Title: Reversal of the effects of deterioration in aged soybean seeds (Glycine max (L.) Merr. Cv. Vicoja)
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Title: Reversal of the effects of deterioration in aged soybean seeds (Glycine max (L.) Merr. Cv. Vicoja)
Alternate Title: Glycine max
Physical Description: xi, 128 leaves : ill. ; 28 cm.
Language: English
Creator: Tilden, Robert Luther
Copyright Date: 1984
 Subjects
Subject: Soybean -- Seeds   ( lcsh )
Seeds -- Viability   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Robert Luther Tilden.
Thesis: Thesis (Ph. D.)--University of Florida, 1984.
Bibliography: Bibliography: leaves 111-126.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099349
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000465274
oclc - 11567164
notis - ACM9378

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REVERSAL OF THE EFFECTS OF DETERIORATION
IN AGED SOYBEAN SEEDS
[GLYCINE MAX
(L.) MERR. CV. VICCJA]






BY


ROBERT LUTHER TILDEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DCCTCR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1984


























lo Martha















ACKNOWLEDGMENTS


During our first meeting, Dr. S.H. West suggested that

with a background in chemistry, I might be interested in

working on a problem related to the plant cell plasma

membrane. Aside from staging the problem, Dr. West

intervened at several critical times during the development

of this study. One of these occasions was to question if it

was possible for the membrane to undergo repair after aging.

This question led to the most significant findings of the

study.

Dr. R.H. Biggs' graduate course stimulated an existing

interest I seared in plant growth regulators. Serving my

committee, he correctly stressed the importance of insight

in research rather than methods. It was with this

orientation that reversal of the age-related effects in

seeds was demonstrated experimentally.

Dr. D.J. Cantliffe's graduate course in seed

physiology/biochemistry was a valuable introduction to

contemporary research on this topic. This knowledge was a

contribution to this dissertation.

During the development of the research proposal, Dr. M.H.

Gaskins insisted that the objectives be as clear and

realistic as those of a grant proposal. The plan which


iii









developed from this approach made organization and

communication of this work much more manageable.

Dr. D.J. Huber was recognized for his knowledge of

membrane biochemistry and of senescence in plants. The

emphasis of this dissertation on homeostasis in seeds was an

outgrowth of interest which can be traced to Dr. Huber's

graduate course in post-harvest physiology.

Non-committee members of the university's research

faculty include Dr. B.C. Aldrich who contributed to the

electron-microscopy. Acknowledged also is Dr. R.H. Berg who

suggested fluid phase partitioning as a possible method to

purify the plasma membrane.

Fellow graduate students helped greatly by sharing their

knowledge and friendship.





























iv


_______________ .J









III. RESULTS . . .. . . . 36

Microflora and Seed Deterioration ...... 36
Histology of Aged Seeds . . . .... . . 36
Tissue Excision Experiment . . .... 38
Automatic Seed Analyzer versus Tetrazolium 38
Cytology of Plasma Membrane Injury ..... . 39
Physiology .. . . .. . . . . . .42
Effects of Rapid Hydration . . ... 42
Effect cf Priming on Electrolyte Leakage . 49
Effect of Aging on Primed Seeds . . .. 51
Priming-Temperature Dependence . . .. 52
Effect of Soaking Temperature on Seed Leakage 53
Biochemistry of Growth Regulators . . .. .55
Age versus ABA ....... . .. . . 55
Age versus Fusicoccin and Ethylene ..... 60

IV. SUMMARY AND CONCLUSICNS . . . ... 65

Microflora of Seeds . . .. . . . 65
Histology of Seed Deterioration . . . .. 65
Symmetrical Necrosis . . . . . .. 65
Tetrazolium Staining Pattern . . .. 65
Cytology of Plasma Membrane Injury .. . . .66
Plasma Membrane Rupture . . . . ... .66
Physiology ........ .. . . . . 66
Controlled Hydration Rate ........ 66
Partial Priming . . . . . . .. 66
Biochemistry of Growth Regulators . ..... 67
Abscisic Acid Studies . . . . .. 67
Fusicoccin Studies . . . . . .. 68
Hypotheses Tested . . . . . . . 68
Homeostasis . . . . . . . . 68
Simon's Hypothesis . . ... . . . 69

V. DISCUSSION . . . . ... . . . . 70

Microflora and Seed Deterioration . . .71
Histology of Aged Seeds . . . ... . . 72
Cytology of Plasma Membrane Injury . . ... 73
Physiology of the Plasma Membrane . . . 74
Test of Hypotheses . . . . .. . 77
Biochemistry of Growth Regulators. . . . .79
ABA versus Accelerated Aging . . ... 79
Interaction of Fusicoccin and Ethephon . 80
Suggestions for Future Research . . . .. .80


APPENDIX

SUPPLEMENTARY EXPERIMENTS . . . . . .. . 81

Microflora and Seed Studies . . . - 81
Fungal Survival .... .. . . . 81

vi


_______ ______ __









Control of Fungal Growth .... . . 82,
Histology of Seed Deterioration . . . .. .83
Location of Seed Fungi . . . . . 83
Location of Hardness in Seedcoats . . . 83
Tetrazolium Staining Studies . . . .. 84
Cytology of the Plasma Membrane . . . .. 85
Physiology of Seed Deterioration . . .88
Priming Experiments . . . . . .. 88
Deterioration of Performance is Permanent .. 94
Effects of Monovalent and Divalent Cations 95
Vacuum Drying to Prevent Seed Deterioration 96
Stimulation of Growth By Slightly Aging Seeds 96
Rates or Uptake and Leakage Compared . . 98
Biochemistry of Growth Regulators . . .. .103
Respiration Experiments . . . . 104
Abscisic Acid Dormancy Induction and
Reversal ...... ...... 105
Antagonistic Experiments . . . . 106
Fusicoccin Stimulation of Seeds . . .. .107
Ethylene Biosynthesis and Stimulation 109

LITERATURE CITED. ........ .. . . . 111

BIOGRAPHICAL SKETCH .. . .... .. . . .127















LIST OF TABLES


TABLE PAGE

1. Literature on Membrane Activity of Growth Regulators 22

2. Literature on Regulatory Activity of Plant Hormones 24

3. Effect of Aging on Rate of Water Uptake . . .44

4. Vigor as a Function of Age and Rate of Hydration .. 47

5. Etiect of Accelerated Aging on Primed Seeds . .. 51

6. Priming-Dependence on Temperature . ... . .. 53

7. Interaction of Age and Abscisic Acid . . ... .58

8. Effect of ABA cn the Vigor of Aged Seeds . . . 60

9. Interaction or Fusicoccin and Ethephon . . .. .61

10. Growth Response to Fusicoccin and Ethephon ... .62

11. Generalized Linear Regression Model . . . . 63

12. Effect of Priming on the Rate of Imbibition . 92


viii
















LIST OF FIGURES


;E


Differential Aging of Seed Tissue . . . . .

Loss of Plasma Membrane Integrity. . . . . .

Microscopic Identification. . . . . . .

Controlled Rate of Water Uptake . . . . .

Electrolytes Lost During Water Uptake. . . . .

Effect of Hydration Rate on Aged Seeds. . . .

Effect of Priming on Electrolyte Leakage. . . .

Effect of Low Temperature on Leakage. . . . .

Dose Response to Abscisic Acid. .. .. . . ..

Age versus Fusicoccin and Ethylene. . . . .

Enriched Plasma Membrane. . . . . . . .

Seed Moisture Effect on Priming. . . . . .

Rates of Hydration versus Leakage. . . . .

Anaerobiosis and Membrane Permeability. . .


FIGURE

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.


AGE

37

40

41

43

46

48

50

54

57

64

87

90

100

102


Pi















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



REVERSAL OF THE EFFECTS OF DETERIORATION
IN AGED SOYBEAN SEEDS
[GLYCINE MAX
(L.) MERR. CV. VICOJA]


By


Robert Luther Tilden


April 1984


Chairman: S.H. West, Ph.D.
Major Department: Agronomy



Deterioration ot seed performance accumulates during

storage. The seeds of most agronomic crops are

significantly affected by this occurrence. Reversal or

rejuvenation of aged seeds is therefore of primary interest.

The objective of this study was to test a contemporary

hypothesis regarding rejuvenation of aged seeds.

Experiments relying heavily on accelerated aging and priming

were designed to test this hypothesis.

Information concerning both seed deterioration and its

reversal resulted from the study. Seed tissue was shown by









tetrazolium staining to age most rapidly at symmetrical

locations on the cotyledons. Aged tissue was in turn

predisposed to imbibition injury which accounted for most of

the loss of performance in aged seeds. When this injury was

avoided by slow hydration, reversal of the sensitivity to

imbibition injury was demonstrated. This reversal was a

temperature dependent process. Slow hydration followed by

dehydration improved the survival of seeds during

accelerated aging demonstrating that loss of seed vigor was

also reversable.

These results were consistent with the hypothesis that

age related deterioration was reversed by pregermination

metabolism. Alternative explanations may account for these

results but the hypothesis tested could not be rejected

based on the observations.

The significance of these findings applies to agronomy

and to seed physiology. New information was learned

regarding seed performance improvement through priming.

This information may have application for increasing the

performance of seeds after long term storage. The

processes under study may relate to natural survival

mechanisms in dry seeds.















CHAPTER I
INTRODUCTION


One of the major problems vith soybean seed production in

Florida is deterioration during storage. The storage

capabilities vary with the cultivar. Vicoja is among the

most stable cultivars whereas Hardee seed loses vigor

rapidly as determined by the accelerated aging seed vigor

test.

This weakness in southern soybean seeds may have arisen

inadvertently during the breeding process since storage

characteristics have not been emphasized as a selection

criteron. The place of origin in Southeast Asia indicates

tropical compatibility in the genetic pool. Through the

adaptation of soybeans to midwestern and northern U.S.A.,

seed lost resistance in tropical and subtropical

environments to deterioration. The problem intensifies as

cultivation of soybeans progresses into the tropics where

there is a demand for high protein production on low

nitrogen soils. Environmental effects of high humidity and

temperature in the tropics preclude seed storage without

expensive refrigeration.









Statement of Objectives

Seed detetioration was analyzed as several contributing

causes including microflora, nonuniform tissue aging and

imbibition injury.

Experimental evidence for hypothetical repair processes

was also an objective. Additionally, investigations were

directed toward providing new evidence to determine if

repair is metabolic or spontaneous as contemporary research

suggests.

In addition to providing a more thorough understanding of

seed deterioration and possible repair processes, knowledge

gained should be useful in efforts to improve seed

performance including agronomic crops.



Literature Review

Original research publications are cited for specific

information. In addition, there are books which provide a

generalized resource for this study. Jensen has edited one

of the most comprehendible texts available on cell biology

(68). Lewin was reviewed for current knowledge of

cytogenetics and cell structure (89).

On the subject of plant physiology, Leopold and

Kriedemanni (88), as well as Salisbury and Ross (148), are

acknowledged. A current text on plant molecular biology by

Smith and Grierson is now available (161). Plant

biochemistry studies are well documented by Wareing (194),









Moore (97) and Nickell (106) for the status of growth

regulator research. A review of plant biochemistry is

provided by Tolbert (178).

Volumes specific for seed interest include the classic

Yearbook on Seeds 1961, published by the USDA (163).

Thompson's six volumes on seed technology were perused, but

were, as the author states, an introductory edition (177).

Kozlowski has three volumes with more emphasis on seed

science but much of the information was dated (78, 79, 80).

To learn the status of Soviet seed science, Ovcharov was

reviewed (112). Perhaps the most elegant atlas on seeds was

the USDA publication Seeds of Woody Plants (153).

Copeland's excellent textbook (40) was used as a lead to

several research publications of interest. For foundation

knowledge in seed longevity research, Justice and Bass (69)

must be mentioned. Khan's two volumes (74, 75) were most

enlightening. For a pragmatic treatment of seeds, the book

by Duffas and Slaughter (47) was of value.

