seedling”
Field or Greenhouse condition
“a seedling emerges from the soil”

Cotyledon
(provide food to the
growing embryo)
Leaves
Hypocotyl
(below the cotyledon)
Basis for the
Epicotyl plant’s stem
(above the cotyledon)
When the radicle has grown out of the covering seed layers,
the process of seed germination is completed
Germination does not include seedling growth after
radicle emergence, which is referred to as Seedling
establishment or Field establishment
Similarly, the rapid mobilization of stored food reserves

that fuels the initial growth of the seedling is considered a
Post germination process

Types of Seed Germination
Epigeal Germination Hypogeal Germination
 Cotyledons are raised out of the soil  Cotyledons remains underground
 Epigeal germination takes place by the  Hypogeal germination takes place by

rapid extension of hypocotyle before the rapid extension of epicotyle and
the growth of the epicotyle the growth of hypocotyle is restricted
 Evolutionary more primitive than  Ex: Paddy, Wheat, Maize, Gram, Pea,
hypogeal Mango, Groundnut etc
 Ex: Bean, Castor, Mustard, Tamarind,
Sunflower, Onion, Papaya, Pine etc
Hypogeal germination of pea seed

Epigeal germination of bean seed Prof. Kumari Rajani, DSST, BAU, Sabour
Radicle emergence: in most of species
Hypocotyle emergence: Bromeliaceae, Chenopodiaceae,
Onagracea, Palmae, Saxifragaceae and Typhaceae
Coleoptile emergence: Maize, Oropetium tomaeum
The mesocotyl
is the first
internode of
the stem
Elongation of the mesocotyle elevates the coloeoptile and its

enclosed inner leaves towards the soil surface
The mesocotyl is the tubular, white, stemlike tissue

connecting the seed and the base of the coleoptile
Phases of Germination
The entire process of germination (water uptake by a
germinating seed) may be divided into three broad phases;
it shows triphasic pattern
• Phase I: Imbibition phase
• Phase II: Active metabolism or Plateau or Lag phase
• Phase III: Cell expansion & radicle protrusion & further

increase in water uptake
The most critical phase is phase II whereas, the
physiological and biochemical processes such as
hydrolysis, macromolecules biosynthesis, respiration,
subcellular structures, and cell elongation are reactivated
resulting in initiation of germination
Phase I : Imbibition phase
 The initial rapid uptake of water by the dry seed during Phase I
is referred to as Imbibition
 It is the first key event that moves the seed from a dry, dormant
organism to the resumption of embryo growth
 The extent to which water imbibition occurs is dependent on three

factors:
composition of the seed
seed coat permeability
water potential
 Species produce seeds with impermeable testa called hard seeds

(hardseededness)
Ex: Leguminosae, Cannaceae, Chenopodiaceae, Convolvulaceae and
Malvaceae Prof. Kumari Rajani, DSST, BAU, Sabour
 Imbibition Phase is relatively shorten and characterized by rapid water
uptake
Chief changes during imbibition phase:

• Absorption of water
• Absorption of other substances
• Release of gases
• Increase in volume of seeds due to swelling
• Leakage of solutes
 The initial period of imbibition induces an immediate and rapid leakage of

solutes such as sugars, organic acids, amino acids, proteins, phenolics,
phosphate and ions, from the seed tissues but it rapidly decreases and
becomes negligible within about 30 min to 1 hr
 The leakage results in loss of enzymes like glucose-6-phosphate

dehydrogenase, glutamate dehydrogenase, cytochrome oxidase and
fumarase
Wetting Burst
“The release of non-respiratory gases as a result of very rapid seed

imbibition”
• It is immediate and last only a few minutes
• It occurs by the release of adsorbed atmospheric gases (Oxygen,

Nitrogen, Carbon dioxide) retained in the dry porous structures
of the seed coats

Imbibitional Injury
Imbibitional chilling injury is defined as sensitivity to a
combination of low seed-water content and imbibition at
cold temperature
The severity of injury depends upon several factors such as

