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16.4B: Germination of Seeds - Biology

16.4B: Germination of Seeds - Biology


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Germination is the resumption of growth of the embryo plant inside the seed.

Requirements:

  • proper temperature
  • Water is always needed to allow vigorous metabolism to begin. It is also sometimes needed to leach away a germination inhibitor within the seed. This is especially common among desert annuals. The inhibitor is often abscisic acid (ABA).
  • oxygen
  • a preceding period of dormancy (often).

    The seeds of many temperate-climate angiosperms will germinate only after a prolonged period of cold. An inhibitor within the seed (probably abscisic acid - ABA) is gradually broken down at low temperatures until finally there is not enough to prevent germination when other conditions become favorable. This mechanism is of obvious survival value in preventing seeds from germinating during an unseasonably warm spell in the autumn or winter.

  • Correct photoperiod (often).

Germination in a Dicot

  • The primary root emerges through the seed coats while the seed is still buried in the soil.
  • The hypocotyl ("below the cotyledons") emerges from the seed coats and pushes its way up through the soil. It is bent in a hairpin shape - the hypocotyl arch- as it grows up. The two cotyledons protect the plumule- the epicotyl ("above the cotyledons") and first leaves - from mechanical damage.
  • Once the hypocotyl arch emerges from the soil, it straightens out. This response is triggered by light. Both red light, absorbed by phytochrome and blue light, absorbed by cryptochrome can do the job.
  • The cotyledons spread apart exposing the epicotyl with the apical meristem at its tip, and two primary leaves
  • In many dicots, the cotyledons not only transfer their food stores to the developing plant but also turn green and make more food by photosynthesis until they drop off.

The above image (courtesy of the Pittsburgh Plate Glass Co.) is a time-lapse photograph showing three stages in the germination of a bean seed.

Germination in Monocots

When grass seeds like corn (maize) or oats (shown here) germinate,

  • The primary root pierces the seed (and fruit) coverings and grows down.
  • The primary leaf of the plant grows up. It is protected as it pushes up through the soil by the coleoptile - a hollow, cylindrical structure.
  • Once the seedling has grown above the surface, the coleoptile stops growing and the primary leaf pierces it.

The coleoptile of grass (e.g., oat) seedlings has been a favorite experimental object for studing phototropism.


The Germination of Seeds

The Germination of Seeds, Third Edition discusses topics concerning seed germination. The book is comprised of seven chapters that tackle subjects relating to the field of germination. Chapter 1 discusses the structure of seeds and seedlings, while Chapter 2 covers the chemical composition of seeds. Chapter 3 tackles the factors affecting germination, and Chapter 4 deals with dormancy, germination inhibition, and stimulation. Chapter 5 talks about the metabolism of germinating seeds, and Chapter 6 discusses the effect of germination inhibitors and stimulators on metabolism and their possible regulatory role. Chapter 7 covers the ecology of germination. The book will be of great interest to botanists, who are particularly concerned with plant physiology.

The Germination of Seeds, Third Edition discusses topics concerning seed germination. The book is comprised of seven chapters that tackle subjects relating to the field of germination. Chapter 1 discusses the structure of seeds and seedlings, while Chapter 2 covers the chemical composition of seeds. Chapter 3 tackles the factors affecting germination, and Chapter 4 deals with dormancy, germination inhibition, and stimulation. Chapter 5 talks about the metabolism of germinating seeds, and Chapter 6 discusses the effect of germination inhibitors and stimulators on metabolism and their possible regulatory role. Chapter 7 covers the ecology of germination. The book will be of great interest to botanists, who are particularly concerned with plant physiology.