On the subject of the plasma membrane, Bittar's three

volumes (27) were read fcr a survey of membrane research.

Tanford's book (169) The Hydrophobic Effect, completed the

membrane literature review.

These references are not intended to represent a complete

survey of the literature. An attempt has been made to

include books and journal articles readily available to

persons with an academic interest in seed science.









Hypothesis: Homeostasis as a Survival mechanism

Seeds have at least two primary functions, propagation

and survival. Survival can be further divided into two

functions. The one more important to agronomic crops is

survival by desiccation, a unique feature of some seeds

(24). This type of seed is referred to as quiescent or

orthodox (137). The second function is dormancy, which is

found among both guiesent and recalcitrant seeds

(110,137,183).

Dormancy provides the natural protection against
seed deterioration while seed are still on the
plant and after they are dispersed. Dormancy is
generally perceived as a mechanism for delaying
germination over time. There would be little
survival value in dormancy, however, if it did not
also reduce the rate of deterioration in seed so
that germination also is distributed over time.
(43, page 16)

The stress applied to seeds during storage is aging which

in turn may be thought of as the spontaneous increase in

disorder (entropy) described by the second law of

thermodynamics (86). Background radiation, including heat,

tends to randomize molecular organization (29, 113). Basic

mechanisms for survival then must minimize entropy by

continuous repair as is thought to be the case in dormancy

(110,188) or to reduce the entropy after imbibition as in

the case of quiescent seed. A mechanism utilized by germ

plasm storage centers is to reduce the entropy by low

temperature storage of very dry seeds (14, 15, 16, 69, 141,

179, 181). Prevention of aging by desiccation and low









temperatures is of practical importance. The metabolic

repair or homeostasisi concept is also of interest since, in

nature, it may be the prevalent mechanism of dealing with

the stress of aging. Detection of this repair activity has

been reported by some investigators but denied by others.


In support of homeostasis. Early physiological support

for dormancy as a survival strategy appeared in a 1953

report by Toole and Toole (180) who showed that the growth

of lettuce was much more vigorous if the seeds were stored

fully imbibed rather than dry. Villiers substantiated this

finding in 1974 (189) with fully imbibed lettuce seeds,

Villiers used thermal dormancy to prevent germination.

Other experiments showed that intermediate imbibition would

restore vigor (17) again supported by Villiers and Edgecumbe

(190). This may be a common phenomenon according to Roberts

and Ellis (141). The temporary wetting extended the vigor

of the seeds after aging. Delouche and Nouyen (44) also

suggested that dormancy may reduce the rate at which seeds

lose vigor during storage. They based this on observations

of moist, dormant rice sealed in glass jars for months

without loss of vitality until its energy reserves were

depleted.


1 Homeostasis a biological tendency to balance anabolism
and catabolism (metabolic turnover and refurbishment)







6

Biochemical evidence for repair was provided by Gichner

et al. (53) who chemically induced single-strand breaks

into DNA. Radioactive thymidine was incorporated into the

DNA in a manner which supported the repair hypothesis.

Osborne and Sen (111, 155) have used similar technology to

find a transient repair of accumulated DNA lesions after

hydration of quiesent, aged seeds.

Microscopic support for the repair hypothesis is based on

electron microscopy of membranes and light microscopy of

metaphase chromosomes. In a series of publications (19, 20,

21, 22) repair of aged corn seeds was viewed as digestion of

membranes by cytoplasmic organelles. Severe aging caused

internal autolysis of the cell which began with collapse of

the plasma membrane which causes it tc shrink away from the

cell wall (188). Microscopy was used by Clowes (38) who

reported replacement of cells in the meristem of corn roots

by replenisher cells following an X ray exposure.

Microscopy has also provided insight into repair of the

genetic damage associated with aging.

Genetic evidence for injury and repair associated with

aging was established in 1931 by Nilsson (107) and confirmed

by Navashin (104) in 1933. Perhaps the first report of

accelerated aging was by Cartledge et al. in 1936 (31).

They used the aging treatment to produce chromosome

aberrations. Others (189,190) have reported that if the

seeds were stored fully imbibed, the chromosomes were









stable. Current wcrk of this type includes that of Murata

(100) and Roos (144), who like Cartledge (31), have shown

that accelerated aging and natural aging produce chromosome

aberrations. Most genetic damage is eliminated during plant

development. Those mutations compatible with development

lead to both chimeras and other phenotypic expressions.



Contradictory views concerninQ the hypothesis.

Roberts' (137) concluded, in a review of the control of

viability during storage, tnat there is no connection

between dormancy and longevity of seeds. In a reiteration

of this position (139), he cites an argument based on the

observation that mcst recalcitrant seeds are short lived.

Perhaps the most emphatic objection to the concept of

homeostasis is that pronounced by Bewley and Black in their

1982 text (26).

We might note, though, that at the present time no
biochemical evidence has been presented for any
turnover, synthetic, or (membrane) repair
mechanisms in either imbibed-stored seeds or those
subsequently germinated, (page 43)

Summarizing the review of literature concerning the

question of repair mechanisms in seeds, the most pertinent

works relative to this dissertation are the reports by Toole

and Toole in 1953 (180), which were substantiated by Villers

in South Africa (189) and by Basu and Dhar (17) in India

during the period 1974 to 1979. Roberts review (140)

further supports the general finding that intermediate









wetting of some orthodox seeds can result in increased

longevity. Still earlier reports suggest that soaking seeds

in a limited amount of water improves their performance even

after they are redried (76). In 1934, Chippindale (35)

found that some grain seeds tolerated drought better if they

were soaked in water before planting. McKee (96), in 1935,

found that slightly sprouted, redried legumes and grasses

grow more quickly. These studies support the results of

this dissertation but since there are contradictory recent

reports in the literature (41,156,157). Reasons for this

discrepancy may include differences in cultivars and/or

methodology.



Microflora and Seed Deterioration

Since bacteria require free water for growth, they are

only evident during seed putrefaction. Even seed-borne

pathogens have little effect on germination according to

Christen (37). For a more comprehensive treatise on seed

pathology, Neergaard should be consulted (105).

Fungi which affect seed deterioration are either genera

specific for field conditions or storage fungi. Field fungi

may affect deterioration before or during harvest. Any

reduction in vigor before storage also increases the rate of

deterioration in storage.

Storage fungi do not affect seeds if the seed moisture

remains below critical levels (37). Relative humidity below









68 percent is sufficient to protect seeds against storage

microflora.

Ultrastructural examination of Asperqillus qlaucus, a

common storage fungus, showed that the attack caused

coalescence of the spherosomes (9). It was suggested that

this event was cytotoxic.

Tao et al. 1974 (170) used chloramphenicol, puromycin

and actinomycin-D tc protect seed during accelerated aging

from fungal and bacterial infection. Similiarly, Royse et

al. (146) used penicillin in concentrations ranging from

200 to 400 parts per million in dichloromethane, to

effectively supress the activity of Bacillus subtilis in

soybean seeds during accelerated aging. This treatment

increased the survival time of the seeds.

Regardless of Tao's previous report on the use of

antibiotics during accelerated aging, its use was not

mentioned in his 1981 study (200) with Woodstock. Also the

use of fungicide treatment before accelerated aging is

usually omitted.



Histoloy _of Aged Seeds

Injury to seed tissue has been studied in relation to

aging as well as the associated injury incurred during rapid

hydration. Among the first symptoms of seed deterioration

is inactivation of the mitochondrial dehydrogenase enzymes.

Tetrazolium staining (55) forms the non-diffusible, formazan







10

pigment in viable cells. Non-viable cells remain colorless.

Evans Blue is another vital stain used to identify patches

of cells on the surface of seed cotyledons which were

ruptured oy soaking the seeds in water with the testa

removed (48,50). This stain is passive in that healthy

cells exclude the pigment. Cells with broken membranes are

flooded with dye and are visible with light microscopy.

Seedlings often display necrotic patches, visible without

magnification. These areas of dead tissue are consistent

with Villiers' (188) description of groups of dying cells

observed with the electron microscope.

Seeds from samples of low vigor, and with only
partially impaired germination, often show these
degenerative changes in isloated cells (or groups
of cells) among apparently normal tissue, and it
is assumed that, when extensive, these relate to
the necrotic spots or areas seen in some low-vigor
germinating seedlings. (188, page 45)

Notwithstanding tais evidence for non-uniform susceptibility

of seed tissue to the effects of aging, Sen (154) came to

the opposite conclusion. By using radioactive precusors and

autoradiography to detect repair after aging, her results

indicated that all seed tissue was affected by aging in a

uniform manner. Perhaps autoradiography failed to detect

repair, whereas vital stains do not depend on this activity.

It is not necessary to reconcile the differences between

Villiers' histologic conclusions and those of Osborne. The

tetrazolium reaction can be used to demonstrate that aging

is uniform or non-uniform when seed tissue has symmetrical







11

areas which degenerate first under natural or accelerated

aging.



Cytology_2f Plasma Membrane Injury

The cell's response to natural and accelerated aging has

been studied on three levels. On the first level,

chromosome aberrations are amenable to study under the light

microscope in rapidly dividing metaphase cells of

meristematic regions (101,104,107,138,139,189). This

abnormality often results in sectorial chimeras (143,144)

but most chromosome lesions are eliminated during plant

development, especially during the pollen haploidd) stage.

Few abnormalities are transmitted to the second generation

(144).

Ultraviolet light microscopy has given visual,

cytological information in dormancy imposed by phytochcome

activation (131). Also the effects of plant hormones on

calcium distribution have been monitored with these methods

(152).

At the electron microscope level, aging during dormancy

has provided another window for observing the repair process

(185, 186,187,188). Organelles and membranes are evidently

phagocytized during dormancy of Fraxinus excelsior, for

example. This cytological view also supports Villiers' and

Osborne's hypothesis of seed repair mechanisms and

correlates with studies which have described the beneficial









effects moisture can have on preventing age related

deterioration (17,180,189),

The most germane cytological study concerning the effect

of aging on the plasma membrane contains electron

micrographs (188) which depict plasmolysis after accelerated

aging followed by collapse of the membrane and ultimate

disoluticn of cell internal structure by autolysis.

Collapse of the plasma membrane after aging could explain

much of the electrolyte loss in imbibition studies.

Few or no cells show signs of possessing a
functional plasma membrane, and the boundary layers
of the cytoplasm have retracted from the walls.
Keeping such seeds imbibed and periodically
sampling for electron microscopy shows rapid
degeneration of the cytoplasm in which the lipid
bodies become confluent, and eventually total
dissolution of the internal structure ensues.
(18B, page 45)

Just as the destruction of the plasma membrane follows an

unexpected scenario, the construction of new membranes is

equally unexpected. Chabot and Leopold (32), using freeze

fracture transmission electron microscopy, indicated that

new membrane material was seen "blebbing" from the cytoplasm

to the plasma membrane. Buttrose (30) also used freeze-etch

to monitor structural changes in seeds during hydration.

Freeze-etch techniques complement earlier fixation which

attempted to preserve the membranes in their dry state.

Microscopy has proven to be a powerful tool for studying

the cytochemistry of aging in situ with vital stains. It

has also offered direct observation of membrane destruction.







13

However, studies concerned with function of the membranes

must use physiological techniques to monitor the

permeability of the membranes as well as a biochemical

approach to determine the activity of the membrane as it

coordinates with the growth processes.



Plasma Membrane Physiology

As aging progresses, the first change observed in the

seed is an increase in membrane permeability. The following

events are in order of increased complexity, corresponding

to a reduction in respiration which is also a membrand-

dependent process (197,198). As the plant's ability to

utilize energy reserves is reduced, there is a cascade of

dependent events. Biosynthesis, such as protein and nucleic

acid turnover, is correspondly slowed, which in turn reduces

growth and resistance of the plant to cope with stress of

the process in emergence.