(i) The species or the cultivars involved
(ii) The initial water content of the seed
(iii) The temperature to which seed is exposed
(iv) The duration of chilling exposure
(v) The period during the course of germination when the chilling
exposure takes place
Ex: Cotton, Soybean, Limabean, Maize

Phase II : Plateau or Lag phase
 Water uptake by imbibition declines and metabolic processes,
including transcription and translation, are reinitiated
 The seed volume may increase as a result embryo expands and the
radicle emerges from the seed coat
 The emergence of the radicle through the seed coat in Phase II

marks the end of the process of germination
 Radicle emergence can be either a one-step process in which

the radicle emerges immediately after the seed coat (testa) is
ruptured, or it may involve two steps in which the endosperm must
first undergo weakening before the radicle can emerge

 Dry seeds contain several enzymes, which are desiccation
tolerant and can become active only after sufficient hydration of
seeds
 Major metabolic pathways affected respiration, protein synthesis,

DNA replication, RNA synthesis

Mobilization of Stored Reserves
 The major food reserves of angiosperm seeds are typically stored in the
cotyledons or in the endosperm
 The massive mobilization of reserves that occurs after germination provides

nutrients to the growing seedling until it becomes autotrophic
 At the subcellular level, starch is stored in amyloplasts in the endosperm of

cereals
 Two enzymes responsible for initiating starch degradation are α- and β-

amylase
 α-Amylase hydrolyzes starch chains internally to produce oligosaccharides

consisting of α (1,4)-linked glucose residues
 β-Amylase degrades these oligosaccharides from the ends to produce

maltose, a disaccharide. Maltase then converts maltose to glucose

 Protein storage vacuoles are the primary source of amino acids
for new protein synthesis in the seedling
 In addition, protein storage vacuoles contain phytin, the K+,

Mg2+, and Ca2+ salt of phytic acid a (myo-inositol hexaphosphate),
a major storage form of phosphate in seeds
 During food mobilization, the enzyme phytase hydrolyzes phytin

to release phosphate and the other ions for use by the growing
seedling

Phase III : Cell expansion & Radicle protrusion
 During Phase III the rate of water uptake increases rapidly due to
the onset of cell wall loosening and cell expansion
 Protrusion of radical during germination is caused by cell

expansion or elongation before cell division
Ex: Maize, Barley, Broad beans, Pea etc
 Pinus lambertiana: cell division and cell elongation occur

simultaneuosly
 Prunus cerasus: cell division precedes cell elongation



Major Events occurring during Germination
STAGE EVENTS
PREGERMINATION (a) Rehydration – imbibition of water.
(b) RNA & protein synthesis stimulated.
(c) Increased metabolism – increased respiration.
(d) Hydrolysis (digestion) of food reserves by
enzymes.
(e) Changes in cell ultrastructure.
(f) Induction of cell division & cell growth.
GERMINATION (a) Rupture of seed coat.
(b) Emergence of seedling, usually radicle first.
POST GERMINATION (a) Controlled growth of root and shoot axis.
(b) Controlled transport of materials from food
stores to growing axis.
(c) Senescence (aging) of food storage tissues.
© 2008 Paul Billiet ODWS

Process: Seed Germination
1. Imbibition
- water uptake, softens
inner tissues
- causes swelling and
seed coat rupture
- more water uptake
2. Gibberelic Acid
- plant hormone
(similar to steroids)
- dissolved & distributed
by water

2. Gibberelic Acid
- arrives at aleurone cells
- activates certain genes
3. Transcription
Transportation
Translation  amylase
4. Amylase accelerates
hydrolysis of starch

Hydrated starch
moves to the
cotyledon and
radicle to initiate
growth

Factors affecting Germination
Internal Factors External Factors
 Seed Vitality  Water