16.4B: Germination of Seeds - Biology

  • Seed germination: Germination of seeds is a complex physiological process triggered by imbibition of water after possible dormancy mechanisms have been released by appropriate triggers (see webpage "Seed Dormancy").Under favorable conditions rapid expansion growth of the embryo culminates in rupture of the covering layers and emergence of the radicle. Radicle emergence is considered as the completion of germination. The definition that a visible protrusion of radicle tip is the completion of germination is not only a definition issue of seed physiologists. This transition point is also characterized by the loss of dessication tolerance and this is a molecular checkpoint (in Arabidopsis regulated by ABI5), a developmental molecular switch from the germination program to the seedling program.
  • Non-dormant seeds: A completely non-dormant seed has the capacity to germinate over the wides range of normal physical environmental factors possible for the genotype (Finch-Savage and Leubner-Metzger, 2006) Besides the basic requirement for water, oxygen and an appropriate temperature the seed may also be sensitive to light and/or nitrate. Germination commences with the uptake of water by imbibition of the dry seed, followed by embryo expansion. This usually culminates in rupture of the covering layers and emergence of the radicle, generally considered as the completion of germination. Radicle protrusion at the completion of seed germination therefore depends on embryo growth driven by water uptake.
  • Cell elongation is necessary and is generally accepted to be sufficient for the completion of radicle protrusion, cell division is not essential. Thus, germination is a process and the completion of germination is an event visible protrusion of the radicle tip through all covering structures is the typical criterion for the completion of seed germination.
  • Uptake of water by a mature dry seed is triphasic (see webpage "Water Relations"), with a rapid initial uptake (phase I, imbibition) followed by a plateau phase (phase II, metabolic preparation for germination). Phase III is a further increase in water uptake which occurs directly after germination is completed. Phase-III water uptake causes hydraulic growth of the embryo and the emerged seedling. The plant hormone ABA inhibits phase III-water uptake.
  • In many species the covering tissues, e.g. endosperm and testa (seed coat), act as a physical barriers which must be overcome by the growth potential of the embryo if the seed is to complete its germination (Linkies et al., 2010). In coat-dormancy, the seeds are prevented from completing germination because the embryo is constrained by the covering layers. The micropylar endosperm is known since a long time as a constraining structure in members of the Asteraceae (e.g. lettuce) and Solanaceae (e.g. tomato, tobacco and Datura spp.). Endosperm weakening, at least limited cell wall hydrolysis, by the action of specific hydrolases seems to be needed for the completion of germination by endosperm rupture. In addition to the endosperm, the micropylar testa also confers a hindrance for radicle protrusion of of tobacco and tomato seeds.
  • Arabidopsis thaliana and Lepidium sativum are Brassicaceae seeds with distinct testa rupture and endosperm rupture. Müller et al. (2006) demonstrated that endosperm weakening occurs prior to Lepidium sativum endosperm rupture and is controlled by the GA-ABA ratio. Linkies et al. (2009) demonstrated that ethylene promotes endosperm cap weakening of Lepidium sativum and endosperm rupture of the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana. Ethylene counteracts the inhibitory action of abscisic acid (ABA) on these two processes.
  • A comprehensive table of Arabidopsis homone mutants summarizes the altered phenotypes regarding germination and dormancy.
  • Recommended reviews on seed germination:

One-step seed germination of Brassica and pea seeds: testa rupture and initial radicle elongation

  • The endosperm is completele obliterated during the seed development of Brassica spp. (see figure below) or pea and the mature seeds of these species are therefore non-endospermic. Uptake of water by a seed is triphasic with a rapid initial uptake (phase I, i.e. imbibition) followed by a plateau phase (phase II). A further increase in water uptake (phase III) occurs only when germination is completed, as the embryo axes elongates and breaks through the testa. Thus, besides radicle elongation, testa rupture is the only visible landmark during Brassica spp. and pea seed germination.
  • Abscisic acid (ABA) does not inhibit imbibition and testa rupture (see figure below), but ABA inhibits phase III water uptake and the transition from germination to postgermination growth (e.g. Schopfer and Plachy, 1984 Manz et al., 2005).

Brassica napus seed germination is one-step. The mature seeds of these species are without endosperm and so testa rupture plus initial radicle elongation result in the completion of germination. ABA does not inhibit testa rupture, but inhibits subsequent radicle growth (Schopfer & Plachy, 1984). Review: Finch-Savage and Leubner-Metzger (2006).

Two-step seed germination of Lepidium and Arabidopsis (Brassicaceae): testa and endosperm rupture

  • For the Lepidium and Arabidopsis seed anatomy see the webpage "Seed Structure".
  • Rupture of the testa (seed coat) and rupture of the endosperm are separate events in the germination of Lepidium and Arabidopsis seeds (see figures below). Arabidopsis (Liu et al., Plant J 41:936-944, 2005) and Lepidium (Müller et al., 2006) exhibit a two-step germination, in which testa rupture and endosperm rupture are sequential events.
  • Such two-step germination is widespread over the entire phylogenetic tree and has been described for many species, e.g. for Trollius (Ranunculaceae Hepher and Roberts 1985), Chenopodium (Amaranthaceae Karssen 1968 Karssen 1976), Nicotiana and Petunia (Cestroideae subfamily of the Solanaceae, Krock et al. 2002 Leubner-Metzger et al. 1995 Petruzzelli et al. 2003).
  • We found that the plant hormone ABA inhibits endosperm rupture, but not testa rupture, of Arabidopsis and Lepidium (Müller et al., 2006). This inhibitory effect of ABA is counteracted by GA, supporting the view that endosperm rupture is under the control of an ABA-GA antagonism (Kucera et al. 2005).
  • We found that ABA inhibits endosperm weakening of Lepidium, and this inhibitory effect is counteracted by GA (Müller et al., 2006). This supports the view that weakening of the micropylar endosperm occurs in Arabidopsis and Lepidium seeds (Brassicaceae, Rosid clade), is under ABA-GA control, and is functioning in controlling the germination of endospermic Brassicaceae seeds.
  • We show that ethylene promotes endosperm cap weakening of Lepidium sativum and endosperm rupture of the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes (Linkies et al., 2009). Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2 ethylene biosynthesis) and constitutive triple response1 (ctr1 ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae (Linkies et al., 2009).