The plasma membrane has been identified as a likely weak

point in the seed's resistance to aging (1,2,3). The

association between the plasma membrane was confirmed by

Parrish and Leopold (119) who demonstrated that aging

decreased respiration and increased the electrolyte leakage

of the membrane. The cause of the leakage was reported to

be oxidation of the phospholipids (133), but this report was

denied (132) by similar work conducted with soybeans.

Peroxidation of the phospholipids has also been implicated







14

(164) in membrane deterioration. Analysis of tocopherol and

the free radical content of seeds led Priestly et al. to

conclude that aging does not affect the plasma membrane of

soybeans in this manner (132,133).

Proteins of the plasma membrane have been overlooked as

part of the problem. According to Keenan et al. (72), 40

percent of the plant plasma membrane is protein on a dry

weight basis. Only recently have purification techniques

been improved (195) sufficiently to characterize the

proteins by two-dimensional electrophoresis (160). Even

more subtle alteration of proteins is suggested by tests

such as the tetrazolium reaction which responds to

dehydrogenase enzyme activity. The activity of proteins

could be altered by aging via partial denaturation without

altering their primary structure. The activity of proteins

of the membranes may be an early event in the catastrophe

cascade brought about by age. More severe aging leads to

the ultimate catastrophe, death of the seed. Along with a

reduction of growth, aging reduces the plant's turgor

(120,204,205) which is dependent on membrane activity.


Physiological injury. A major part of injury to an aged

seed occurs during imbibition (94,95). Predisposition of

aged seeds to soaking injury was reported by Woodstock and

Tao in 1981 (200). At the same time Parrish and Leopold

(119) reported that aging of seeds decreased respiration and

increased electrolytes resulting from soaking. They







15

concluded that aging compromised the ability of the membrane

to reform during hydration. Woodstock cited earlier reports

in which seeds had been protected from imbibition injury by

slowing the rate of water uptake. Pollock et al.

(125,126,127,128) found that moist seeds, conditioned with

water vapor, were less susceptible to chilling injury.

Obendorf and Hobbs (108) also found that preimbibed seeds

were protected from chilling injury. Another pertinent

report was by Powell and Matthews 1978 (130), who used

polyethylene glycol (PEG) to slowly hydrate pea embryos

which resulted in less injury from this process. Woodstock

and Tao (200) recognized that dry soybean embryos were also

injured less if they were hydrated on germination paper

moistened with 30 percent PEG rather than with water alone.

The benefits to low vigor and accelerated aged (42) embryos

were especially dramatic. Aged embryos recovered most nut

not all of their vigor following the PEG treatments. These

authors thought that by slowing the water uptake, the plasma

membrane had time to "repair" itself. This inference was

not supported by data and must have related to Simon's

hypothesis (158,159).

Cell injury can also result from membrane rupture during

rapid imbibition (28,48,49,85,196), although rupture alone

is probably an incomplete explanation (49). The structure

of a large seed such as the soybean may incur structural

damage as the external tissue swells more rapidly than the









center. It is unlikely that seeds in nature undergo the

extremely rapid hydration found in this type of membrane

study. The extreme situations may result in experimental

artifacts. Nevertheless, insight can be derived from

imbibition experiments concerning the permeability of the

membrane as influenced by aging.


Spontaneous physiologicalrepair. The "repair"

hypothesis was based on a an extensive literature review

concerning membrane permeability (159). The explanation

contended that the phospholipids of dry seeds assumed a

quasi-laminar gel structure which required several hours to

reorganize as the seed was hydrated. Such phase transition

was investigated by Parrish and Leopold (118), but further

studies by the same group were unable to support this

concept (109).


Physloloqical_)rimin_ and_desiccation. These two seed

treatment practices are reviewed together because they are

often used in combination. There is no obligate

relationship between them and each can be used separately as

dictated by the circumstances.

The term priming was coined by Hedecker and Coolbear

(63,64) who are credited with discovering its usefulness in

invigoration of seeds. Priming is making a major

contribution to agriculture and seed science, although the

principle is not new. More than 60 years ago, in 1918, Kidd







17

and West (76) described the practice of slow seed hydration

before planting as a treatment which improved productivity.

Other historical reports include those of Chippindale (35)

and McKee (96). Chippindale found that "soaking" increased

the vigor of some Graminae. As an extreme example, McKee

reported that, in addition to grasses, slightly sprouted,

redried legumes grew more rapidly. A more recent report on

the use of priming in agronomic practice describes the

application of polyethylene glycol (PEG) to improve the rate

and uniformity of cereal emergence (7). Priming has been a

more common practice with vegetable seeds (201) and has been

combined with the advent of fluid drilling technology.

Priming may not be limited to the use of PEG or other

practices which may be difficult for both industry and farm

managers to utilize. A new technique was reported by Perl

and Feder (122), who found that water vapor alone could be

used to prime pepper seeds. This report also helps to

explain why Woodstock and Tao (200) found that accelerated

aging for very short intervals of time actually improved

soybean seed performance.

Since vigor is a controversial term, its 1979 AOSA

definition follows:

Seed vigor comprises those seed properties which
determine the potential for rapid uniform
emergence and development of normal seedlings
under a wide range of field conditions. (94, page
785)









The author (RLT) has used vigor as a relative term which

allows growth-related measurements between treatments and

controls to be compared. This concept does not conflict or

contradict the defined concepts of rapid and uniform

emergence; it does, however, consider these measurements on

a relative basis.

While desiccation or redrying is a common practice, not

all seeds are tolerant of this treatment. Adegbuyi et al.

(4) found that treatment with PEG resulted in no beneficial

effect on normal germination of herbage seed and redrying

negated most effects. Most recently, a group of

investigators (156,157) found that desiccation was injurious

to soybean seeds after 36 hours of hydration.

Desiccation tolerance in higher plants is unique to

seeds. Bewley reviewed this topic in 1979 (24). The plasma

membrane of recalcitrant seeds cannot tolerate moisture

levels below 40 percent (18). This is consistent with

Bewley's belief that the plasma membrane may be a key factor

in desiccation (24). Others (41) suggest that the

reorganization of genetic material is a critical factor.

Klein and Pollock (77) developed their views of desiccation

through use of the electron microscope.

The point of no return has been studied as a means of

determining what events in the seed prevent redrying. A

likely associated event is the beginning of cell division

(24). Cell division and cell expansion can be separated







19

experimentally (57), but no reports were found during this

literature review which have used this technique to test

Bewley's hypothesis (24).

The literature concerning priming and related fields of

research is only beginning to emerge and should prove

fertile. The simplicity of priming should promote its use

as a common practice. Basu and Dhar (17) have been using

moisture to invigorate seeds in India since 1974. They have

found the practice to be applicable to a diverse variety of

genera including grains and vegetables. For a subject so

central to seeds with economic potential, priming is clearly

a promising topic for research.



Growth Regulator Bicchemistry

Biochemical processes of the plasma membrane are affected

by aging just as is the permeability. Stimulation of this

activity with growth regulators can increase the germination

of aged seeds (61,123). One important growth regulator is

fusicoccin which is reported by Marre' (92, 93) to stimulate

proton extrusion. The nature of this activity is suggested

by previous studies on plants with fusicoccin. The benefit

of monovalent cations (123) is consistent with the mechanism

of K+/H+ exchange during active transport.

The plant hormone ethylene is endogenously produced

during germination (5,150,151). Like fusicoccin, ethylene

is antagonistic to abscisic acid (ABA). Also like









fusicoccin, ethylene has been reported to improve the

germination of aged seeds. Both fusicoccin and ethylene

break dormancy, demonstrating a common mode in their

activities. Fusiccccin has been referred to as "super

auxin" (97), and the association between ethylene production

and auxins has been established (202).

Previous reports have examined the activity of ethylene

on membrane permeability of the stomata (115,116). Stomata

have been shown to open under the control of 2-chloroethyl

phosphoric acid (ethephon), a water soluble source for

ethylene (191). Since both fusicoccin and ethylene open

stomates, their activity seems to be similar to, or perhaps

synergistic, with respect to proton transport.

An impairment in respiration and oxidative

phosphorylation, both mitochondrial membrane-dependent

processes, would in turn limit energy to support membrane

dependent processes. Abscisic acid inhibits active

transport by the plasma membrane of germinating seeds (39),

but since abscisic acid does not inhibit respiration (23),

it must antagonize fusicoccin by affecting membrane

permeability. While this may be a logical statement, it

constitutes only an untested hypothesis.

Etaylene may have some activity in common with

fusicoccia. Unlike fusicoccin, whose activity is limited to

proton extrusion, ethylene is associated with other

cellular events including sugar transport and metabolism









(176), but not starch hydrolysis (70). The synergism

reported for ethephon (2-chloro-ethyl phosphoric acid) and

kinetin in breaking dormancy in small cocklebur seeds (171)

suggest possible synergism between ethephon and fusicoccin

in low vigor, aged seeds as well as abscisic acid treated,

dormant seeds. Tested alone, fusicoccin and etuephon

antagonize abscisic acid induced dormancy in soybeans.

Testing for synergism between fusicoccin and ethephon would

provide new information regarding mechanisms of action they

have in common. If synergism cannot be demonstrated, this

information precludes practical applications of this

combination. Antagonism between fusicoccin and ethephon

would indicate that they are activating mutually exclusive

or competitive events.

Table 1 relates references concerning the plasma membrane

activity of seeds to the mode of growth regulator activity.

The growth regulators include fusicoccin, ethylene and

abscisic acid. The events starting with membrane-bound

receptors and membrane-bound enzyme activity move upward in

the table. As the proton pump is activated, growth is

promoted by wall softening and increased turgor of the cell.

If these events dominate, dormancy is broken and germination

commences.

It is interesting to speculate that if ethylene is

required by germinating seeds, then abscisic acid may induce

dormancy by antagonizing the action of ethylene. According





6, 46, 66, 73,
134, 168, 192


121,


-I


TABLE 1

Literature on Membrane Activity of Growth Regulators


_--- -
System, Activity and References


Germination
stimulation
5, 61, 123, 151
Dormancy
173
Absicisic acid
23, 25, 91
Ethylene
5, 67, 70, 71, 90,
171
Osmoregulation
Hormonal
165, 166
PM permeability
62, 65, 114, 167
Stomatal aperture
115, 116, 172, 176,
191
Membrane Activity
Adsorption of Ions
33, 117, 162
Proton Pump
33 34, 117, 135,
165, 166, 174
Growth
135, 174, 182
Fusicoccin
8, 12, 13, 39, 83,
84, 92, 93, 103,
123, 135, 147
Receptors









to this idea, inhibitors of ethylene synthesis such as

amino-oxyacetic acid, AOA, should produce effects similar

to ABA.

It is recognized that the activity of growth regulators

extends beyond regulation of the membrane activity. Table 2

summarizes these regulatory activities in seeds with

supporting references. Two reports (114,167) suggest that

the permeability or the membrane is primarily affected by

growth regulators. This concept is based on artificial

membranes. The permeability of phospholipid membranes can

be altered with physiological concentrations of gibberellins

(114) and kinetin (167). This would explain why, with the

exception of ethylene, plant hormones are active in the 10-3

to 10-6 molar range.









TABLE 2

Literature on Regulatory Activity of Plant Hormones



System, Activity and References
r

Energy Regulation
Sugar uptake


Hormonal Regulation






Biosynthesis











Genetic


Synergism

Antagcnism




Hormone Synthesis

Induction

De Novo Synthesis


Membrane Synthesis



Second Messanger


Cell Division


8, 60, 70, 129, 175,
176



170, 171

34, 36, 58, 124,
136, 172, 191



11, 52, 151, 193

52, 54, 59, 150, 202

10, 36, 114, 124,
167, 203

51


61, 81,
152

83, 203


84, 92, 93,


r----- ---- -- --- ----------~















CHAPTER II
MATERIALS AND METHODS



Microtlora of Seed Deterioration

Seed Materials

Locally grown soybean seeds [Glycine max serr. cv.

