Citation
Reversal of the effects of deterioration in aged soybean seeds (Glycine max (L.) Merr. Cv. Vicoja)

Material Information

Title:
Reversal of the effects of deterioration in aged soybean seeds (Glycine max (L.) Merr. Cv. Vicoja)
Added title page title:
Glycine max
Creator:
Tilden, Robert Luther ( Dissertant )
West, S. H. ( Thesis advisor )
Biggs, Robert H. ( Reviewer )
Cantliffe, Daniel J. ( Reviewer )
Gaskins, Murray H. ( Reviewer )
Huber, Donald J. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1984
Language:
English
Physical Description:
xi, 128 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Accelerated aging ( jstor )
Cell membranes ( jstor )
Dormancy ( jstor )
Electrolytes ( jstor )
Germination ( jstor )
Imbibition ( jstor )
Seedlings ( jstor )
Seeds ( jstor )
Soybeans ( jstor )
Water uptake ( jstor )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Seeds -- Viability ( lcsh )
Soybean -- Seeds ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Deterioration of 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 tetrazoiium staining to age most rapidly at symmetrical locations on the cotyledons. Aged tissue was in turn predisposed to imbibition injury which accounted tor 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 reversible. These results were consistent with the hypothesis that age related deterioration was reversed by pre-germination 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.
Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 111-126.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Luther Tilden.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030447801 ( alephbibnum )
11567164 ( oclc )
ACM9378 ( notis )

Downloads

This item has the following downloads:


Full Text












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.




Full Text

PAGE 1

REVERSAL OF THE EFFECTS OF DETERIORATION IN AGED SOYBEAN SEEDS [GLYCINE MAX (L.) HEfifi. 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 DOCTCfi OF PHILOSOPHY UNIVERSITY OF FLORIDA 1984

PAGE 2

To Martiia

PAGE 3

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. E.H. Biggs' graduate course stimulated an existing interest I soared in plant growth regulators. Serving my committee, ne 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. 8. B. Gaskins insisted that the objectives be as clear and realistic as tiiose of a grant proposal. The plan which

PAGE 4

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 1 s graduate course in post-harvest physiology. Non-committee members of the university's research faculty include Dr. H. C. Aldrich who contributed to the electronmicroscopy. 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.

PAGE 5

TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS iii LIST OP TABLES .................... viii LIST Of FIGURES ix ABSTRACT ............... ... X CHAPTER I. INTRODUCTION ..................... 1 Stateaent of Objectives 2 Literature Review ............... 2 Hypothesis: Homeostasis as a Survival Mechanism ............... t Microflora and Seed Deterioration 8 Histology of Aged Seeds ........... 9 Cytology of Plasma Membrane Injury 11 Plasma Membrane Physiology ......... 13 Growth Regulator Biochemistry 19 II. MATERIALS AND METHODS ................ 25 Microflora of Seed Deterioration ........ 25 Seed Materials ..25 Experimental Design-General ......... 25 Antimicrobial Chemicals 26 Histology of Aged Seeds . . ... ... . . . .27 Cytology of Plasma Membrane Injury ....... 27 Injury and Fixation ............. 27 Microscopy ......... ..28 Physiology of the Plasma Membrane ....... 29 Accelerated Aging ......... 29 Controlling Kater Uptake Rate ........ 29 Automatic Seed Analyzer ..30 Partial Priming ...............31 Biochemistry of Growth Regulators 31 Age versus ABA ...............31 Age versus Fusicoccin and Ethylene ..... 33

PAGE 6

III. RESULTS . . 36 Microflora and Seed Deterioration ....... 36 Histology of Aged Seeds ...36 Tissue Excision Experiment ......... 33 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 CONCLUSIONS 65 Microflora of Seeds ...... ...65 Histology of Seed Deterioration ........ 65 Symmetrical Necrosis .. 65 Tetrazoliuffi 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 SOPPLEMENTARY EXPERIMENTS ................ 8 1 Microflora and Seed Studies 81 Fungal Survival »...•..»•>*•>* .81