Two-step germination of Lepidium sativum. (C-F) During the two-step germination of Lepidium testa rupture (C,D) is followed by endosperm rupture, which occurs after 16 h under control conditions (E). Due to the microphotographic settings the transparent outer mucilage layer is not visible. (F) ABA specifically inhibits endosperm rupture, the radicle remains covered by the micropylar endosperm even after 60 h incubation in the presence of ABA. (G) Drawing of a mature Arabidopsis seed the seed anatomy that is very similar to that of Lepidium. (H-J) Arabidopsis seeds also germinate with testa rupture (H) preceding endosperm rupture (I). Also during the two-step germination process of Arabidopsis, ABA specifically inhibits endosperm rupture (J). Seeds were incubated in continuous light without (control) or with 10 µM ABA added to the medium. From Müller et al., (2006).

Two-step germination of Arabidopsis thaliana. (G) Drawing of a mature Arabidopsis seed the seed anatomy that is very similar to that of Lepidium. (H-J) Arabidopsis seeds also germinate with testa rupture (H) preceding endosperm rupture (I). Also during the two-step germination process of Arabidopsis, ABA specifically inhibits endosperm rupture (J). Seeds were incubated in continuous light without (control) or with 10 µM ABA added to the medium. From Müller et al., (2006).

A comprehensive table of Arabidopsis hormone mutants summarizes the altered phenotypes regarding germination and dormancy.

Two-step seed germination of Nicotiana spp. (Solanaceae): testa and endosperm rupture

  • For the tobacco seed anatomy see the webpage "Seed Structure".
  • Rupture of the testa (seed coat) and rupture of the endosperm are separate events in the germination of tobacco seeds and there is strong evidence that both, testa rupture and endosperm rupture are the limiting factors in the germination of these seeds (reviewed by Leubner-Metzger 2003).
  • Electron microscopic studies support the view that the endospermic hole of the germinated tobacco seed, which is always at the micropylar end, is formed by "dissolution" rather than by "pushing" action (Arcila and Mohapatra, Tobacco Science 27: 35-40, 1983). Endosperm weakening is a pre-requisite for the completion of germination of many Solanaceous seeds.
  • In photodormant varieties of tobacco that do not germinate in darkness, both the testa rupture and endosperm remain intact in the photodormant seeds. However, when the testa and endosperm are mechanically removed, there is radicle growth even in the absence of light. This shows that in tobacco coat-imposed dormancy is more important than embryo dormancy.
  • Treatment of tobacco seeds with 10 µM abscisic acid (ABA) greatly delays endosperm rupture, but not testa rupture, and results in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue.

Testa rupture (b) and endosperm rupture (c) are separate events during the germination of tobacco seeds.

ABA delays endosperm rupture, but not testa rupture (e and h).

Figure 2: Stages in the germination of tobacco seed homozygous for the GLB-GUS transgene. Seeds were germinated in continuous light at 24 °C with 10 µM ABA (ABA) and without ABA (control) added to the medium. At the times indicated after the start of imbibition, seeds were stained for GUS activity. The blue staining is indicative of transcriptional activity of the class I ß-1,3-glucanase B promoter. (a) Stage I (control, 3 h): intact seed prior to seed-coat rupture. (b) Stage II (control, 60 h): seed with ruptured seed coat and protruding endosperm. (c) Stage III (control, 72 h): seeds with ruptured endosperm showing GUS staining at the rupture site and emerging radicles, which do not stain for GUS. (d) Stage III (control, 96 h): seed with ruptured endosperm and elongating radicle. GUS staining is localized in a collar of endosperm tissue at the site of radicle penetration. (e) Stage II (ABA, 144 h): ABA treatment markedly delays endosperm rupture and results in a novel structure consisting of the enlarging radicle completely enclosed in a sheath of intact endosperm, which does not stain for GUS. (f) Stage II (control, 60 h): endosperm dissected prior to rupture showing GUS stain localized in the micropylar region. (g) Stage III (control, 96 h): endosperm dissected after rupture showing that the radicle penetrates the region which stains for GUS. (h) Stage II (ABA, 144 h): a seed arrested in stage II by ABA treatment dissected to show that the elongated radicle is enclosed in a sheath of endosperm. Magnification: 40X.


Physiology of Seed Germination

In this article we will discuss about:- 1. Subject-Matter of Seed Germination 2. Factors Influencing in Seed Germination 3. Mobilization of Reserves during Seed Germination 4. How to Test Seed Viability?.

Subject-Matter of Seed Germination:

Seeds develop and mature within the fruits. Once the fruit attains maturity and ripens it is shed and the seeds inside it undergo period of dormancy. In some of the succulent fruits even though the seeds are provided with moisture they do not germinate. This is because of lack of other germination factors.

Dormancy is imposed by several inhibitors present in the seed coat or the seed itself. Sometimes seed coat is thick and highly impervious to water and oxygen. In the latter part of our discussion we shall discuss some of the factors which cause seed dormancy and also how this dormancy is overcome.