Vicoja], were used in all experiments. The seeds were

stored in an air-conditioned room (65 percent relative

humidity), which resulted in an equilibrated seed moisture

content of 11 percent on a fresh weight basis.

Control samples of seeds used as internal standards were

important considerations in the experimental design.

Factorial experiments included these controls.



Experimental Design-General

Most experiments were factorial with various levels of

treatment, two for tae chemicals and four for accelerated

aging. The variables consisted of the chemicals, mode of

application and application sequence, before or after

accelerated aging. Controls were routinely used and served

as a basis of comparison for data analysis.









Antimicrobial Chemicals

Streptomycin' was the only antibiotic evaluated. Several

fungicides were tested singly and in combination. These

fungicides were botran,2 captain 3 maneb,4 and benomyl.s

Benomyl was used alone and mixed with captain and maneb.

Other powdered mixtures which were used were captan/botran,

captan/maneb, and streptomycin/captan/botran.

Several techniques were used for application. Chemicals

were infiltrated into the seeds by imbibing them with

saturated solutions of fungicides and streptomycin (400

p.p.m.). This application was essentially a combination of

priming with chemical incorporating.

The application technique used as a routine was to tumble

the seeds in captan/botran. Streptomycin and fungicides

were applied to the seedcoats and were effective in

preventing signs of fungal growth.








1 Streptomycin structural name: 0 2 dioxy 2 -
(methylamino) alpha L glucopyranosyl (1 to 2) 0
5 deoxy 3 C formal alpha 2 lyxofuranosyl (1
to 4) N, N' (aminoiminomethyl) D streptamine

2 Botran structural name: 2,6 dichloro -4- nitro-aniline

3 Captan structural name: 3a, 4, 7, 7a tetrahydro -2-
[ (trichloromethyl) thio] -1H- isoindole 1,3 (2H) dione

* Maneb structural name: [[1, 2 ethanediylbis
[carbamodithioato]] (2-)] manganese

s Benomyl structural name: [1 [(butylamino) carbonyl] -1H-
benzimidazol -2- yl] carbamic acid methyl ester







27

Additional protection from microflora was obtained by

limiting the severity of accelerated aging. Selection of

seeds was based on their growth relative to other sources.



Histoloqy_or Aged Seeds

Control seed samples consisted of high vigor, Vicoja

seeds and were obtained from the Agronomy Department's seed

storage facility. Other control seeds were subsaaples of

seeds which had been subjected to accelerated aging for 24

and 48 hours. Included also were other sources of Vicoja

seeds from 1980 and 1982. Some experiments included other

cultivars tested at various degrees of deterioration. Seeds

harvested at maturity were also compared to postharvested

seeds. Additionally, seeds primed after accelerated aging

were tested.

Tetrazolium was used as a vital stain (55). It

differentiates tissues according to dehydrogenase enzyme

activity. Five dimensions of the tetrazolium experiments

included natural aging, accelerated aging, priming,

postharvest deterioration and cultivar differences.



Cg2olo1__lof Plasma Mlembrane Injurl

Injury_and Fixation

Injury was accomplished by soaking seeds in water at 4 C.

After two hours of excessively rapid hydration, the embryos

were removed and fixed overnight in cacodylate buttered







28

glutaraldehyde (4 percent) at 4 C. The low temperature was

used to slow temperature dependent processes within the

cells.

The tissue was then rinsed three times with 0.1 M sodium

cacodylate buffer, pH 7.2-7.5. Osmium tetroxide (1 percent)

post-fixation also proceeded overnight in the cold.

Embedding was preceded by an alcohol dehydration series,

including an overnight exposure to uranyl acetate. Acetone

dehydration completed this process before the embryos were

embedded in plastic using the Mollen-Hauer formulation. The

maximum size of the tissue block was one cubic millimeter.

The plastic resins were infiltrated in three stages,

starting with 70 percent acetone and 30 percent plastic.

This process allowed one hour for each stage. Finally, the

samples were put into a 60 C vacuum oven and allowed to

bubble until no longer foamy. The vacuum was released and

the 60 C incubation continued overnight.



AicroscoY

Embedded embryos were "blocked off" with razor blades

then ultramicrotome sectioned with a diamond knife. Thick

sections were cut through the central axis of an embryo and

viewed without stain using a phase contrast microscope.

This showed the orientation of tissue in the block. Thin

sections were then cut and post-fixed with lead citrate.

The Jewel electron microscope (JEM) was used for photography

on film plates.









Physiology of the Plasma Membrane

Accelerated Agin

Just before the accelerated aging process, samples ot

seed were coated with captain and botran fungicides.

Standard equipment and conditions of 100 percent relative

humidity and 41 C were used for aging (42). However,

nonstandard methods were used to expose the seed to this

environment. Petri dishes (25 x 150 mm) were filled with a

single layer of seed but remained uncovered in the aging

chamber. An aluminum foil "tent" protected seeds from

condensation. Treatment intervals were 20, 30, 40 and 50

hours. Moisture of 20 to 30 percent was obtained due to the

aging process. This treatment was not severe enough to

affect seed vitality but did affect the growth rate curve.

After this treatment, the seeds were equilibrated to the

original 11 percent moisture, by air-drying them for a weex

under storage conditions.



Controlling Water Uptake Rate

The rate of water uptake was controlled by varying the

number of germination papers (Anchor, Inc.) wetted with a

constant amount of water. Deionized water (20 ml) was

placed in (25 x 150 mm) petri dishes; then in progression,

one paper was placed in the first dish and five were placed

in the last dish; 20 seeds were added to each dish. Cne

paper served as the check, in that the water potential was









zero at the point of contact between the seed and paper.

Each additional layer lowered that potential by reducing the

availability of water. Imbibiticn was monitored by weighing

the seeds at various time intervals. Transfer of seeds to

petri dishes containing 20 milliliters of water and three

papers allowed germination to begin in one day at 25 C.

Four additional days were allowed for growth before

measuring seedling weights or lengths. Each experiment was

repeated and replicated at least twice for confirmation.



Automatic Seed Analyzer

In principle, the seed analyzer (ASA) records the

electrical conductivity of a steep solution. Conductivity

of the solution increases as the seeds are soaked. The

electrolytes are of cytoplasmic origin consisting mainly of

potassium salts of organic compounds.

The method used to determine electrolyte leakage as a

function of hydration rate Figure 5, follows. Electrolytes

absorbed by the germination papers during imbibition were

measured after one hour of soaking the papers with a total

of 60 millilters of water. The automatic seed analyzer

(ASA-610, Agro Sciences, Inc.) was used to measure the

conductivity of 4 milliliter aliguots of the leached water.

The conductivity or the solution was then expressed in

microamperes. Differences in conductivity, due to

treatments, were relative to controls. It was necessary to









soak the control paper, using the same methodology, to

correct for background electrolytes.



Partial Priming

In some experiments seeds were partially primed by slow

imbibition on four layers of paper for various time

intervals then dried in open containers for at least 4 days.

Imbibition was controlled at 25 C and 100 percent relative

humidity unless otherwise indicated.

The term vigor is used to express the relative growth of

treated to that of the control seedlings during the same

time and under the same conditions. Growth was measured as

fresh weight and expressed as the ratio of the root-

hypocotyl to total seedling weight. Vitality refers to the

percent of live seed based on germination or chlorophyll

development. A new term, partial priming, is introduced to

emphasize the short (24 hours) time required to fortify

redried seed against soaking injury.



Biochemistry of Growth Requlators

Aye versus ABA


Experimental design. The design was (5 X 2) factorial,

replicated twice. Five levels of aging were tested against

two levels of abscisic acid (ABA).







32

Preparation of the ABA solution. Cis-trans abscisic acid

(molecular weight 264 and 95 percent purity) was the

starting material. The free acid was not soluble in water

but could be dissolved in an alkaline solution which

converted the acid to a salt. In approximately 300

milliliters of water, contained in a 500-milliliter

Erlymeyer flask, 200 my of sodium hydroxide was dissolved.

Racemic ABA (66 mg) easily dissolved in the alkaline

solution. Afterwards, sulfuric acid (0.01 M) and a glass

electrode pH meter were used to titrate the solution to

neutrality. The neutral solution was diluted to 500

milliliters. The pH of the final solution was 7.0.


Protocol. To each of the petri plates (25 X 150 mm) was

added 2 milliliters of ABA (5 X 10-4 M), or 2 milliliters of

a sham, followed by 10 milliliters of water. After mixing,

the ABA concentration was 4 X 10-s M. Two brown paper disks

imbibed the liquid and 20 seeds were placed thereon. Four

days at 25 C were allowed for germination and growth.

Measurements were made on the total fresh weight cf the

seedlings in each plate and for the detached root-

hypocotyls.

Data reduction used the percentage of root-hypocotyl of

the total as a growth index. Since ABA reduced the rate of

growth, it was necessary to normalize these growth

percentages as a percentage of the unaged controls. This

was done for the ABA treated and untreated series. The







33

interaction of ABA and accelerated aging could be determined

by using the percentage of normalized growth with ABA to

normalized growth without ABA, for each age category.

It was necessary to use triple ratios to correct tor seed

sampling error, ABA growth reduction and for reduction in

growth due to aging. Using this method, the results then

can be expressed in pure numbers (no units of measurement).

The final percentages represent the effect ABA has on age.

If the percentage decreases with age, then the isolated

interaction is negative. The desirable outcome would be for

the response to increase with age.



Aae versus Fusicoccin and Ethylene


Preparation of the fusicoccin solutions. The publication

of Cocucci and Cocucci (39) was used as a reference for this

procedure. Fusicoccin (mol. wt. 670) was dissolved in

ethanol (10-1 M). A stock solution 10-3 M was obtained by

diluting the ethanolic solution with water.

Twenty-five milligrams of fusicoccin were dissolved in

0.5 milliliters of ethanol and then diluted to 38

milliliters with deionized water. A working solution of

10-s a was prepared from this stock solution. Concentration

at the tinal dilution was in the 10-6* range.


Dose response. Concentration of fusicoccin was 1.5 X

10-6 M at final dilution. This value was based on 10-6 M, a







34

common concentration used by other investigators (39), and

on the author's preliminary experiments. This concentration

produced some dwarfing and also stimulated secondary root

development. The concentration of ethephon (125 ppm) at

final dilution (1:8,000) was selected by reducing the

concentration until obvious stunting of the seedlings was

stopped. The pH was not affected by this concentration of

the acidic ethephon.


Protocol. Twenty seeds were used for each observation.

The aged seeds were exposed to 24 hours of 100 percent

relative humidity and 41 C, in an open petri dish protected

from condensation by an aluminum "tent." Each petri dish

also included two "brown" germination papers and a total

volume of 12 milliliters. An additional five milliliters

was added after seven days of incubation at 25 C.


ExperiEmental desqnig. The design was two-cubic, two

concentrations for each of the three variables: age,

fusicoccin and ethylene.


Statistics. Statistical analysis can be obtained from

the design. Since the experiment was balanced, analysis of

variance (ANOVA) may be applied to the data. Each of the

eight treatments was run in triplicate and the experiment

was repeated three times.







35

Data Analysis. Data were recorded after nine days of

incubation. Three methods were used to interpret the data.

The first was to use a graph. The second method was to

tabulate the data as percent control for two groups, aged

and unaged. The graph gives information in the third method

(ANOVA) but statistical interpretation would not be as

readily comprehended and would nct be as appropriate for

small differences due to treatments.















CHAPTER III
RESULTS



Microflora and Seed Deterioration

In general, putrefaction was not common and streptomycin

was limited to applications where bacterial activity was

indicated by odor. Very high concentrations of streptomycin

inhibited seedling growth. Another problem with coating

seeds with streptomycin powder, also because this antibiotic

is very hydroscopic, it increased the seed miosture when

applied as a powder. The captan/botran mixture gave

consistent protection from fungal growth in the petri dish

germination test.



Histolc y_f_ ASed Seeds

Tetrazolium stain defined areas of dead cotyledon tissue

present on deteriorated seeds (Figure 1). These white

areas or dead tissue were symmetrical between cotyledons.