PAGE 7

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 of Uptake and Leakage Compared .... 98 Biochemistry of Growth Begulators ...... 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

PAGE 8

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 aater Uptake 44 4. Vigor as a Function of Age and Rate of Hydration . . 47 5. Effect of Accelerated Aging on Prised Seeds . ... 51 6. Priming-Dependence on Temperature ...... ...53 7. Interaction of Age and Abscisic Acid ........ 58 8. Effect of ABA en the Vigor of Aged Seeds ...... 60 9. Interaction or Fusicoccin and Ethephon ........ 61 10. Growtn Response to Fusicoccin and Ethephon ..... 62 11. Generalized Linear Regression Model ........ 63 12. Effect of Priming on the Sate of Imbibition .... 92 vxn

PAGE 9

LIST OF FIGUHES FIGURE PAGE 1. Differential Aying of Seed Tissue. ......... 37 2. Loss oi Plasma Membrane Integrity 40 3. Microscopic Identification. ............ 4 1 4. Controlled Bate of Hater Uptake. .......... 43 5. Electrolytes Lost During Hater Uptake 46 6. Effect of Hydration Eate on Aged Seeds. ...... 48 7. Effect of Priming on Electrolyte Leakage .50 8. Effect of Low Temperature on Leakage 54 9. Dose Response to Abscisic Acid. .......... 57 10. Age versus Fusicoccin and Ethylene. ...64 11. Enriched Plasma Membrane. .............37 12. Seed Moisture Effect on Priming ..90 13. Rates of Hydration versus Leakage. ........ 100 14. Anaerobiosis and Membrane Permeability. ..... 102

PAGE 10

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 r GLYCINE MAX (L.) MERECV. VICOJA] By Robert Luther Tilden April 1984 Chairman: S.H. West, Ph.D. Major Department: Agronomy Deterioration of 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

PAGE 11

tetrazoiium staining to age most rapidly at symmetrical locations on the cotyledons. Aged tissue was in turn predisposed to imbibition injury which accounted tor 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 perrormance of seeds after long term storage. The processes under study may relate to natural survival mechanisms in dry seeds.

PAGE 12

CHAPTER I INTRODUCTION One of the major problems with 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 aidwestern 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.

PAGE 13

2 Statement of Objectives Seed deteti oration 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 Beview 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 Kriedemana (88), as well as Salisbury and Boss (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),

PAGE 14

3 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 Ye arbook 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 wi th 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 Duff as 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 Hydropho bic Effect, completed the memorane 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.

PAGE 15

*4 Hypothesis: Homeostasis asa _Suryiy_al_Hechjinisffl 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 unigue 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 distribured over time. (43, page 16) The stress applied to seeds during storage is aging which in turn may be thougnt of as the spontaneous increase in disorder (entropy) described by the second law of tuermodynamics (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 guiescent seed. A mechanism utilized fcy 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

PAGE 16

5 temperatures is of practical importance. The metabolic repair or homeostasis 1 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 homeo stasi s. 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, Deiouche 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 tendancy to balance anaholism and catabolism (metabolic turnover and refurbishment)

PAGE 17

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 DM in a manner which supported the repair hypothesis. Osborne and Sen (111, 155) have used similiar 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 alsc 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

PAGE 18

7 stable. Current wcrk of this type includes that of Murata (100) and Boos (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. Contradi ctory vi ews concerning the hypothesis. Roberts' (137) concluded, in a review of the control of viability during storage, that 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 most 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 ^i2£^.§Si£§i 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 Soutn 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

PAGE 19

8 wetting of some orthodox seeds can result in increased longevity. Still earlier reports suggest that soaking seeds in a limited amount or water improves their performance even after they are redried (76) , In 1934, Chippindale (35) found that some grain seeds tolerated drought better if tuey were soaked in water before planting, McKee (96), in 1935, found that slightly sprouted, redried legumes and grasses grow more guickly. These studies support the results of this dissertation but since there are contradictory recent reports in the literature (4 1,156,157), Seasons for this discrepancy may include differences in cultivars and/or methodology, Hi cr or 1 o r a _an d _ Seed Pet erio ration 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