 Seed Age or Maturity  Air
 Seed Dormancy (Oxygen & Carbon dioxide)
 Mechanical Damage  Temperature
(The effect of mechanical  Light
injury is greater when it
affect the embryo)
Water
 Water is clearly the most important factor in germination
 An adequate continuous supply of water is necessary for assumption of the

physiology, metabolism and molecular processes that drive germination
 Water functions as triggering enzyme for starch conversion into sugar, turgor
pressure for moving the radicle root down and the cotyledons up, and for
transporting nutrients and enzymes within the seed
 Recalcitrant seeds usually do not require external water for germination since
their natural water content is sufficient for them to complete germination
 Germination on parent tree before shedding: Spp. of Mangrove swamps such as

Rhizophoraceae, Rhizophora spp, Bruguiera gymmorrhiza, Cerops tagal, Avicennia
marina
 In fleshy fruits within which they are enclosed: Mango
 Excess of water is harmful and seeds don not germinate when immersed in
water
 Sugarbeet seeds: A thin layer of water around the seed inhibits

germination
 Barley: Germination is affected by excess water, which is called as water

sensitivity (Excess of water intervenes indirectly by depriving the embryo of
oxygen)
 Typha latifolia (Aquatic plant) and Paddy: Germinate well when covered
with water (under reduced oxygen levels)
 Cynodon dactylon: Germinate in low oxygen levels
 Oldenlandia corymbosa (tropical weed): Germinate only when

completely immersed

Oxygen
 Oxygen in presence of enough moisture causes respiration to start
metabolism and it creates energy for the germination process
 Respiration rates for germinating seeds are very high; adequate

oxygen is necessary to complete respiration
 Oxygen concentration higher than air: promotes germination
 Carbon dioxide concentration higher than air: retards germination

Lettuce and Timothy grass (Phleum pratense)
 If oxygen supply is limited during germination, emergence may not

occur due to inhibited growth

 The germination percent of most seeds will be retarded if the
oxygen percent goes below 20 percent (Normal air is 20 percent
oxygen)
 Typha latifolia (Aquatic plant) and Paddy: Germinate well

under reduced oxygen levels
 Cynodon dactylon: Germinate in low oxygen levels
 Oxygen removes metabolic waste from the cell; without oxygen,

waste is not removed and the cellular metabolism is slowed

Light
• Light is another key germination factor; it can either stimulate or
inhibit seed germination
• Both light quality (light intensity) and quantity (duration of

exposure) influence seed germination
• Promotion of germination is generally through breaking the seed

dormancy
• Some crops have a requirement for light to assist seed germination

(e.g.Tobacco, Lettuce, Petunia, Begonias, Impatiens)

Photoblastic: Seeds respond to light for germination
Three categories of photoblastic seeds:

(a) Positive photoblastic: Seeds that are stimulated to germinate
by light
Ex: Lettuce, Tobacco, Poa pratensis, Poa nemoralis,
mistletoe, Petroselinum crispum (Parsley) etc.
(a) Negative photoblastic: Seeds whose germination is inhibited

by light
Ex: Onion, Lily, Amaranthus, Nigella, etc.
(a) Non-photoblastic: Seed which germinates in light as well as

dark

Visible light radiation is required by seed for germination
Maximum promotion of
germination occurs at 660 to
670 nm with a peak at 670 nm
(red area) since phytochrome
has an absorption maximum
at this wavelength
Wavelengths >700 nm and

<290 nm: inhibit germination

Photoreversible Germination
 Phytochrome is a plant pigment found in cytoplasm that senses
the presence of red light
 Phytochrome absorbs light in two inter-convertible forms
1. Phytochrome-red (Pr) is metabolically inactive & absorbs red

light (660 nm)
2. Phytochrome-far red (Pfr) is metabolically active and gets
transformed from Pr
 The Pfr promotes germination and other phytochrome-

controlled processes in plants
 Pfr reverts back to Pr after absorbing far-red (730 nm)

Inactive form Active form
This reversible effect of red to far-

red light was first reported in 1952
in lettuce and also shown by other
plant spp. like tobacco, pepper grass,
elm, birch etc
The light intensity should be approx 750 to 1250 lux in seed

germinator for light requiring seeds and 250 lux is sufficient for
non-dormant seeds
This is determined by how the seed would naturally be sown
 Small seeds must sprout on the surface of soil because they lack a
suitable endosperm to supply the needed nutrients; these are
typically aided by light exposure
 Large seeds contain enough nutrition to grow underground when

photosynthesis is not possible. These seeds are more likely to
germinate in dark conditions