Seeds will only germinate if given appropriate environmental conditions including water, air, temperature, free from high salt concentrations, inhibitors and sometimes specific spectral quality of light.

Seeds are highly dehydrated and naturally require water before germination. The first phase of seed germination is water imbibition till critical level of water is attained. Once the imbibition is completed, seeds begin to germinate and seedling emerges out. Radicle or root penetrates the seed coat and is followed by shoot emergence.

This is phase of emergence. Clearly in this phase root and shoot systems develop. Thus germination is preceded by imbibition and followed by emergence. The period of the two vary in different species and may be spread over several days or weeks.

Factors Influencing in Seed Germination:

Several environmental factors influence seed germination and these are described below:

Dry seeds can withstand diverse temperatures but once water is imbibed and germination begins they are sensitive to high temperatures. For every seed minimal, maximal and optimal temperatures exist and can be conveniently worked out. The minimal and maximal temperatures vary for different species and no reasons can be ascribed to such a variability.

Oxygen is essential for seed germination. The initial phase of seed germination may involve anaerobic respiration but immediately it shifts to aerobic state. In a seed where testa is retained the oxygen consumption is much higher than in the seeds where testa has been removed. Several other gases like CO2, CO, N2, H2S and ozone also affect germination by affecting several metabolic processes.

Several different types of compounds are known to affect seed germination and these include phenols, cyanides, alkaloids, herbacides, fungicides, salts of some metals, diverse acids, etc.

Some of the seeds are responsive to light. Light may trigger or inhibit seed germination. Lettuce seeds germinate rapidly when exposed to brief red light period.

The age of seeds is an important factor in germination.

Mobilization of Reserves during Seed Germination:

Seeds may be endospermic or non-endospermic depending upon the state whether endosperm is retained or consumed by the cotyledonary leaves of the embryo. Following seed germination several different types of metabolites e.g. starch, proteins, fats or other polysaccharides have to be hydrolysed and mobilized for the nutrition of the growing embryo and then seedling.

Clearly during early stages of seed germination hydrolytic enzymes are activated or synthesized. Seemingly gibberellins play a very vital role in their enhancement. In cereal grains the endosperm is starchy and is surrounded by a cellular tissue called aleurone layer. Several of the hydrolases are increased or secreted in this tissue.

β-amylase enzyme concerned with starch digestion is already present in the seed. However, α- amylase and protease appear soon after germination. Several investigators have shown that removal of embryo led to non-appearance of amylases and the addition of GA could replace the embryo removal effect.

It has been concluded that β-amylase was activated whereas α-amylase was synthesized de novo. Both the processes were mediated by gibberellin. Using l4 C-amino acids it was shown that they were incorporated in α-amylase indicating its fresh synthesis.

Gibberellins seemingly act at the molecular level and derepress the genes which cause α-amylase synthesis. Further embryo provides the requisite GA needed to initiate the synthesis or activation of amylases.

On the contrary seeds which have fats as the stored material convert fats into sugars and the latter are translocated to the growing embryo (Fig. 24-1). In such seeds fats are converted to acetyl-CoA through β-oxidation pathway. Acetyl CoA enters glyoxysomes and undergo glyoxylate cycle. In this cycle two molecules of acetyl CoA are converted to one of succinate.

Succinate is converted to oxaloacetic acid (OAA) which gives rise to phosphoenolpyruvate (PEP). Through reversal of glycolysis PEP is converted into sugars. ATP and reducing power needed in reverse glycolysis are obtained from the oxidation in the glyoxylate cycle and during succinate conversion to OAA and also from β-oxidation of fats when NADH is formed.

Figure 24-2 shows diagrammatic representation of mobilization of different nutrients in a germinating seed.

Areas of new growth and translocation of sugars, amides, etc. is clearly shown from the seed to the new centres of growth. The point to be noted is that the nitrogen transported compounds are reassembled in the growing embryo using carbon skeleton obtained from transported sugars. Thus amino acids are constituted and these are used during protein synthesis in the growing embryo.

Seed has been shown to have diverse types of storage products like fats, starch or proteins. The operation of different pathways is clearly indicated. It may be observed that ultimate products of translocation are sucrose, amides, amino acids, etc.

In summary we may state that during seed germination, the following types of metabolic processes are noticed:

(iii) Subcellular organization of the embryo or endosperm,

(iv) Alterations in the activity of phytochrome (if operative)

(vi) Enzymes synthesis de novo,

(vii) Hydrolysis of metabolites e.g. fats, starch, proteins etc.

(viii) Formation of organic molecules and their translocation to the new centres of growth,

(ix) Synthesis of nucleic acids and proteins,

(x) Oxygen uptake and respiration,

(xi) Enlargement of cell and cell division,

(xii) Upsurge of phytohormones,

(xiii) Synthesis of membranes and other cellular constituents,

(xiv) Variation in CO2 and O2 levels.