PAGE 20

9 68 percent is sufficient to protect seeds against storage microflora. Ultrastructural examination of Aspergillus glaucus, 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 rungal and bacterial infection. Similiarly, fioyse et al. (146) used penicillin in concentrations ranging from 200 to 400 parts par 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. Histology of Aged S eeds Injury to seed tissue has been studied in relation to aginy as wall 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

PAGE 21

10 pigment in viable ceiis. Non-viable cells remain colorless. Evans Blue is another vital stain used to identity patches of cells on tne surface of seed cotyledons waich were ruptured oy soaking the seeds in water with the testa removed (48,50) . Ihis 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 1 (188) description of groups of dying cells observed with the electron microscope. Seeds froiB samjles 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 sfots 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 1 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

PAGE 22

11 areas waich degenerate first under natural or accelerated aging. Cytology of_.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 aeristematic 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 (haploid) stage, Few abnormalities are transmitted to the second generation (144). Ultraviolet light microscopy has given visual, cytological information in dormancy imposed by phytochrosae activation (131). Also the effects cf 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 pnagocytized 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

PAGE 23

12 effects moisture can have on preventing age related deterioration (17,180,189), The fflost germane cytclogicai study concerning the effect of aging on tae 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 or 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 boundry layers or 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. (188, 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 technigues complement earlier fixation which attempted to preserve the memfcranes 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.

PAGE 24

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 membranddependent 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

PAGE 25

14 (164) in membrane deterioration. Analysis or 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 catastropne 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

PAGE 26

lb concluded that aginy 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 ai. (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 aydrate 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 but 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 suca as the soybean may incur structural damage as the external tissue swells more rapidly than the

PAGE 27

16 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 physi ological repair. The "repair" hypothesis was based on a an extensive literature review concerning membrane permeability (159). The explanation contended that tae phospholipids of dry seeds assumed a guasi-laminar gel structure which required several hours to reorganize as the seed was hydrated. Such phase transition was investigated uy Parrish and Leopold (118), but further studies by the same group were unable to support this concept ( 109) . Physiological priming 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 oy 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 agrxculture and seed science, although the principle is not new. More than 60 years ago, in 1918, Kidd

PAGE 28

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 ficKee (96). Chippindale found that "soaking" increased the vigor of some Graminae. As an extreme example, HcKee 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 tarm managers to utilize. A new technigue was reported by Perl and Peder (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)

PAGE 29

18 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 en 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 unigue to seeds. Bewley reviewed this topic in 1979 (24). The plasma meaorane 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

PAGE 30

19 experimentally (57) , but no reports were found during this literature review which have used this technique to test Bewley's hypotnesis (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 nave 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 Biochemistry 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

PAGE 31

2J 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 similiar 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 tne 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. Ethylene may have some activity in common with fusicoccin. Unlike fusicoccin, whose activity is limited to proton extrusion, ethylene is associated with other cellular events including sugar transport and metabolism

PAGE 32

21 (176), but not starca 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 etaephon antagonize abscisic acid induced dormancy in soybeans. Testing for synergism between fusicoccin and ethephon would provide new information regarding mechanisms of action they nave 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

PAGE 33

2 2. TABLE 1 Literature on Membrane Activity of Growth Regulators i System, Activity and References i Germination Dormancy Osmoregulation Membrane Activity Stimulation Absicisic acid Ethylene Hormonal PM permeability Stomatal aperture 5, 61, 123, 151 173 23, 25, 91 5, 67, 70, 71, 90, 171 165, 166 62, 65, II**, 167 115, 116, 172, 176, 191 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 6, 46, 66, 73, 121, 134, 168, 192

PAGE 34

23 to this idea, inhibitors of ethylene synthesis such as aaiino-oxyacetic acid, AOA, should produce effects similiar 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 of the membrane is primarly affected by growth regulators. This concept is based on artifical 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.