Temperature
 A favorable temperature is necessary to allow for plant growth
 Temperature not only affects the germination percentage but also the
rate of germination
 For every species of seed, there is an optimal temperature for

germination; at that temperature, the maximum number of seeds will
germinate and in less time than at any other temperature
 The optimum temperature for most seeds is between 15°C and

30°C
 Kharif crops: 25°C and Rabi Crops: 20°C
 The maximum temperature for most species is between 35°C and

40°C
Trigger and Germination Agents
 At some point, the seed becomes sensitive to the presence of

“trigger” agents
 A “trigger” agent can be defined as a factor that elicits

germination but whose continued presence is not required
throughout germination
 A “trigger” agent such as light or temperature alterations shift

the balance of inhibitors to favor promoters such as gibberellins
 In contrast, a “germination” agent is a factor that must be

present throughout the germination process; an example is
Gibberellic Acid

Seed Germination Testing
Seed Germination
Physiologist
“the emergence of radicle and
plumule”
Seed Testing
“Seed Germination is the
emergence and development of
the seedling to a stage where the
aspect of its essential structures
indicates whether or not it is able
to develop further into a
satisfactory plant under favorable
conditions in the field”
(ISTA, 2015)

Materials Required for Germination Testing
Germination paper/sand
Wax or butter paper
Petri plate
Seed germination chamber
Plastic boxes or tray or pots
Rubber band
Seed counting board
Marking pencil/pen

Growing Media
Composition: the growing medium can be paper, pure sand or
mixtures of organic compounds with added mineral particles
Characteristics of germination paper

 It should be porous in nature
 It should have maximum water holding capacity to ensure
continuous supply of water during the test period
 Free from bacteria, dirt, fungi and toxic substances
 Made out of 100% cellulose
 pH should be 6-7.5
 Paper should posses sufficient strength to the prevent penetration
of root in to the paper
 Paper size is 46 X 29 cm
 It should have reasonable cost
 Should not serve as suitable media for saprophytic Fungi
a) Paper substrata
 The paper substrata are used in the form of top of paper (TP) or
between paper (BP) tests
 In most of the laboratories, paper-towel method (Roll towel test) is

most commonly used for medium sized and bold seeds
 The paper substrata are not reusable
b) Sand substrata
 The sand substrata have advantage of being relatively less
expensive and reusable
 The results in sand media are more accurate and reproducible in

comparison with 'roll towel‘ tests especially in case of seed lots
that are aged or heavily treated with chemicals
Methods of seed germination using paper
A. Top of paper (TP): the
seeds are germinated on top
of one or more layers of
paper which are placed
B. Between paper (BP): the

seeds are germinated between
two layers of paper
C. Pleated paper (PP): the

seeds are placed in a pleated
paper strip with 50 pleats,
usually two to a pleat
Methods using sand or organic growing media
i)Top of sand (TS), Top of organic growing medium (TO):

the seeds are pressed into the surface of the sand or the organic
growing medium.
ii) Sand (S), Organic growing medium (O): the seeds are
planted on a level layer of moist sand or the organic growing
medium and covered with 10–20 mm of uncompressed substrate,
depending on the size of the seed

Methods using a combination of paper and sand
i) Top of paper covered with sand (TPS): the seeds are

germinated on top of a moistened sheet of cellulose paper
which is covered with a 2 cm layer of dry sand
ii) Soil: Soil is generally not recommended as a primary growing

medium
However, it may be used as an alternative to organic growing

media when seedlings show phytotoxic symptoms or if
evaluation of seedlings is in doubt on paper or sand