Areas most prone to aging were grouped loosely into several

patterns. Similar patterns and symmetry were also observed

on seedlings as necrotic spots. It was found that both

accelerated aging and natural deterioration produce these

results. There may be variations on this process. One

exception was Vicoja 1980 which had a more uniform

deterioration.










r ----- ----- -- -------- -- -






I i









I I































Figure 1: Differential Aging of Seed Tissue.
Tetrazolium staining indicated that aging was not
uniform in seed tissue. The pattern of aging was
symmetrical. These symmetrical patterns occurred in
several prevalent locations on the cotyledons. Both
natural and accelerated aging produced this effect.

L -_____1












Tissue Excision Experiment

An additional facet of this histological examination of

seed deterioration was to determine if the parts of the

cotyledon most often affected by aging were important to the

growth of the seedling. Parts of the cotyledons were

excised with a scalpel and the wounds were coated with

fungicide. These excisicns removed approximately one-third

of the moist seed mass. The most extreme treatment was to

remove one of the cotyledons.

Compared to the controls, the rate of seedling growth

after five days was not affected by removing part of the

cotyledons. Removal of one cotyledon reduced growth.

Location of the excision was not critical.



Automatic Seed Analyzer versus Tetrazolium

Results of this test were in agreement with the

expectation that seeds which had the greatest extent of dead

surface also had the highest ASA values. This observation

was made by comparing two extreme categories of seeds, those

with little evidence of dead tissue based on tetrazolium and

those with extensive damage. The ASA values were consistent

with the tetrazolium results, on an individual seed basis.







39

Cytolog_ of Plasma embrane Inri

Figure 2 snows the plasma membrane retracted from the

cell wall. Ribosomes have escaped through the membrane but

larger organelles remain inside the membrane.

Figure 3 confirms the particles as ribosomes because they

form poly-ribosomes. The plasma membrane was also seen

extending into the plasmodesmata in the cell walls.

Electrolytes escape rrom the symplastic to the apoplastic

compartment. Membrane rupture is likely the basis of the

seed analyzer (page 30).
















1 .






































Figure 2: Loss of Plasma MebL-rane Inteyrity.
Excessively rapid water uptake burst the plasma
I mebri:ne (f) allowing elec.trolytes to escape.
:Iibosomes (2) also escape but organelles remain as the
imeibrane is plasnoiysed.
I _-


















^^^*^**^ *\?y- '*1t* *
^ -* **- *A So T*.*" *' '?
'' 'I'

it a.


'r ^ -* *- *. i 4'r' -i "'
***''** ~:. t^ .*** .* '*', -









*<. 't .f^ . ~*: 'i1, f 1,- *-

t -'- >a k
A.~
"~' "," '. '




*""-
a-E"~


Figure 3: Microscopic Identification.
Particies were determined to be ribosomes (R) Ly
id.itiiyii, them with polyribosome (P) formation. The
me.tLrane was also determined to be plasma membrane (M)
)y its association witi pidsmodesmata (D) in the cell
wall.


1 _______ _______I _____I__~_ ~___ _______














Effects of RanidBdration

Water uptake was effectively slowed by additional layers

of germination paper (Figure 4). Twenty milliliters of

water completely saturated one layer of paper. Thus, one

layer served as a control with the same matric potential as

water. The uptake profiles were effectively modeled with a

logarithmic linear transform corresponding to the following

general equation:


Y = I + S log X


The percent moisture on a fresh weight basis was the

ordinate (Y). Hours of uptake were represented by the

abscissa (X). The linear regression coefficient was at

least 0.94. The terms (I) and (S) refer to the intercept

and slope. Excluded from the model was the plateau portion

of imbibition, 50 percent moisture and above.


Effect of_agqe on_ufIake rate. The effect of aging on the

rate of water uptake was determined from graphs similar to

Figure 4 and was prepared from data on unaged seeds and

seeds aged 30, 40 and 50 hours (Table 3). This information

was essential for determining if aging predisposed seed to

injury by weakening the plasma membrane by the velocity of

water uptake. Since the uptake rates were logarithmic, one-












65,


60-


55-


50-




o 40-



3- 35
0.


25


20


15-



10. HOURS O
0 5
HOURS OF


PAPER LA
0 1
*2
03
*4
*5


IM
IMBIBITION


Figure 4: Controlled Rate of Hate
Soybean seed with an initial moisture
were imbibed in petri dishes (25 x 150
20 milliliters of deionized water. E
contained 20 seeds and one to fi
germination paper. The graph symbol
number or paper disks per petri dish.


IYERS








20 25





er Uptake.
of 11 percent
mm) containing
Each plate also
ve layers of
represents the


~------


~- I -- ------------.







44

half maximum imbibition was used as a reference point to

compare the rates of uptake versus aging treatments.

Soybean seeds were found to reach 60 percent moisture before

germinating; therefore, 30 percent moisture was equivalent

to one-half maximum uptake. The data in Table 3 indicate

that aging treatments did not influence the rate of

imbibition. As the age ot the seeds increased, there was no

apparent reduction in the time required to reach one-half

maximum imbibition for any of the five uptake rates tested.



TABLE 3

Effect of Aging on Rate of Water Uptake



Age Layers of Paper
r 1 -------------
1 2 3 4 5

hours

00 1.18 2.0 2.6 6.0 7.0

30 2.0 2.3 3.1 6.5 9.0

40 1.9 2.2 3.6 6.6 10.0

50 2.0 2,6 4.9 7.5 9.7


MEAN 1.9 2.3 3.6 6.7 8.9

S.D. 0.1 0.3 1.0 0.6 3.0







45

Electrolyte Leakaqe. Electrolytes were measured from the

papers used to imbibe seed. The data in Figure 5 indicate

that aging increased the leakage of seed that experienced

rapid water uptake. As the uptake rate was lowered with two

and three papers, there was also a reduction in electrolytes

lost by the seed. Four and five papers reduced the leakage

of the most severely aged (50 h) to that of the unaged

control.


Effects on growth. As uptake rate was controlled and

electrolyte leakage was reduced, a concomitant improvement

in vigor and vitality resulted. The extent to which growth

could be increased depended on the extent of deterioration.

The data of TaDle 4 correlate with the electrolyte data

(Figure 5). Seed growth, after three levels of aging, was

expressed as a percent of the growth of the unaged control.

The term "vigor" was used for this variable. In general,

vigor increased as the uptake rate was slowed with

additional layers of paper. This dependence of leakage on

imbibition rate increased as the age of the seeds increased.

Treatments that resulted in greater leakage also

expressed the least growth. Growth of the seed then

depended in part on the injury sustained at the time the

seed was imbibed. Thus the elimination of imbibition injury

was partitioned from the residual deterioration associated

with aging.










I


Figy
The
imbibi
rate
repress
uptake
imbibi
millil
soluti


ELECTROLYTE LEAKAGE



-
































1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
LRTERS OF PRPER
LEGENO: ---- 0 30 Z-- -- 40 -a- 50


ire 5: Electrolytes Lost During Water Uptake.
concentration of electrolytes recovered from
tion papers is a function of seed age and the
of water uptake. Symbols for each curve
sent the hours of accelerated aging. The rate of
e is controlled by varying the layers of
ition paper. Each reading represents a 4
liter sample taken from 60 milliliters of leached
on, replicated four times.
I









TABLE 4

Vigor as a Function of Age and Bate or Hydration


rI---- I

Layers of Paper

AA 1 2 3 4 5

h growth as Eercent of control

30 66 45 55 90 66

40 15 13 35 62 43

50 02 14 28 35 44


AA = accelerated aged (hcurs)



In addition to the growth of seedlings from aged seed

being reduced by imbibition injury, the vitality or

percentage of germination was also affected. Both aging and

injury from soaking resulted in loss of vitality. The

characteristic sigmoid curves of Figure 6 showed percentage

of germination to be a function of age as well as the rate

of hydration.













100- c-

90-


80

2 70-


60-


50-


40- PAPER LAYERS
0 22

*4
.5
20-

10-I

0I
IO-

0- --------r------------
0 10 20 30 40 50
HOURS OF ACCELERATED AGING






Figure 6: Effect of Hydration Rate on Aged Seeds,
The vitality (percent germination) as a function of
the hours of rapid aging and as a function of the
control used to reduce the uptake rate. Each curve
symbol represents the number of paper disks (one to
Fivee.
1 I












Effect or Priming on Electolyte Leakage

Reversal of plasma membrane predisposition to imbibition

injury was demonstrated (Figure 7). Seeds first primed,

then redried, had one-third the conductivity of the

controls. This reduction in electrolyte leakage during

soaking was observed for the unaged control and each of the

three levels of aging.

Possible explanations for the reduction of leakage in

Figure 7 were tested to determine whether redrying was

effective because it hardened the seed sufficiently to

reduce the rate of water entry. Priming produced no

detectable differences in the uptake rate (see Table 12).

Reduction in leakage could not, therefore, be attributed to

modification or the seedcoat or to other histological

variables.

Additional evidence suggested that the plasma membrane

was altered during partial priming. A primed versus aging

factorial experiment similar to that represented in Figure 7

was conducted with beans split between the cotyledons. This

removed the seedcoat as a moderator of water adsorption.

Results of the experiment with seed halves were similar to

those with whole seeds (Figure 7). An exception to this was

that the electrolyte loss was greater for half seeds than

tor waole, because of greater soaking injury,







50

r-------- ------------ __--^----_----__^--------















50 ;./ 5^/
171











ACCELERATED 0



rated agi 988(0, 20, 301ours) ith

20
Z f862 251


/ 786 262
N Y
PARTIAL PRIMING










Figure 7: Ertect o Primingy cn Electrolyte Leakage.
A tactorial experiatiint consisted of rive levels of
dcccelrated agiiiy (0, 20, 30, 40 and 50 hours) with
San without partial priming. All seeds were
aeuiiibrated to 11 percent moisture during storage.
ASA data were ccilected alter 9 nours of soaking and
IexLressed as tne sum or the microamperes, resulting
trom ASA data, 1or each or the 20 seeds per 10
treatments.












Effect of Aging on Primed Seeds

Table 5 shows taat the vitality of seeds which had been

primed was 140 percent that of the unprimed seeds, after 24

hours of accelerated aging. All seeds exposed to 48

hours of accelerated aging were much more severely injured

but the vitality of primed seeds was increased to 320

percent that of the unprimed. All treatments were

replicated twice. Each test plate contained approximately

85 seeds (10 grams). The vigor of seeds which

survived all four treatments was noted two days after the

vitality data were recorded. In general, the survivors of

the primed seeds grew considerably taller than the surviving

seeds of the unprimed controls.



TABLE 5

Effect of Accelerated Aging on Primed Seeds

C------- ------- -----
Treatment Results

No. Primed AA % Live % Increase

1 n 24 65
2 y 24 92 140

3 n 48 24
4 y 48 77 320


AA accelerated aging (hours)
% Increase in vigor over control









Pria i e-niTeerat ure De edence

Reversal of the predisposition to soaking injury was

tested to determine whether temperature dependence could be

established (Table 6). The aged (34 h) and unaged seeds

were put into two groups exposed to imbibition at 4 C and at

25 C. The low temperature was expected to suppress

metabolic activity. The low and high temperature treatments

were again put into two drying conditions. Drying at 4 C

minimized metabolism. Drying at 25 C allowed the moist seed

to activate metabolic processes during this phase. The

results are consistent with the hypothesis that repair was

involved. Table 6 expresses the ASA data relative to those

of the unprimed controls. Total suppression of repair, 4 C

imbibition followed by 4 C drying, resulted in seeds that

were statistically like the controls. The second and third

pair of treatments gave similar results. The 4 C uptake and

25 C drying benefited both aged and unaged seed. The

reverse, 25 C uptake and 4 C drying, was as effective. The

most beneficial combination was 25 C uptake followed by 25 C

drying. Accelerated aged and unaged (natural age) had the

same ranks associated with the priming combinations but the

aged seed had consistently higher values.