PAGE 35

24 TABLE 2 Literature on Regulatory Activity of Plant Hormones System, Activity and References r 1 Energy Regulation Sugar uptake 8, 60, 70, 129, 175, 176 Hormonal Regulation Synergism Biosyntnesis Genetic 170, 171 Antagonism 34, 36, 58, 124, 136, 172, 191 Hormone Synthesis 11, 52, 151, 193 Induction 52, 54, 59, 150, 202 De No vo Synthesis 10, 36, 114, 124, 167, 203 Membrane Synthesis 51 Second Messanger 61, 81, 84, 92, 93, 152 Cell Division 83, 203

PAGE 36

CHAPTER II MATERIALS AND METHODS Ii££2£i5£§_2£_J>eed_peterioratigii Seed Materials Locally grown soybean seeds [Glycine max Merr. 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 tne 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. 25

PAGE 37

2 6 Antimicrobial Cnemicals Streptomycin 1 was tiie cnly antibiotic evaluated. Several fungicides were tested singly and in combination. These fungicides were notran, 2 captan, 3 maneb, 4 and benci»yl. s Benomyl was used alone and mixed with captan and maneb. Other powdered mixtures which were used were captan/botran, captan/maneb, and streptomycin/captan/botran. Several technigues were used for application. Chemicals were infiltrated into the seeds ny imbibing them with saturated solutions of fungicides and streptomycin (400 p. p.m.). This application was essentially a combination of priming with chemical incorporation. 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: 2 dioxy 2 (methylamino) alpha L glucopyranosyl (1 to 2) 5 deoxy 3 C formal alpha 2 lyxof uranosyl (1 to 4) N, N* (aminoiminomethyl) D streptamine 2 Botran structural name: 2,6 dichloro -4nitro-aniline 3 Captan structural name: 3a, 4, 7, 7a tetrahydro -2£ (tricaloromethyl) thio] -1Hisoindole 1,3 (2H) diona Maneb structural name: [[1, 2 ethanediylbis [carbamodithioato ] J (2-) J manganese 5 Benomyl structural name: [1 [ (butylamino) carbonyi] -1Hbenzimidazol -2yl ] carbamic acid methyl ester

PAGE 38

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. Hist ology of Aged See ds Control seed samples consisted of high vigor, Vicoja seeds and were ootained from the Agronomy Department's seed storage facility. Other control seeds were subsamples 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. Clf2i2ai_o£._£i§^BS_l?^l££§5S_l5.J^£I 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 fxxed overnight in cacodylate buttered

PAGE 39

23 giutaraldehyde (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 H sodium cacodylate buffer, pfl 7.2-7.5. Osmium tetroxide (1 percent) post-fixation also proceeded overnight in the cold. Embedding was preceded by an alconoi dehydration series, including an overnigxit exposure to uraayl 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. 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.

PAGE 40

29 E^ZSJQlpgy .of the Plasma Membrane Acc elerated A ging Just before the accelerated aging process, samples ot seed were coated tilth captan 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, tu and 50 hours, Hoisture 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 tae growth rate curve. After this treatment, the seeds were equilibrated to the original 11 percent moisture, by air-drying them for a wees under storage conditions. Controlling Hater .Uptake gate 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

PAGE 41

30 zero at the point of contact between the seed and paper. Each additional layer lowered that potential by reducing the availability of water. Imbibition 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. A utomatic Seed Anal yz e r 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 of the solution was then expressed in microamperes. Differences in conductivity , due to treatments, were relative to controls. It was necessary to

PAGE 42

31 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 tour layers o± 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 roothypocotyl 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 empnasize the short (24 hours) time reguired to fortify redried seed against soaking injury. Biochemistry _of _ Growth Regulators Age versus _AB A Experimental design. The design was (5 X 2) factorial, replicated twice. Five levels of aging were tested against two levels of abscisic acid (ABA) .