The accuracy and reproducibility of the germinator result
are very much dependent on the quality of the substrata
(paper and sand) used for germination testing
The germination substrata must meet the following basic

requirements:
 It should be non-toxic to the germinating seedlings
 It should be free from mould sand other microorganisms
 It should provide adequate aeration and moisture to the
germinating seeds
 It should be easy to handle and use
 It should make good contrast for judging the seedlings
 It should be less expensive

Important facts to be remembered…
pH: the growing medium must have a pH value within the range 6.0–7.5 when
checked in the substrate
Conductivity: the salinity must be as low as possible and no more than 40

millisiemens per metre
Measurements of conductivity can be replaced by biological tests
Cleanness and freedom from toxicity: the growing medium must be free
from seeds, fungi, bacteria or toxic substances, which may interfere with the
germination of seeds or the growth or evaluation of seedlings
Seed sample: 400 seeds are used for germination testing
Seedlings evaluation: is done on two days: (Different for various crops)

1. First Count 2. Second or Final Count
Re-use of substrates: it is strongly recommended that the
growing medium is only used once
Counting boards: Counting boards are often used for large seeds
such as Zea, Phaseolus and Pisum
Vacuum counters: Vacuum counters can in principle be used for

all species, but are mostly used for species with regularly shaped
and relatively smooth seeds such as cereals or species of Brassica
or Trifolium

Procedures of Routine method
(Between Paper)
Place 100 seeds on soaked paper Place another soaked paper Roll in wax paper
at equal distance in 8 rows
(12 seeds: 1, 3, 5, 7
13 seeds: 2, 4, 6, 8)

Evaluation of germination test
 Normal Seedlings: Seedlings that possess essential structures that is
indicative of their ability to produce useful mature plants under favorable field
conditions
 Abnormal Seedlings: Seedlings that exhibit some form of growth but have
insufficient plant structures to maintain a healthy plant, such as missing roots
or shoots
 Fresh Seeds: Seeds that have failed to germinate but have imbibed water.
They appear firm, fresh and capable of germination, but remain dormant
 Dormant Seeds: Viable seeds (other than hard seeds) that fail to germinate
when given the prescribed or recommended germination conditions
 Hard Seeds: Seeds that remains hard at the end of the prescribed test
period, because their seed coats are impermeable to water
 Dead Seeds: Seeds that cannot produce any part of a seedling

Evaluation of seedlings
Normal Seedlings
AS
Seedlings with all

essentials structures, well
developed, proportionate
root and shoot, healthy

Abnormal Seedlings
NS
Damaged Seedlings: missing essential structures

Deformed Seedlings: unbalanced development
Decayed Seedlings: diseased
Dead Seeds
Hard Seed

Calculations and Reporting of Results
Replication No. of No. of Ungerminated Seed
Germination
(100 Normal Abnormal No. of Hard No. of Fresh No. of Dead (%)
seeds) Seedlings Seedlings Seeds Seeds Seeds
R1 89 4 3 0 4 92 (89+3+0)
R2 92 2 1 2 3 95 (92+1+2)
R3 90 3 2 2 3 94 (90+2+2)
R4 87 3 5 0 5 92 (87+5+0)
Average % 89.5 3 2.75 1 3.75 93.25
The results of the germination test are reported as percentage of normal seedlings,
abnormal seedling, hard seeds, fresh seeds and dead seeds
The sum of the normal, abnormal and ungerminated seeds must be 100 (90+3+3+1+3)
Germination percentage is calculated based on number of normal seedling
The percentage are rounded to the nearest whole number
Normal seedlings + Hard seeds + Fresh seeds
Germination (%) = X 100
Normal seedlings + Abnormal Seedlings + Hard + Fresh + Dead Seeds
Minimum Seed Certification Standard for Seed
Germination Percentage Recommended in Field Crops
Field Crops Foundation and Certified

Maize Hybrid (Sweet Corn Hybrid, Synthetic, 90
Composite, OPV)
Barley, Wheat, Triticale, Bengal gram, Rapeseed, 85
Mustard
Paddy, Maize (Inbred lines, Single cross FS), 80
Horse gram
Sorghum, Pearl millet, Minor millets, Black 75
gram, Cowpea, Green gram, Indian bean,
Lathyrus, Lentil, Moth Bean, Pea, Rajmash
Castor, Groundnut, 70