TABLE 6

Priming-Dependence on Temperature


Treatment Results
f !-
temp aged unaged
r----- r r
wet dry rank ASA SD %C rank ASA SD %C

4 4 1 85 8.6 111 1 68 12.9 113

4 25 2 57 0.9 74 2 50 3.7 84

25 4 3 50 8.6 65 3 38 9.2 64

25 25 4 47 4.7 61 4 28 2,9 47


ASA automatic seed analyzer (microamperes
per seed per 4 ml); SD standard deviation (n=2);
%C percent control



Effect of Soaking_Temmpera ture on Seed Leakage

Figure 8 contains the results from a test of Simon's

phase transition hypothesis. Microampere values represent

the averages or 20 seeds. The top curve (H) shows the

increase in electrolyte leakage with age, when seeds were

soaked at ambient temperatures. The second curve (Hp) was

constructed from the ASA values of seeds primed after aging.

Priming reduced the plasma membrane permeability which was

consistent with other experiments.

The third and fourth curves repeat the profiles of the

first and second but have much lower electrolyte values.

The third curve (L) represents the aged treatments imbibed

at 4 C in the refrigerator. Compared to the first curve

































--- -----------
I I

















;-.---.----"
0 o








15 .'Hp




ar. i.not..d y (L) ad (Lp) rspec y.
... ... ......... .. i----- ..---- - - -


03 5 12 1s I 2 L '4 27 30 3j -i 39 U3 2 Ua 5 ,3




Figure 8: Eftect of Low Temperature on Leakage.
Soaking temperature (25 C) was represented by an (H)
Seeds primed arttar aging then soaked at 25 C are
represented by (Hp). Low temperature (4 C) soaking of
five age categories, unprimed and prime after agijing
dare denoted jy (i) aid (Lp) respectively.







55

(H), it was apparent the effect of the lower temperature was

to decrease leakage of the seeds. The fourth curve

represented seeds soaked at the lower temperature and primed

(Lp) was also consistent in that the effect of the lower

temperature was to reduce the leakage of primed seeds.

Additional measurements were taken at the conclusion of

this experiment. Tae individual weights of the first 20

seeds from curve (i) were compared to the individual weights

of the first 20 seeds from the (L) curve; the weight

differences were highly significant using the Student's "t"

statistical criteria. Seeds soaking for nine hours under

refrigeration still had the wrinkled appearance indicating

that imbibition was not complete. Seeds soaked at the

higher temperature appeared smooth. Therefore, the lower

temperature slowed the rate or water uptake.



Biochemistri of Growth Regulators

Age versus ABA

The objective of this experiment was to determine if ABA-

induced dormancy has a beneficial effect on the performance

of aged soybean seeds. The rationale was that ABA would

delay germination sufficiently to allow the seeds'

metabolism to be channeled into repairing the deleterious

effects of aging. Aging treatments were sufficient to

influence growth while gentle enough to minimize the effects

on percent germination.









ABA dose response. In order to determine the

concentration of ABA which would delay germination but would

not require additional treatments to break dormancy, a dose

response curve was generated. This curve, Figure 9, was

modeled with a log-logit linear transformation. The dose of

ABA (10-3 M) was expressed as the log of the ABA dose used

in each test.

logit = In [P/(100-P) ]

Linear regression analysis had a coefficient of the

determination or (2) equal to 0.99. This analysis provided

a new and effective mathematical model, judging from various

alternatives described by Moore (97).


Isolation of ABA and age interaction. In Table 7 are

treatments and primary data. Root-hypocotyl weights, in

general, decrease with age when treated with ABA, but in

this experiment the weight actually increased with age up to

30 hours, then started to decline. No effect of ABA was

apparent on the unaged seeds. In both cases the root-

hypocotyl was 22 to 23 percent of the total weight. After

seven days of growth, the total weights (T) and root-

hypocotyl weights (RH) were measured. The mean refers to

the average or the two (RB/T) ratios.

In order to interpret Table 7 it was necessary to reduce

the primary data as described in the methods protocol.

Table 8 was constructed using the procedure previously

described (page 32). Except for the unaged controls, ABA












LOGIT Y = -0.059 -2.09 LOG X
R2= 0.99




\M9





1
L






.
T
G


N

2












LOG RBR InL)


Fiure 9: Dose Response to Abscisic Acid.
Dose response tc AEa was modeled with a log-logit
linear rej session. Each observation contained 50
Iseds. Tota volume of liquid was constant (15 al),
Lil the ABA dose varied from 0 to 8 ml. The
conversion fact: for milliliters added to the final
molar concentration is (6.6 X 10-) Data were
Srcorded fter Live days of incubation at 27 C.
L ---------- ------------------------









TABLE 7

Interaction of Age and Abscisic Acid




TREATMENT RESULTS


TRT# AA ABA REP T RH RH/T MEAN SD CV
-, r 1


1 00 Y 1 6.23 1.37 22.0
2 6.20 1.44 23.2 22.6 0.8 3.7

2 20 Y 1 5.91 1.22 20.6
2 6.05 1.22 20.6 20.4 0.3 1.4

3 30 Y 1 5.67 1.17 20.6
2 5.97 1.16 19.4 20.0 0.9 4.3

4 40 Y 1 6.23 1.04 16.7
2 5.81 0.84 14.5 15.6 1.6 10.3

5 50 Y 1 5.72 0.86 15.0
2 15.0

6 00 N 1 6.65 1.50 22.6
2 6.66 1.49 22,4 22.5 0.1 0.6

7 20 N 1 7.11 2.09 29.4
2 6.86 2.20 32.1 30.8 5.5 17.8

8 30 N 1 7.19 2.10 29.2
2 7.40 2.53 34.2 31.7 3.5 11.0

9 40 N 1 6.76 1.64 24.3
2 7.09 2.19 30.9 27.6 4.7 17.0

10 50 N 1 5.37 1.04 19.4
2 19.4


T total weight; RH root hypocotyl weight; MEAN of two
replications; CV coefficient of variation







59

results in less growth as expected since ABA reduces growth

as well as inducing dormancy. If a variable called vigor is

postulated and defined as growth relative to the unaged

control, then this compensates for the reduction ABA has on

growth relative to the unaged control. Vigor expressed as a

percent then was corrected for the ABA effects on growth

irrespective of age. To correct for age, aged ABA

treatments were divided by the aged treatments without ABA

and again expressed as a precentage. This new variable was

called "vigor ratios" since it was the ratio of two vigor

terms (with and without ABA). Vigor ratios then represent

the isolation of the ABA age interaction. These data do not

show a substantial interaction of ABA and accelerated aging.

Perhaps a higher concentration of ABA would have given more

definitive information. Statistical analysis by the

generalized linear model may demonstrate significant

interaction.










TABLE 8

Effect of ABA on the Vigor of Aged Seeds


RESULTS
r --- --

GROWTH VIGOR
GROWTH RATIO RATIO


TREATMENT
I -I


ABA AGE
r~----~-----

Y 00
N 00

Y 20
N 20

I 30
N 30


Age versus Fusicoccin and Ethylene

The rationale of this experiment was to determine if

fusicoccin and ethylene, or its substitute, ethephon,

interact synergistically. A synergistic response would

indicate that each growth regulator has independent but

complementary mechanisms cf action. If, however, fusicoccin

stimulates ethylene production, then it would be difficult

to assign effects to a specific regulator. The objective

was to find effective chemical means for low vigor seed

stimulation.


r________~__l~~ __ I


1







61

After five days of growth, seeds were weighed before and

carter detaching the cotyledons and Table 9 contains the

results.


Interaction


TABLE 9

of Fusicoccin


and Ethephon


OBS observation;
hypocotyl weight


TRT treatment; REP replicate; BOOT root-
(grams); AA accelerated aged


Table 10 summarizes the results relative to each of the

two controls. In this experiment, ethephon stimulated root

mass development. The roots appeared thick, short and root-


ROOI

0.39
0.37

0.45
0,64


0.43

0.50
0.49

0.70
0.67

1.09
1.10

0.55
0.42

0.89
0.90


r -- ---------------~


~----------- --- ------------------







62

hairs were prominent. Seeds grown on two percent sucrose

developed roots similar in appearance. Fusicoccin partially

neutralized ethepnon's effect indicating antagonism rather

than the synergism expected. Fusicoccin did not reduce

growth compared to the controls. At the end of five days of

incubation, the total Fresh weights and the root-hypocotyl

weight were recorded for each petri plate. The percentage

root-hypocotyi to total weight (ITot) was used as the

result. Each observation was relative to the aged or unaged

control (%C), (Table 10).



TABLE 10

Growth Response to Fusicoccin and Ethephon




Treatment Results
r 1--- 1--- -
Aged Unaged

%Tot %C %Tot %C

Ethephon 13.0 159 19.0 202

Interaction 10.0 122 13.4 143

Control 8.2 100 9.4 100

Fusicoccin 8.3 101 9.1 97


%Tot percent of total weight (20 seeds); %C percent of
control



Since the data in Table 9 contains a missing value, the

generalized linear model was used for statistical analysis







63

(Table 11). No significant interaction was attributed to

Lusicoccin, with age and ethylene. All other interactions

and main effects were significant or highly significant.


TABLE 11

Generalized Linear Regression Model


SOURCE
MODEL
ERROR
CORRECTED TOTAL


R-SQUARE
0.968362

SOURCE
AA
FC
ET
AA*FC
AA*ET
FC*ET
AA*FC*ET

MODEL F


C, V,
9.7680

DF
1
1
1
1
1
1
1

30.61


SUiM O S
0.83559333
0.02730000
0.86289333


ROOT MSE
0.06244998

TYPE I SS
0.23937190
0.03544835
0.45825016
0.00000019
0.06627273
0.03402778
0.00222222


MEAN SQARE
0.11937048
0.00390000


ROOT MEAN
0.63933333

F VALUE PR > F
61.38 0.0001
9,09 0.0195
117.50 0.0001
0.00 0.9946
16.99 0.0044
8.73 0.0213
0.57 0.4750

0,0001


Variance alone did not give positive or negative

information. Therefore, it was useful to use the graphic

interpretation. As shown in Figure 10, no treatment

produced less growth than the control. Etaephon was

beneficial to both aged and unaged seedlings. Fusicoccin

did not demonstrate a positive effect under these

conditions. A combination or fusicoccin and ethephon

reduced tne benefit of ethephcn alone.


r ----- -----












IIO -





S. 10
l:.












1.095-





T 0.375-
H






0.30 \"
N 0.780












0 0 S 25 30
0 0 .75-
N 0.70-

RI"


















0 5 0 15 00 25 30 35
ACCELERATED GED (H)




Figure 10: Aye versus Fusicoccin and Ethylene.
Tne root mass of eCch observation (20 seeds) was
recorded atter rive days of growth at 25 C.
Fusicoccin (F) ethylene (E), and their combined
interaction (I) were applied to ajed (314 h) and unaged
seeds.

L-------------- -----------















CHAPTER IV
SUnMARY AND CCNCLUSICNS



Microflora of Seeds

Control of microtlora prevented fungi from inhibiting the

growth of seedlings. Use of at least two fungicides in

combination, was a necessary protocol for these studies

because aging weakened seeds and promoted fungal activity.

Antibiotics have been used during accelerated aging, but use

of fungicides for this purpose was not apparent during the

literature survey.



.istoloiogof Seed Deterioration

SImetrical _Necrosis

Black spots are frequently observed on emerging

seedlings. Tne nature of these necrotic lesions was, in

general, symmetrical. This injury may be related to

differential aging ot seed tissue as described below.



Tetrazolium Staininq_Pattern

Use of this stain on naturally aged and accelerated aged

seeds showed symmetrical differential aging of the cotyledon

tissue. This finding helps explain the nature of seed aging

at the histologic level. The source of most seed leakage is

probably from the unstained areas.