PAGE 43

32 Prepara tion of the ABA solution . Cistrans abscisic acid (molecular weight 264 and 95 percent purity) was the starting material, The tree 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 rlask, 200 my of sodium hydroxide was dissolved. Bacemic ABA (6b my) easily dissolved in the alkaline solution. Afterwards, sulfuric acid (0.01 K) 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 K) , or 2 milliliters of a sham, followed by 10 milliliters of water. After mixing, the ABA concentration was 4 X 10-* 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 of the seedlings in each plate and for the detached roothypocotyls. 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

PAGE 44

3J interaction of ABA and accelerated aging could be determined by using tne percentage of normalized growth with ABA to normalized growth without ASA, for each age category. It was necessary to use triple ratios to correct for seed sampling error, ABA growth reduction and for reduction in growth due to aging. Using this metood, 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. Age yersus_fusicoccin_and_gthylgne Preparation .of the f usicoccin so lut ions. The publication of Cocucci and Cocucci (39) was used as a reference for this proceedure. Fusicoccin (mol. wt. 670) was dissolved in ethanol (10 _1 P!) . 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 ethauol and then diluted to 38 ailliliters with deionized water. A working solution of 10 -5 a was prepared from this stock solution. Concentration at the rinal dilution was in the 10~ 6 H range. Dose response. Concentration of fusicoccin was 1,5 X 1Q& H at final dilution. This value was based on 10~ 6 K r a

PAGE 45

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. Tne 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, Experimental .design. 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 (ANQVA) may be applied to the data. Each of the eight treatments was run in triplicate and the experiment was repeated three times.

PAGE 46

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. Tne graph gives information in the tnird method (ANOVA) but statistical interpretation would not be as readily comprehended and would net be as appropriate for small differences due to treatments,

PAGE 47

CHAPTER III RESULTS Microflora a^j_5ged_ Deteriorat ion 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 growthAnother problem with coating seeds with streptomycin pcwder, 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 tne petri dish germination test, Histology of Aged Seeds Tetrazolium stain defined areas of dead cotyledon tissue present on deteriorated seeds (Figure 1). These white areas of dead tissue were symmetrical between cotyledons. Areas most prone to aging were grouped loosely into several patterns. Similiar 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 1960 which had a more uniform deterioration, 36

PAGE 48

37 wnnr Figure 1: Diff erentiai 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.

PAGE 49

is Ti§§ue_£xj:isJ^on_Exj>e^iffient 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 excisions 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 ol 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 See d Analy zer versus Tetrazolium Heaults 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 tne tetrazolium results, on an individual seed basis.

PAGE 50

3 9 Cytology of Plasma Mem^ranelnjury Figure 2 snows the plasma membrane retracted frosa the ceil wall. Ribosomes have escaped through the membrane tut larger organelles remain inside the membrane. Figure 3 confirms the particles as ribosomes because they form poly-rinosomes. The plasma membrane was also seen extending into the plasmodesmata in the cell walls. Electrolytes escape from the symplastic to the apcplastic compartment. Membrane rupture is likely the basis of the seed analyzer (page 30) .

PAGE 51

40 • » FT~v' >: *??% /#i fc ^5--,V Figure 2: Lcsa of Plasma Membrane Integrity. Excessively rapid water uptake burst the plasma membrane (i1) , allowing electrolytes to escape, Hibosomes (d) also escape but organelles remain as the membrane is plasmolysed,

PAGE 52

in « . ,.

PAGE 53

42 Physiology Effects of Rapid Hydration Water uptake was effectively slowed by additional layers of germination paper (Figure 4). Twenty milliliters of water coapletely saturated one layer of paper. Thus, one layer served as a ccntrol with the same matric potential as water. The uptake profiles were effectively modeled with a logarithmic linear transform corresponding to the rollowing general eguation: 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. Ef f ect_of_age_on_u£take_rate, Tne 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-

PAGE 54

43 5 10 15 20 HOURS OF IMBIBITION 25 Figure 4: Controlled Rate of Hater Uptake. Soybean seed with an initial moisture of 11 percent were imbibed in petri dishes (25 x 150 am) containing 20 milliliters of deionized water. Each plate also contained 20 seeds and one to five layers of germination paper. The graph symbol represents the number of paper disks per petri dish-

PAGE 55

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 6 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 of tne seeds increased, there was no apparent reduction in the time required to reach one-half maximum imbibition foe any of the five uptake rates tested. TABLE 3 Effect o£ Aging on Rate of Water Uptake 1 " Age

PAGE 56

45 Electrolyte Leakage. Electrolytes were measured from the papers used to imbibe seed, The data iu 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. Effect s 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 Taole 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.