In the following we shall briefly discuss the chemical changes during germination of maize (monocot) seeds:

Maize seed is a grain filled up with starch and some amount of protein as well. Endosperm is surrounded by aleurone layers. It has one cotyledon which is modified into scutellum. Scutellum cells secrete hydrolases which digest endosperm metabolites.

Once immersed in water, maize seeds imbibe water and increase in diameter. Imbibition phase is completed within 12-14 hours of soaking. This is followed by enlargement of radicle and coleorhiza.

Seed coat is ruptured by the coleorhiza within 20-24 hours of imbibition and soon after radicle emerges out of seed or grain. In maize given favourable conditions and environments, germination is accomplished within a day or so. Biochemically changes begin after 24 to 48 hours of radical emergence. There is change in dry weight indicating loss of some metabolites from the endosperm.

Both fats and starch are digested after 72 hours of germination. Nitrogenous substances change after radical growth. It has been shown that after water imbibition by the seeds there is high metabolic activities and then the second upsurge of activity takes place after 72 hours. In the first phase there is high nucleic acids, protein and enzymes synthesis and activity.

How to Test Seed Viability?

The percentage of viable seeds can be determined through several methods and some of these are briefly mentioned below:

(i) Direct germination. A desired sample of seeds is germinated and viability percentage computed.

(ii) Seeds are soaked in distilled water and the electric conductivity of surrounding medium is determined. If there is high proportion of non-viable seeds then the conductance will increase.

(iii) Seeds soaked in potassium permagnate dilute solution also provide some indication on viability. If the number of seeds in a given sample is large then the KMnO4 solution will rapidly decolorize. Non-viable seeds are permeable and release high amounts of electrolytes and reducing substances into the surrounding medium.

(iv) In seeds with prolonged dormancy embryo is removed from the cotyledons or endosperm and put on sterilized nutrient medium. The viability is known within a week or so.

(v) Some seeds are split open and immersed in some redox dye (TTC) or tested for peroxidase using histochemical staining reaction. The loss of peroxidase or some dehydrogenase reactions also indicate dead nature of the seeds.


Table of Contents

List of Contributors
Preface Contents of Other Volumes
1 Importance and Characteristics of Seeds
I. Introduction
II. Importance of Seeds as Foods
III. Other Uses of Seeds
IV. Structure of Seeds
V. Seed Variability
VI. Plant Propagation by Seeds
References
2 Development of Gymnosperm Seeds
I. Introduction
II. Prepollination Phase
III. Pollination Mechanism
IV. Male Gametophyte
V. Postpollination-Prefertilization Phase
VI. Fertilization
VII. Embryogeny
VIII. Maturation of Seed
IX. Development in Relation to Time
X. Conclusions
References
3 Development of Angiosperm Seeds
I. Introduction
II. Ovule
III. Female Gametophyte
IV. Pollination and Fertilization
V. Endosperm
VI. Embryo
VII. Polyembryony
VIII. Seed Coat
IX. Mature Seed
X. Conclusions
References
4 Anatomical Mechanisms of Seed Dispersal
I. Introduction
II. Abscission
III. Dispersion
IV. Zoochory
V. Anemochory
VI. Hydrochory
VII. Autochory
VIII. Conclusion
References
5 Seed Germination and Morphogenesis
I. Introduction
II. Overview of Germination
III. Germination of Zea mays L.
IV. Germination of Pinus
References
6 Seed and Seedling Vigor
I. Introduction
II. Expression of Vigor
III. Evaluation of Vigor
IV. Seed Development and Vigor
V. Mechanical Damage and Vigor Reduction
VI. Other Factors Influencing Vigor
VII. Modification of Vigor
VIII. Summary: Present Status and Future Developments in Seedling Vigor
References
Author Index
Subject Index


Answer Key to Questions Asked on the Student LabSheet

The following is based on a student investigation of planting depth.

List below some factors you think may influence seed germination and seedling growth, and give a brief explanation of the expected influence.

Planting depth If planted too deep, seedlings will not be able to reach the surface and will die. If planted too near the surface they may dry out and not germinate.

Water If the soil is too dry, seeds may not be able to absorb enough water to germinate. If the soil is too wet, the seeds may rot.

Temperature If the soil is too cold, seeds may not germinate.

Weeds If there are too many weeds, seeds may germinate but the seedlings may be weak.

Fertilizer If seedlings do not have fertilizer, they may grow poorly. If there is too much fertilizer, the seedlings may be burned.

Light If there is not enough light, seedlings may grow poorly.

Choose a factor from your list and develop a question about seed germination that you can answer through experimentation.

The question we will investigate is Does planting depth affect how well seeds germinate?

State a hypothesis for your experiment. State your hypothesis in the form of if …, then … because (i.e., if this variable is changed in this way, it will produce this change for this reason). A hypothesis is not a guess it is a predicted outcome based on prior knowledge.

If we plant seeds at different depths, the seeds planted deepest may germinate, but the seedlings will not reach the surface. There will be an optimal planting depth.