65









Ltolo oyf Plasma Membrane Injury

Plasma Membrane Rupture

Electron-micrograpts were obtained of cells with ruptured

plasma membranes following rapid hydration. Some of the

riboscmes were located between the membrane and the cell

wall. Therefore, it is likely that cytoplasm would escape

from these cells and constitute electrolyte leakage.



Physiology

Controlled Hydration Rate

A novel method was developed, allowing the hydration rate

to be varied. Rapid water uptake injured seeds, especially

when they were previously aged. By controlling the rate of

hydration, injury during hydration could be varied and even

prevented. Thus it was possible to partition imbibition

injury and accelerated aging.



Partial Priming

A method was developed for priming seeds. It was also

found tnat redrying could be applied to soybean seeds even

after germination had begun.

The mechanism of priming was temperature dependent and

probably metabclically dependent. Rejuvenation or seeds was

indicated by at least three criteria: (1) Priming reduced

electrolyte leakage (60 to 20 percent of controls). (2)

Priming more than doubled the survival of seeds exposed to







67

accelerated aging. (3) Priming followed by redrying at 4 C

did not affect leakage in aged or unaged seeds.

Interpretation or this data was that metabolism suppressed

by the low temperature was not sufficient for seed repair.



Biochemistry of Growth Requlators

Abscisic Acid Studies


Lo-jlojit dose response of ABA. This mathematical model

effectively described the inhibition by exogenous ABA on

germination. Similar models were not found during the

literature survey.


Antagonistic chemicals to ABA. Ethylene was the most

effective treatment for reversing exogenous ABA dormancy.

Other effective antagonist included thiourea and fusicoccin.


Effect on respiration by ABA. ABA was found to prevent

an increase in respiration in imbibed seeds. Respiration

increased during hydration until seed saturation, but ABA

prevented an additional increasein respiration associated

with germination.


Age interaction with ABA. The experiment was conducted

with the hope that ADA could be used to improve the vigor of

aged seeds by allowing the seed to channel metabolism into

repair before starting the growth process. Onfortunatly, no

improvement in seed vigor could be detected as a result of







68

ABA treatment of aged seeds. Ethylene, on the other hand,

did have a positive age interaction.



Fusicoccin Studies

Seedling morphology was affected by fusicoccin in the

10-6 M range and higher. With this evidence of biological

activity, fusicoccin was shown to reduce the seedling growth

attributable to ethylene stimulation. This activity was

interpreted as antagonism between fusicoccin and ethylene.

This activity was interpreted as antagonism between

fusicoccin and ethylene. It seems reasonable that

fusicoccin does not stimulate ethylene production, as is the

case with some other growth regulators with auxin like

activity.



Hylpotheses Tested

Homeostasis

Several experiments in this study did support the

homecstasis hypothesis metabolicallyy dependent repair, page

4). Conversely, no evidence could reject the hypothesis.

Until this hypothesis fails to explain experimental

observations, or until a more complete description is

provided, this explanation must be accepted.







69

Simon's Hypothesis

This hypothesis (spontaneous repair, page 16), was found

unsatisfactory when applied to observations encountered

during this investigation.

Another report failed to support Simon. O'Neill and

Leopold, 1982 (109) found no detectable phase transition in

soybean phosphloipids over the 0 C to 50 C temperature

range.















CHAPTER V
DISCUSSION


This study analyzed seed deterioration and repair using

five approaches (microflora, histology, cytology, physiology

and biochemistry). The analytical methods were

progressively more detailed and on smaller scales.

Chronologically this work began with the use of growth

regulators in an effort to refurbish the performance of aged

seeds. Dormancy induced by abscisic acid was used to

determine if repair took place during this period. The use

of abscisic acid was complicated by additional steps to

reverse the process. The very brief dormant period

preceding germination was enough "dormancy" to improve

performance. This led to a type of priming which was

distinguished from the conventional technique (64) and was

initially referred to as partial priming.

Beneficial effects of priming were evident with reduction

in membrane permeability. Plasma membrane leakage was

identified with cell injury and death. Prevention of

membrane rupture improved seed vigor and vitality. Part of

this improvement was explained by prevention of injury but

repair processes were also evident.









Microflora and Seed Deterioration

Control of microbial activity with chemicals had a

limited objective, which was to suppress fungal growth on

seeds in the petri dish germination test. The results of

these tests were used to establish a protocol which used

captan/botran on all seeds unless otherwise noted.

This treatment effectively eliminated most signs of

fungal growth. Avoidance of seed deterioration also reduced

the fungal problem.

Questions remaining include the extent to which

microrlora affect the vigor or seeds before accelerated

aging. Another question is the influence of microflora on

the symmetrical, histological patterns visualized by

tetrazolium staining.

It was assumed that the presence of most fungi was

limited to dead tissue of the seedcoat. These saprophytic

fungi may not penetrate the living aleurone layer. Another

assumption was that absence of visual signs of fungi also

indicated an arrest of fungal activity.

The design of the experiments used control treatments to

minimize the unknown influence of microflora and reduce

confounding due to their possible influence. Fungicides

were used in earlier studies by Tao (170) and by Royse,

Ellis and Sinclair (146). Few reports have considered the

microflora component in relation to accelerated aging or its

influence on the vigor of seed development.









Although microflora were of interest to the present

investigation, it was not practical to pursue all of the

avenues suggested. Emphasis was confined to the

permeability of the plasma membrane in relation to aging.



Histology of Aged Seeds

This was integrated with other phases of the study and

with information in the literature. For example, the

contribution of microflora to the differential aging was

unknown. Since most of the high vigor seeds developed

symmetrical necrotic spots when accelerated aged for 24

hours, most of the effects are assumed to be physiologic.

Microflora injury to the seed tissue may have been indicated

only in selected seeds.

The correlation between tetrazolium analysis and seed

analyzer results was consistent with the interpretation that

dead, disrupted cells make major contributions to the ASA

results rather than alternative concepts such as phase

transitions of the phosphalipids.

Certain inconsistencies of these tetrazolium results were

also noted. Sen and Osbcrne (154,155) found that aging was

uniform in rye embryos. Their materials and methods

(monocots, isotopic precursors) were radically different

from those used in the present study.

Another inconsistency within these experiments should

also be mentioned. 9hen seeds were aged then primed and







73

finally stained with tetrazolium, they developed areas of

tissue without dehydrogenase activity. These tissues then

may have lost enzyme activity without disruption of the

cells. Osborne's (110) review credited the basis of

tetrazollum staining to mitochondrial dehydrogenase enzymes.

This raised another question: can cytoplasmic denydrogenase

enzymes also affect the tetrazolium stain?

The anatomical locations of the cotyledons most

susceptable to aging were not critical to seedling

development. The tissue excision experiments demonstrated

that it was not the loss of necrotic tissue which affected

growth of the seedling but only represented the more

profound aspects of generalized deterioration.



Cytology o Plasma Membrane Injury

Knowledge obtained from cytological observations was

integrated with the tetrazolium experiments as well as non-

visual, physiological experiments dependent on the ASA.

An explanation consistent with tne experiments of this

study relies heavily on Villiers' (188) report. The

description beginning on page 39 and the figures 2 and 3 are

similar to that of Villiers'. His description of cell

disruption was one accepted for this experiment. A basic

difference between the present observation and Villiers'

experiment was that he used soybean seeds rendered non-

viable by accelerated aging. Rupture of the plasma membrane







74

by rapid water uptake probably occurred in both experimental

situations. Villiers described collapse of the membrane

followed by hydrolytic digestion of the cell contents.

Severe cellular injury would explain electrolyte leakage

during soaking without invoking Simon's phase transition

hypothesis (159,158).

If the force of water ruptured the membrane, then the

osmotic potential would tend to force electrolytes out of

the cell. This process would shrink or collapse the

membrane.

Additional questions concern the effect of predisposing

the membrane to soaking injury and how this process can be

reversed. These questions are discussed in the physiology

sections.



Physioloy_ of the Plasma Membrane

The results of the present study support the findings of

Woodstock and Tao (200), in that accelerated aging (42)

predisposed seed tissue to imbibition injury. The present

study differed in its experimental approach and in extending

previous work to gain insight into the reorganization of the

plasma membrane. Evidence for this process was observed in

both aged seeds and high vigor seeds.

Experimentally, hydration was slowed with matric

potentials rather than an osmotic potential. This technique

emphasized that the desired effect of slow hydration







75

resulted in the avoidance of physical injury to the seed.

This technique also ruled out the possibility that

polyethylene glycol was directly responsible for membrane

restoration. In fact, polyethylene glycol probably slowed

water uptake by increasing viscosity rather than by osmotic

effects. Another consideration was that this chemical

treatment is not entirely innocuous. Once imbibitional

injury was avoided, the seed annealed the membrane.

Annealing1 was evident when primed and redried seed were no

longer predisposed to soaking injury after accelerated

aging. Furthermore, the repair process was temperature

sensitive and probably metabolically dependent. It was of

both practical and basic interest that the effects of aging

was extrapolated from excised embryos (200) to whole seeds.

The information gained has made it possible to increase seed

vigor and resistance to the effects of aging.

Hypotheses considered in the course of these experiments

included the following: First a hydrophilic/hydrophobic

interaction of water with the plasma membrane phospholipids

might bring about spontaneous reorganization. This

explanation was not supported. The presence of water did

not restore the membrane integrity at low temperatures.

Although the temperatures may have prevented the

phospholipid reorganization, it was expected that metabolism

was the more sensitive to temperature.



1 Anneal to temper or toughen seeds against age induced
predisposition to soaking injury.









Secondly, chilling was found to decrease electrolyte

leakage, indicating that injury did not result from

chilling. Therefore, the data in Table 6 are interpreted as

evidence for the metabolic dependence of repair. Seeds that

were imbibed and dried at low temperatures did not differ

significantly from the controls for aged and unaged seed.

The uptake of warm water followed by warm drying was the

most beneficial treatment. This allowed the longest time

and most favorable condition for metabolism. Protein

activity may be implicated as part of the plasma membrane

reorganization. On a dry weight basis, 40 percent of the

plant plasma membrane is composed of proteins (72).

Activity of these proteins may have affected membrane

priming.

Thirdly, avoidance of injury during water uptake would

explain the benefit of gradual hydration (125,130,200),

without postulating a repair mechanism. Importantly, it was

possible to separate plasma membrane repair from the

confounding effects of imbibition injury.

It has been suggested that imbibition does not allow time

for the membrane to reorganize (200). This rate-dependent

process then results in leakage. Since leakage of redried

seed was reduced by one-third, hysteresis was suggested.

Once the membrane was organized by priming, it did not lose

all of its organization and could organize more rapidly.

Evidence against this hypothesis was that seeds had lower







77

ASA values at 4 C than at 25 C. If reorganization was the

limiting factor, cold water should have decreased the rate

of organization and subsequently increased leakage. Cold

water did imbibe more slowly (102), but this treatment also

decreased leakage. This evidence does not favor the

hypothesis of Simon (158).



Testof_ Hypotheses


metabolic repair. Interpretation of these results

answered two guesticns. The first is "Does priming have an

effect on aging?" The obvious answer was "yes." The second

question was "How does priming affect aging?" The answer to

this second question provides information about the priming

process. Priming apparently does more than restore the

plasma membrane. These results show that there was a total

improvement in the vigor of the seeds which emphasizes the

metabolic basis of priming and further supports the

homeostasis concept of anabolic repair. Anabolisn increased

the vigor of the seeds. The accelerated aging treatment

then became a vigor test and demonstrated the increase in

vigor due to priming (82). That unaged seeds can benefit

from priming was plausable since these seeds may have lost

some of their maximum potential after a year of storage.

Other chemicals considered for use during priming include

fusicoccin and ethylene. These chemicals may enhance seed

vigor under stress conditions.







78

Spontaneous repair. Simon's concept of plasma membrane

permeaoility has stimulated seed research for ten years

(158). Experimental evidence supporting this concept

includes that of Parrish and Leopold (118). Their results

were based on protein leakage which occurred during soaking.