PAGE 57

46 1.0 1.5 2.0 2.5 3.0 3.5 4.0 LflTERS OF PFIPEH Lc^cNO: flfl » . « v » < 30 Z J a 40 4.5 5.0 Figure 5: Electrolytes Lost During Water Uptake, The concentration of electrolytes recovered from imbibition papers is a function of seed age and the rate of water uptake. Symbols for each curve represent tue hours of accelerated aging, The rate of uptake is controlled by varying the layers of imbibition paper. Each reading represents a 4 milliliter sample taken from 60 milliliters of leached solution, replicated four times.

PAGE 58

47 TABLE 4 Vigor as a Function of Age and Bate of Hydration Layers of Paper AA 1 2 3 4 5 i" 30 66 45 55 90 66 40 15 13 35 62 43 50 02 14 28 35 44 AA = accelerated aged (hours) 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.

PAGE 59

i48 10 20 30 40 HOURS OF ACCELERATED AGING 50 Figure 6: Effect of Hydration Bate 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 five) .

PAGE 60

a 9 Effect ox Priming on Electrolyte 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 tc 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 of 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 ansorption. Results of the experiment with seed halves were similar to those with whole seeds (Figure 7) . An exception to this was taat the electrolyte loss was greater for half seeds tnan for waole, because of greater soaking injury.

PAGE 61

50 ACCELERATED AGING N Y PARTIAL PRIMING Figure 7: Effect of Priming en Electrolyte Leakage. A factorial experiment consisted of live levels of accelerated aging (0, 20, 30, 40 and 5 nours} with ana without partial priming. All seeds were equilibrated to 11 percent moisture during storage. ASA uata were collected after 9 nours of soaking and expressed as tne sum of the microamperes, resulting from ASA data, for eacu of the 20 seeds per 10 treatments.

PAGE 62

51 E ffec t of Agi ng _gn Prime d Se e d s Table 5 shows tnat 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 tne vitality of prised 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 ox Accelerated Aging on Primed Seeds Results Treatment

PAGE 63

52 Priming -Temperature pep en den ce 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 ayain 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 unpriced 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 c£ 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.

PAGE 64

53 TABLE 6 Priming-Dependence on Temperature Treatment Results temp aged unaged wet dry rank ASA S D %C fanJS ASA SD %C 4 4 1 65 8.6 111 1 66 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 l^I§£t_oi_Soakina_Tem£erature_on_Seed_Leakage Figure 8 contains the results froe a test of Simon's phase transition nypothesis. Microampere values represent the averages or 20 seeds. The top curve (H) shows the increase in electrolyte leakage with age, wnen seeds were soaked at ambient temperatures. The second curve (Hp) was constructed from the ASA values or 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

PAGE 65

[ 35^1 c 3 54 4 -Hp «Lp 9 12 15 13 2L 24 27 30 33 36 3^ ACCELERATED AGING Figure 8: affect of Low leaperature on Leakage. Soaking temperature (25 C) was represented by an (fl) , Seeds prised after aging then soaked at 25 C are represented by (Hp). Low temperature (4 C) soaking of five age categories, unprimed and primed after aging are denoted ay (L) dud (Lp) respectively,

PAGE 66

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 (H) were compared to the individual weights or the first 20 seeds from the (1) curve; the weight differences were hignly significant using the Student's "t" statistical critera. Seeds soaking for nine hours under refrigeration still had the wrinkled appearance indicating that imbibition was not complete. Seeds soaked at the higner temperature appeared smooth. Therefore, the lower temperature slowed the rate of water uptake. 5i ochgmistry of _ G r o w t h Regulators Age versu s A6 A The objective ot this experiment was to determine if ABAinduced 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 whxle gentle enough to minimize the effects on percent germination.