Experimental Procedure

We will plant radish seeds at different depths and record data on the number of seedlings that emerge above the soil level for each planting depth. Data will be displayed in a data table and as a histogram. Our research indicates that radish seeds germinate in 2 days under good conditions. On this basis, we plan to allow 10 days from planting to the end of our trial. Since radish is a small seed, we predict that the seeds will germinate best at a shallow planting depth.

Materials

radish seeds
potting soil
7-oz cups for planting seeds
rulers

Variables that we will control

number and type of seed planted
temperature
moisture
light
type of potting soil
size of planting container


Seed Germination

The process of awakening of the embryo at the end of the dormancy period is called seed germination. The embryo lies dormant within the seed for a considerable period and germinates to give rise to seedling, which grows further and develop to produce an adult plant. Seed germination is the process of seeds developing into new plants. First, environmental conditions must trigger the seed to grow. Usually, this is determined by how deep the seed is planted, water availability, and temperature.

Condition for seed germination

There are both external and internal factors which are necessary for the germination of seeds. Those are as follows:

External factors of seed germination

There are four external factor for the seed germination those are:
i) Air (or oxygen) supply: - Constant oxygen supply is necessary for increasing the respiratory rate of germinating seeds.

ii) Water or moisture : - Water is essential for bringing about the changes in the vital activities of a germinating seed, which comprise hydrolysis of the stored organic substances in the cotyledons. So, Water is crucial to seed germination. The seed must go through imbibitions to activate root growth.
iii) Light: - The process of seed germination can even occur in the darkness, so light has no direct effect on the seed germination, but it is quite necessary for the growth and development of the seedling.
iv) Temperature: - The optimum temperature is required to carry out certain fundamental activities in the protoplasm of the germinating seeds. Some seeds germinate when it is cold, such as plants in northern environments. Other seeds only germinate when the weather reaches spring temperatures, which is why we see so much plant growth in the spring like temperate climates. Other seeds only germinate after extreme temperatures.

Internal factors of seed germination

Three main internal factors for the germination of the seeds are as follow:
i) Dormancy period :- Each individual seed has a definite period of dormancy and seed germination takes place only after the dormancy period is over.


ii) Food : - The stored food is converted to simple soluble forms at the onset of seed germination by specific enzymes.
iii) Hormones : - The gibberellin hormone is responsible for the conversion of insoluble stored food into soluble forms. On the other hand, Auxin hormone is responsible for the growth and development of the seedling.

Important Events of Seed Germination

The various events that take place in a process of seed germination, those are as follows:
a) The hydrophilic colloid present in the seed coat absorbs water.
b) The seed swells up due to imbibitions of water by the inner tissue.
c) The seed coat ruptures under the pressure of the swelling seed.
d) The cell wall and protoplasm of the inner cells are hydrated.


e) The hormone gibberellin is activated.
f) De-novo synthesis of the enzyme a amylase takes place, which converts storage starch into soluble sugar.
g) Increase in osmotic potential causes greater absorption of water.
h) The soluble sugar is assimilated by the growing embryo.
i) The emergence of radical takes place and thus the seed germination is take place.


Seed Fecundity, Persistence, and Germination Biology of Prairie Groundcherry ( Physalis hederifolia ) in Australia

Prairie groundcherry [ Physalis hederifolia (A. Gray) var. fendleri (A. Gray) Cronquist] is an invasive perennial weed with the potential to become a significant summer weed across 409 million hectares in Australia. Current management practices do not provide effective control of established populations. A better understanding of the seed biology is needed to effectively manage this weed. A series of field and laboratory studies were conducted to determine plant fecundity, soil seedbank longevity, and the factors that affect seed germination. Physalis hederifolia has the capacity to produce 66 to 86 berries plant −1 , 51 to 74 seeds berry −1 , and approximately 4,500 seeds plant −1 , with the seeds potentially able to persist in the soil seedbank for 20 yr if buried in an intact dry berry pod. The bare-seed component of the soil seedbank can be virtually exhausted within 3 yr if cultivation is minimized to avoid burial of seed. Optimal temperature for germination is diurnal fluctuations of 15 C within the temperature range of 10 and 30 C. Increasing osmotic stress levels reduced the germination under all temperature regimes, with less than 6% germination occurring at −0.96 MPa. Physalis hederifolia seed germination was not significantly affected by substrate pH 4 to 10 or salt levels less than 160 mM, while the germination was significantly reduced at NaCl concentrations above 160 mM. These results suggest that P. hederifolia can adapt to a range of substrate conditions. Stopping seed set, avoiding grazing plants with viable seeds, and minimizing seed burial in the soil are some effective strategies to control this weed.


These 142-year-old seeds sprouted after spending more than a century underground

These resilient plants are part of a centuries-long experiment at Michigan State University.

David Lowry, Assistant Professor of Plant Biology and Marjorie Weber, Assistant Professor of Plant Biology, dig for the Beal Bottle, to continue the seed germination study, first done over 140 years ago.