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Concept

Prof. Kumari Rajani

Seed coat : Provides protection

 The nonliving starchy endosperm consists of thin walled cells filled

 Living cells of the aleurone layer, which surrounds the endosperm,

 As a consequence, the stored food reserves of the endosperm are

 The isolated aleurone layer, consisting of a homogeneous population of

Prof. Kumari Rajani, DSST, BAU, Sabour

Prof. Kumari Rajani, DSST, BAU, Sabour

Similarly, the rapid mobilization of stored food reserves

Prof. Kumari Rajani, DSST, BAU, Sabour

 Epigeal germination takes place by the  Hypogeal germination takes place by

Hypogeal germination of pea seed

Elongation of the mesocotyle elevates the coloeoptile and its

The mesocotyl is the tubular, white, stemlike tissue

• Phase I: Imbibition phase

• Phase II: Active metabolism or Plateau or Lag phase

• Phase III: Cell expansion & radicle protrusion & further

 The extent to which water imbibition occurs is dependent on three

 Species produce seeds with impermeable testa called hard seeds

Chief changes during imbibition phase:

 The initial period of imbibition induces an immediate and rapid leakage of

 The leakage results in loss of enzymes like glucose-6-phosphate

“The release of non-respiratory gases as a result of very rapid seed

• It is immediate and last only a few minutes

• It occurs by the release of adsorbed atmospheric gases (Oxygen,

Prof. Kumari Rajani, DSST, BAU, Sabour

The severity of injury depends upon several factors such as

Ex: Cotton, Soybean, Limabean, Maize

Prof. Kumari Rajani, DSST, BAU, Sabour

 The emergence of the radicle through the seed coat in Phase II

 Radicle emergence can be either a one-step process in which

Prof. Kumari Rajani, DSST, BAU, Sabour

 Major metabolic pathways affected respiration, protein synthesis,

Prof. Kumari Rajani, DSST, BAU, Sabour

 The massive mobilization of reserves that occurs after germination provides

 At the subcellular level, starch is stored in amyloplasts in the endosperm of

 Two enzymes responsible for initiating starch degradation are α- and β-

 α-Amylase hydrolyzes starch chains internally to produce oligosaccharides

 β-Amylase degrades these oligosaccharides from the ends to produce

Prof. Kumari Rajani, DSST, BAU, Sabour

 In addition, protein storage vacuoles contain phytin, the K+,

 During food mobilization, the enzyme phytase hydrolyzes phytin

Prof. Kumari Rajani, DSST, BAU, Sabour

 Protrusion of radical during germination is caused by cell

 Pinus lambertiana: cell division and cell elongation occur

 Prunus cerasus: cell division precedes cell elongation

Prof. Kumari Rajani, DSST, BAU, Sabour

Prof. Kumari Rajani, DSST, BAU, Sabour

Prof. Kumari Rajani, DSST, BAU, Sabour

© 2008 Paul Billiet ODWS

Prof. Kumari Rajani, DSST, BAU, Sabour

Prof. Kumari Rajani, DSST, BAU, Sabour

Prof. Kumari Rajani, DSST, BAU, Sabour

Internal Factors External Factors

 Seed Vitality  Water

 An adequate continuous supply of water is necessary for assumption of the

 Germination on parent tree before shedding: Spp. of Mangrove swamps such as

 Sugarbeet seeds: A thin layer of water around the seed inhibits

 Barley: Germination is affected by excess water, which is called as water

 Cynodon dactylon: Germinate in low oxygen levels

 Oldenlandia corymbosa (tropical weed): Germinate only when

Prof. Kumari Rajani, DSST, BAU, Sabour

 Respiration rates for germinating seeds are very high; adequate

 Oxygen concentration higher than air: promotes germination

 Carbon dioxide concentration higher than air: retards germination

 If oxygen supply is limited during germination, emergence may not