PAGE 67

56 ABA dose response. In order to determine the concentration ox 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 (103 H) 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 (H 2 ) equal to 0.99This analysis provided a new and effective mathematical model, judging from various alternatives described by Moore (97) . Isolati on of ABA and age interac tio n. In Table 7 are treatments and primary data. Koot-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 roothypocotyl was 22 to 23 percent of the total weight. After seven days of growth, the total weights (T) and roothypocotyl weights (RH) were measured. The mean refers to the average 01 the two (EH/I) 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

PAGE 68

57 -si LOGIT Y = -0.059 -2.09 LOG X R 2 = 0.99 0.0 LOG flBfl (ML) Figure 9: Dose Response to Abscisic Acid. Dose response tc ABA was modeled with a log-logit linear regressionEach observation contained 50 seeds. Total volume of liquid was constant (15 al) , waile rhe ABA dose varied rroiu to 8 ml. The conversion factor for milliliters added to the final fflolar concentration is (6.6 X 10 _s ). Data were recorded after live days of incubation at 21 C.

PAGE 69

TABLE 7 Interaction of Age and Abscisic Acid 1

PAGE 70

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.

PAGE 71

60 TABLE 8 Elfect of ABA on tne Vigor of Aged Seeds r TREATMENT ABA

PAGE 72

61 After five days of growth, seeds were weighed before and alter detaching the cotyledons and Table 9 contains the results, TABLE 9 Interaction of Fusicoccin and Ethephon OBS TRT REP AA FC El ROCI 1

PAGE 73

62 hairs were prominent. Seeds grown on two percent sucrose developed roots similar in appearance. Fusicoccin partially neutralized efhephon'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-hypocotyl to total weight («Tot) 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

PAGE 74

6 3 (Table 11). Mo significant interaction was attributed to fusicoccin, with age and ethylene. All other interactions and main effects were significant or highly significant, TABLE 11 Generalized Linear Regression Model 1 SOURCE

PAGE 75

64 C 0.8Cr\ o w T 0.75G ft A Q.6515 20 25 ACCELERATED AGEO (H! Figure 10: Aye versus Fusicoccin and Ethylene. Tne root mass of each observation (2 seeds) was recorded attar live days of growth at 25 C. Fusicoccin (F) , ethylene (E), and their combined interaction (T) were applied to ajed (34 h) and unaged seeds.

PAGE 76

65 CIIAPTES IV SUMMARY AND CONCLUSIONS iliS£5flora_of_Seeds Control of microflora 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. fi^tology_of_Seed_peterioration Symmetrical Necrosis Black spots are freguently observed on emerging seedlings. Tne nature of these necrotic lesions was, in general, symmetrical. This injury may be related to differential aging of seed tissue as described below. Tetrazolium Staining Pattern Use of this stain on naturally aged and accelerated aged seeds showed symmetrical differential aging of the cotyledon tissue. Tnis finding helps explain cue nature of seed aging at the histologic level. The source of most seed leakage is probably from the unstained areas. 65

PAGE 77

oft CztolOHY_oj _gldsaia Membrane .Injury Plasma Me mbr an e R upture Electron-micrographs 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. Physi ol ogy C2££E2ii§il_*II
PAGE 78

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 iow temperature was not sufficient for seed repair. Biocnemistry of Growtn Regulators A bsc isic Acid Stu dies Log-lo git 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. Antagqnistic_chemicals _.t.o_ABA. Ethylene was the most effective treatment for reversing exogenous ABA dormancy. Other effective antagonist included thiourea and fusicoccin. Ef f ect_on_resgirat ion_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_in teraction_*i th_ABA . The experiment was conducted with the hope that ABA could be used to improve the vigor of aged seeds by allowing the seed to channel metabolism into repair before starting the growth process. Dnf ortunatly, no improvement in seed vigor could be detected as a result of

PAGE 79

68 ABA treatment of aged seeds. Ethylene, on the other hand, did have a positive age interaction, F usic occin Studies Seedling morphology was affected by fusicoccin in the 10 -6 8 range and uigher. lith 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, iII£Otheses_Tested Homeostasis Several experiments in this study did support the homeostasis hypothesis (metabolically 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.