Under the cover of darkness, a team of scientists set out into a cold Michigan night in April, armed with flashlights and a map from 1879. It marked the secret location of an unlikely treasure: bottles of seeds from two centuries past, buried underground and patiently waiting for their turn to come to life.

All of these mystic items are part of an ongoing biology experiment at Michigan State University (MSU) called the Beal seed experiment, which seeks to find out how long seeds can remain viable in soil—and this recent successful germination of 13 seeds shows that the answer is at least 142 years. Named for botanist William James Beal, who started the experiment, the project has become an ongoing and living legacy of the university’s botanists.

To test his question all those years ago, Beal buried 20 bottles of seeds underground on the MSU campus, almost upside down but at a slight angle to avoid water collecting in them, while still allowing moisture and soil to enter. Each bottle contained 50 seeds from 21 plant species, for a total of 1,050 seeds per bottle. The intention was that a bottle would be dug up every five years, and the seeds within would be planted to see what could grow.

When Beal retired in 1910, he passed the experiment to fellow botany professor H.T. Darlington to continue his work, the first in a series of handoffs. In 1920, the length of time between unburyings was extended to 10 years, and in 1990 it was further extended to its current cadence of 20 years. The current bottle was originally scheduled to be unearthed in 2020, but the pandemic meant that the campus and the growth chambers within it were closed, so it was postponed a year.

Now, Frank Telweski, a professor of plant biology at MSU, is the most senior member of the team presiding over the bottles, having also unearthed a bottle in 2000. Teleski recently made the decision to expand the team beyond just one successor to represent a diversity of expertise on seed viability.

“Because of the diverse team, people have different perspectives and bring their different knowledge to it,” Telewski says. “We can begin to expand some of the things that Beal did, and really build upon the 140 years of science that has accumulated since he designed and started the experiment.”

The current team includes fellow plant biologist David Lowry, restoration ecologist Lars Brudvig, evolutionary ecologist Marjorie Weber, and evolutionary and molecular biologist Margaret Fleming, who previously worked in the US’s National Laboratory for Genetic Resource Preservation at Fort Collins. Weber and Fleming are the first two women involved in the experiment.

The 13 seeds that defied the odds this year and germinated appear to be the same species: a weedy, fairly common plant with a yellow flower called Verbascum blattaria. The verbascum’s continued success proves that the resilient seed is a great choice for future experimentation on seed viability.

“I would have assumed that everything with really small seeds, naively, would not survive very long,” Lowry says. “You usually think that these larger seeds that are well provisioned with resources that would be able to survive for long periods of time in the soil, but that doesn’t seem to be the case. Now we know that there’s probably lots of things with very small seeds that can survive multiple centuries in the soil.”

Frank Telewski spreads seeds from the Beal Bottle in a tray in the growth lab.

If no more plants germinate in about the next week, the team of scientists will try to jump-start growth through an eight-week cold treatment to simulate winter, a technique that yielded one additional seed germination in 2000.

Then, they will try some treatments new to this experiment, like a liquid smoke treatment that they predict could trigger the growth of fireweed seeds (which has never grown in all the years of the experiment). The idea is to simulate the chemicals in smoke that trigger germination for some species in environments with a regular burn frequency. They will also treat the seeds with gibberellic acid, a plant hormone, to stimulate growth.

Finally, Fleming will test the metabolic and nucleic acid activity of any seeds that didn’t germinate, as some seeds still could have some functional piece of their metabolism that has broken, making them unable to germinate.

“They’re kind of like zombie seeds, if you will,” Telewski says. “[Fleming is] going to see if she can’t find any of these that still show some level of life, but not enough to actually permit them to germinate. And that’s something that Beal probably never would have dreamt of being able to do.”

The successfully germinated plants will later be on display at the W. J. Beal Botanical Garden, an outdoor laboratory for students founded by Beal and where Telewski is the director.

Even so, the original question of seed viability remains unanswered, and only the continuation of the project could provide an answer.

There are four more bottles left in the ground, and at this pace the experiment is set to end in 2100. While the scientists have considered widening the gap between unearthings to a longer time like 50 years, if nothing grows it would be harder to know when exactly the seeds stopped being viable.

A longer cadence between unearthings would also bring up challenges with maintaining continuity between generations of scientists. Recent events have demonstrated the tenuous nature of the project.

“Frank handed me the map to the bottles and he’s like, ‘Just in case something happens to me, I want you to have the map,’” Lowry says. “The next month he had a stroke. Fortunately, he’s mostly recovered from it and that’s been amazing, but it was this realization that these things are fragile and you do have to have a number of people know where the bottles are. You also don’t want to stretch it out too long because there might not be that institutional memory that will allow you to keep the experiment going.”

What’s much more likely for the future is that the team will embark on a “Beal 2.0,” a second version of the experiment. But there are many questions to consider while deciding on the design of the new study, like what species of seeds to use, the location of the bottles and the pace for opening them.