Contradictory results were later found by O'Neill and

Leopold (109). These investigators used differential

scanning calorimetry to directly measure enthalpic changes

in phospholipids during hydration. Interpretation of the

current findings was also inconsistent with the phase

transition concept.

According to that concept, the expected results would be

an increase in electrolytes at the lower temperature. The

rationale for these predicted results was that the lower

temperature would slow the phase transition and hold the

membrane in the permeable formation.

Simon's publication (158) calls attention to several

deficiencies, wnich are apparent in retrospect. The

application of the concept to seeds was extrapolated from

research on brain phospholipids. Simon thought that in the

dry state, the phcspholipids formed the equivalent of

micelles, although no microscopy supports this conjecture.

His scholarly and stimulating report was based on a

literature survey only, unsupported by experiments directed

at determining its validity.







79

The role of water uptake in influencing the leakage of

seed tissue was emphasized by the results of this experiment

(Figure 8). Several experiments were subsequently performed

to determine if priming reduced the leakage of seeds by

reducing the rate of uptake. These experiments also failed

to support this explanation for the priming phenomenon.

Results of physiological experiments are consistent with

the homeostasis hypothesis. Conversely, Simon's hypothesis

was not supported.



Biochemistry of Growth Regulat ors

ABA versus Accelerated Aging

Ultimately, use of this hormone was not necessary since

other experiments in this study have shown that dormancy can

be induced by redrying the seeds before they germinate or by

reducing the availability of water. These other approaches

have demonstrated the benefit "dormancy" has on seed

performance.

Another consideration, evident in retrospect, is that

both the control and ABA treatments would probably reverse

some of the effects of aging in both treatments. Therefore,

although ABA did not improve the "vigor" of aged seeds, this

experiment does not contradict the homeostasis hypothesis.









Interaction of Fusicoccin and Ethephon

The antagonism between fusicoccin and ethylene may be

limited to the concentrations used in these experiments.

Perhaps the combined effect was excessive and became

inhibitory. The potential for fusicoccin in stimulating

rapid seedling emergence has been well documented by Khan

(74). In defense of these results, the experiment improved

on previous practices in which the same molar concentrations

were used for each growth regulator (58). These practices

failed to recognize the differences in dose responses.

Perhaps dose selection should be based on the optimized

value. Alternatively, the use of 50 percent maximum

response would give a reference concentration which would

prevent overdoses. Combining optimal concentrations could

possibly have a negative effect by exceeding the limits of

productive activity.



Suggestions for Future Research

This work tested an existing hypothesis and was unable to

reject the possibility that dry seeds reverse the effects of

deterioration after aging through metabolic turnover as a

pregermination event. Additional future testing would

attempt to inhibit protein synthesis chemically to determine

if protein synthesis is required for the reversal of aging

effects by priming.















APPENDIX
SUPPLEMENTARY EXPERIMENTS



Microflora and Seed Studies

Several interesting observations on microflora were

obtained in some cases from formal experiments and on other

occations informally during work directed toward unrelated

problems.



FunqalISurvival

A grey mold was found that could grow on dead seeds at 41

C. This was the only seed fungus that was able to grow

under the high temperature which is standard for the

accelerated aging test. Seeds accumulated sufficient

moisture (40 percent) to support fungal growth. The growth

of fungi during aging was minimal since no signs were

evident on seeds immediately alter this treatment.

In another experiment, seeds with 11 percent moisture

were sealed in a jar for 30 days at 41 C. The seeds did not

survive but the fungus did. Seeds had more fungal growth

after aging. This was probably because fungal growth was

favored by nutrients which leaked from the seeds during

imbibition.









Control of Fungal Growtn

The use of fungicides was necessary for petri dish

germination of aged seeds and for priming seeds. The reason

for this requirement was that aging weakened the seeds and

may also have made more nutrients available which stimulated

fungal growth. Priming seeds at 15 C without fungicides

resulted in severe fungal growth. Fungal activity in a

petri dish reduced the growth of most seedlings not only

those directly infected. Dead seeds would, at times,

support fungal growth even though the seedcoat had been

treated with fungicide.

Conversely, removal of nutrients leached from seeds and

prevention of imbibition injury may explain why it was

possible to germinate seeds without signs of fungi and

without fungicides as is commonly done in growing bean

sprouts. The seeds were rinsed with water several times a

day, then drained. Care was taken to prevent evaporation.

In this test germination could be controlled by limiting the

seed moisture below 60 percent on a fresh weight basis.

Large numbers of seeds could be treated as in the above

experiment for commercial production of bean sprouts in the

food industry. The seed industry could also use this

technology to prime seeds on a commercial scale without the

use of PEG. Seed testing, with and without fungicides, may

become a means for detecting harmful effects of microflora.









Histology of Seed Deterioration

Location of Seed _Funi

The location of most of the fungi was probably limited to

the seedcoat. These fungi were probably saprophytes.

Dusting the seeds with a dry powder prevented signs of

rungal growth. Coating seeds with a fungicide slurry was

effective and would be expected to penetrate the seedcoat.

Fungal control was similar for the slurry and the dusting

techniques.

Possible fungal growth within the live seed tissue was a

topic of interest. It was possible by staining seed tissue,

to locate fungus above the living aleurone layer. The

organisms could be seen with a dissecting microscope. Only

pathogens would be expected to be able to penetrate into

living tissue.



Location of Hardness in Seedcoats

WUen a hard seedcoat was removed from the seed and

imbibed with stain, the dye penetrated all but one cell

layer, starting from the inside. Excluded from the stain

was the exterior palisade layer. Only after some time was

the stain able to penetrate these cells as they lost their

impermeability.









Tetrazolium StaininqStudies

The dead areas cn deteriorated seeds may be related to

microflora. The relationship of fungus to dead tissue is

not understood at this time. For instance, does aging kill

parts of the seed tissue and allow microflora to enter this

dead tissue or are the dead areas predisposed to aging by

microorganisms?

One experiment in which seedlings were grown on a dilute

solution of tetrazolium showed that not all white areas were

necessarily dead. The root and hypocotyl did not stain

except for the root-tip and the junction between the root

and hypocotyl. White, symmetrical and clearly defined

patches developed on the cotyledons, indicating a lack of

dehydrogenase activity in the unstained cells. Necrotic

spots on seedlings were also symmetrical and may be the same

areas identified with the tetrazolium stain. The extent of

the dead tissue only indirectly indicates that injury had

occurred in the other tissue as well. Surgical excision of

parts of the cotyledons comparable to the necrotic areas had

little effect on seedling growth. Removal of one cotyledon

reduced growth, however. The presence of the insoluble

formazan dye in the solution used to soak seeds indicated

that cellular rupture was due to soaking injury.

Priming seeds after aging did not affect the subsequent

tetrazolium stain. Penetration of the stain was observed by

splitting the cotyledons and by making cross sections with a







85

scalpel. For the stain to penetrate the entire seed, it is

suggested that the seeds be split between the cotyledons

then imbibed directly in a tetrazolium solution.



Cytologyof the Plasma Membrane

The cause of seed leakage was the question addressed by

several preliminary experiments, suggested by current

concepts, that aging causes deterioration of the membranes

which results in loss of cytoplasmic electrolytes. Since

about half of the membrane consists of protein, on a dry

weight basis, perhaps there are alterations in membrane

proteins which were associated with aging.

Proteins rather than phospholipids were selected for

study because previous reports (132,133) indicated that

phospholipids were not likely to be associated with the

aging problem. Proteins were also selected for study

because they are labile and therefore could be expected to

be sensitive to environmentally imposed deterioration.

Experimentally, the object of this experiment was to

characterize plasma membrane proteins in the embryos of two

cultivars, Hardee and Vicoja, before and after accelerated

aging. Protein characterization was ideally based on two-

dimensional electrophoresis following ultracentrifuge

purification. Aging was expected to produce an alteration

in the protein composition. Also since cultivars differ in

their aging rates, perhaps protein composition could account

for tnis difference.









Membrane enrichment was accomplished by use of the

discontinuous density gradient ultracentrifugation

technique. Determination of purity was based on electron

microscopy. Plasma membrane was distinguished from other

membrane material by use of the Boland stain (142).

Membrane preparations were successfully extracted first

from embryos and then from bean sprouts (Figure 11). Even

though sufficient membrane quantity was obtained, the 70

percent purity was insufficient for further progress.

Technology at the time limited preparations to "enriched"

fractions. Since that time, a report has been published

which makes it possible to obtain pure plasma membrane

fractions (195).

Now that it is technically practical to obtain pure

plasma membranes, the objectives of this early work may be

attainable. Since experiments with both animal and plant

tissues has identified "heat shock" proteins, it seems

entirely likely that aged seeds would have a similar de novo

synthesis response to injury (160). Additionally, evidence

was obtained using other approaches (priming) which

supported the conceit of homeostasis or anabolic repair in

pregerminated seeds.













4--- -- V-- 142





W I
WL' r '.















'* "





,P W, ,




Figure 11: Enriched Plasma Membrane.
Seed embryos were used as a source of membrane. After
the tissue was homogenized, differential
centritugation was used to sequentially remove tissue
debris, nuclei and mitochondria. Plasma membrane was
separated from other membranes by discontinuous,
gradient ultracentrifugation. A purity of 70 percent
was obtained with this method based on electron
m icrogra ph s.
I -
F~: 5 i

Figue 11 Enrche Plama Mmbrae.
I Sed mbros ereuse asa surc ofmemran. ftr
I ~ ~ ~ th ise a ooenzd ifeeta
I ~r cetrtuato wa sdt eunial eoetsu
I ebis ncei n miocodri. Pam mmrn a
I searatd frm oher embrnes y dsconinuos,
I graientultrcentifugtiol. A uiyo 7 ecn



I ee emiroyrd s.vr sda oreo ebae fe












Phksjioloyof Seed Deterioration

Priming Experiments

At least five points can be made which supplement an

understanding of the priming effects based on physiology

experiments. Taese experiments were conducted to answer the

following questions: [1] What is the relationship between

the maximum seed moisture used for priming and the leakage

of redried seeds? [2] Does a divalent cation, such as

calcium, benefit the priming process? This is a reasonable

expectation since calcium had been shown by Gracen, in 1970,

to stabilize the plasma membrane (56). [3] What is the

"point of no return" for redrying seeds without loss of

vitality? [4] Rather than repair of the plasma membrane,

what other possible mechanisms may be responsible for the

reduction in seed leakage after priming? For instance, is

there sufficient moisture in the redried seed to slow the

rate of hydration? [5] If seeds are redried to their

original weight, do they return to their original moisture

content?


Moisture versus priminq effect. The relationship between

maximum seed moisture used for priming and leakage was

obtained for aged and unaged seeds. Figure 12 displays

these results. The curves reflect the statistical analysis

but more questions were raised by this experiment. The







89

first question is what determines the maximum priming effect

once 60 percent moisture is attained? For instance, if 60

percent moisture produced the maximum priming effect then

the line would be asymtotic to the X-axis. The second

question or perhaps expectation is that the curve should be

sigmoid because very low amounts of moisture would be

proportionly less effective. Two separate experiments

failed to obtain sufficiently consistent data to answer

these questions with certainty. The limitation was finding

a technique which gave the desired results. The first

technique was to imbibe seeds to pre-selected moisture

percentages, letting the time required vary. This was

tedious and gave disappointing results. The second

experiment was simplified in that time was controlled and

the moisture was variable. There was some indication that

moisture up to 30 percent increased seed leakage, but more

than this amount lowered leakage. Additional factors

contributing to the uncertainty of these findings were that

only two points were affected, 20 percent and 30 percent

moisture. Also the differences were not large.


Desiccation of bean sprouts. Experiments did show with

certainty that when half of the seeds had germinated, it was

possible to maintain vitality after redrying the seeds. The

sprouted seeds survived, grew secondary roots, and

demonstrated geotropic growth.




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