What the researchers are pretty much set on, however, is having two bottles that will be dug up for each unearthing. The idea is to plant the seeds from one bottle, like in the original experiment. The second set would be used for destructive sampling, which is the type you need to properly analyze how DNA, RNA, and other metabolic processes are breaking down.

Although Telweski has already started to gather seeds that could be soon placed in new bottles, as of now, planning is still underway.

“What we’ve realized is that it’s the long game with this thing,” Lowry says. “These experiments are so long that it’s worth planning and making sure you’ve thought of everything you can before going forward.”


16.4B: Germination of Seeds - Biology

Tomato seed has become a model system of seed germination research. Tomato seed provides an excellent system for seed germination research, because it has the embryo and the endosperm, which is essential for analyzing physical and chemical interactions between these two tissues. Its size is relatively larger than seeds in other plant species, such as tobacco and Arabidopsis seeds, making it feasible to dissect into different seed parts, and small enough for population analyses such as germination tests and biochemical assays.

Mechanisms of Seed Germination

In germinating seeds, radicle emergence is determined by the balance between embryo growth potential and the mechanical resistance of the endosperm. This basic concept of seed germination has been greatly advanced by studies on tomato seeds.

Different forms of mannanase appear during and following germination. In the endosperm of tomato seed, the cell wall galactomanans are major carbohydrate reserves. Galactomannans are degraded in the endosperm of germinated seeds to support seedling growth. This stage is called the post-germinative stage. The post-germinative stage-specific endo- b -mannanase (LeMAN1) is expressed in the whole endosperm of germinated tomato seeds. In contrast, the germination-specific mannanase (LeMAN2) is expressed exclusively in the endosperm cap region which is adjacent to the radicle tip. The physiological role of the geminative mannanase is to degrade the cell walls and subsequently weaken this tissue for the radicle to penetrate it. Tissue print and RNA hybridization experiments clearly show that these two mannanase genes are expressed in spatially and temporally different manners.

Tomato seed tissue prints. Dark areas indicate the localization of endo- b -mannanase mRNA. Left and right, germinating seed probed with LeMAN2 and germinated seed probed with LeMAN1, respectively.

Tissue-Specific Gene Expression in Seeds

GeneChip Analysis

Tomato seed was dissected into the micropylar region and lateral regions, from which embryo parts were removed. The micropylar and lateral endosperms were termed endosperm cap (EC) and lateral endosperm (LE) (Figure below). The embryo was divided into the radicle-half (R) and cotyledon-half (C). RNA was extracted from the four different tissues and used for GeneChip analysis.

GeneChip analysis identified 150 EC-, 135 LE-, 72 R-, and 29 C-enriched genes with more than 2-fold enrichment compared to other tissues. Thirty-four EC-enriched genes exhibited more than 5-fold enrichment in the EC compared to other tissues, with some of them showing more than 15-fold enrichment. The major groups of the EC-enriched genes were pathogenesis-related (PR), cell wall- and hormone-associated genes, suggesting that there are EC-specific pathogenesis/wounding response, cell wall modification, and hormone metabolism and signaling.

The promoter regions of the EC-enriched genes contained DNA motifs recognized by ETHYLENE RESPONSE FACTORs (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the EC, suggesting an involvement of the ethylene response cascade in this process: the EC-enriched PR genes, NP24, P23 and PR5-like, contained the consensus DNA motifs including the AGC box (AGCCGCC), which is known to be bound by TERF1, suggesting an involvement of ethylene response in EC-specific gene expression.

The known EC-specific enzyme endo-b-mannanase (MAN) is induced by gibberellin (GA), which is thought to be the major hormone inducing the other EC cap-specific genes. Analysis of the mechanisms of MAN induction by GA using isolated, embryo-less seeds suggested that GA might act indirectly on the endosperm cap. Two hypotheses are proposed based on previous reports and the results of our experiments (see below).

Direct induction of the EC genes by GA. GA which is produced in the embryonic axis is secreted to the endosperm cap and induces EC-specific transcription factors (TFs), which directly or indirectly induce the EC- genes. How GA exclusively stimulates EC without affecting the other part of endosperm needs to be explained. It is possible that GA receptors are present exclusively in EC and only EC can respond to GA diffused in a whole seed. Alternatively, non-diffusible secondary messenger(s) can be produced in the embryo by GA and move to EC.

Indirect induction of the EC genes by mechanosensing. GA does not stimulate the EC directly, but does induce EC gene expression through its effects on cell expansion in the embryonic axis. In this hypothesis, the growth potential of the embryo, which is generated through GA biosynthesis, provides pressure to the EC. This triggers mechanosensing by the EC, which mimics wounding or pathogenesis response, a major consequence of which is ethylene response including the activation of TERF1. While TERF1 involvement in EC gene induction was verified, evidence for mechanosensing remains to be shown. Note that the major role of ethylene signal transduction in the EC in this hypothesis and the well-known EC gene induction by GA are not mutually exclusive.


(Martinez-Andujar et al., 2012 The Plant Journal 71, 575-586)



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