PAGE 80

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 phosphioipids over the C to 50 C temperature range.

PAGE 81

CHAPTER V DISCUSSION This study analyzed seed deterioration and repair using five approaches (micro! lora, 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 technigue (64) and was initaliy 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 memorane rupture improved seed vigor and vitality. Part of this improvement was explained by prevention of injury but repair processes were also evident. 7J

PAGE 82

71 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 microflora affect the vigor of seeds before accelerated aging. Another guestion 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.

PAGE 83

72 Althougn 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, H istol ogy .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 tne 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 ASK results rather than alternative concepts such as phase transitions of the phospholipids. 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 tae present study. Another inconsistency within these experiments should also be mentioned. Shen seeds were aged then primed and

PAGE 84

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 tetrazolium staining to iaitochondrial dehydrogenase enzymes, This raised anotaer 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_ot_Plasaa_Membrane_ln jury Knowledge ootained from cytological observations was integrated witn the tetrazolium experiments as well as nonvisual, physiological experiments dependent on the ASA, An explanation consistent with tne experiments of this study relies heavily on Villiars' (188) report. The description beginning on page 39 and the figures 2 and 3 are similiar to that of Villiers 1 . His description of ceil disruption was one accepted tor this experiment, A basic difference between the present observation and Villiers 1 experiment was taat he used soybean seeds rendered non?iable by accelerated aging. Supture of the plasma membrane

PAGE 85

74 by rapid water uptake probably occurred in both experimental situations, Villiers described collapse of the membrane followed by nydrolytic 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 guestions 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. Physiolo gy 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 technigue emphasized that the desired effect of slow hydration

PAGE 86

75 resulted in the avoidance of physical injury to the seed. This tecnnigue 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. Annealing 1 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 miyht 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. * Anneal to temper or toughen seeds against age induced predisposition to soaking injury.

PAGE 87

76 Secondly, chilling «as 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 hypotnesis was that seeds had lower

PAGE 88

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 tiypotliesis of Simon (158). Test_of_Hyp_o theses 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 memorane. Ihese 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. Anabolisa increased the vigor of tne 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.

PAGE 89

7a S ponta n eous repa ir. Simon's concept of plasma membrane permeaoility has stimulated seed research for ten years (158). Experimental evidence supporting this concept includes that or 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 fee an increase in electrolytes at the lower temperature. The rationale tor these predicted results was that the lower temperature would slow the pnase transition and hold the membrane in the permeable formation. Simon's pubxication (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 phospholipids formed the eguivalent of micelles, although no microscopy supports this conjecture. His scnolarly and stimulating report was based on a literature survey only, unsupported by experiments directed at determining its validity.

PAGE 90

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 subseguently 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, jioche mi st ry of G to w th __R eg ul at or s ABA versus Accelera te d Agin g 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 probanly reverse some of the eftects of aging in both treatments. Therefore, although ABA did not improve the "vigor" of aged seeds, this experiment does not contradict the homeostasis hypothesis.

PAGE 91

80 Interaction of „.Fusicoccin and Bthephon The antagonism between f usicoccin 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 nave a negative effect by exceeding the limits of productive activity. Suggestions for Future Research This work tested an existing aypothesis 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 reguired for the reversal of aging effects by priming.

PAGE 92

APPENDIX SUPPLEMENTARY EXPERIMENTS Microflora and Seed Studies Several interesting observations on microflora were obtained in some cases from formal experiments and on other occations xnformaliy during work directed toward unrelated problems. Fungal_Survival 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 hign 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 after 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. 81

PAGE 93

82 C ontrol of Fungal Growth 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 growtn. Priming seeds at 15 C without fungicides resulted in severe fungal growth. Fungal activity in a petri disa 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 nelow 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.

PAGE 94

83 Histology of Seed Deterioration k 2 cat i o n_ o f _S e e d_ F u n g i The location of most ox the fungi was probably limited to the seedcoat. Tnese fungi were probably saprophytes. Dusting the seeds with a dry powder prevented signs of fungal 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 iaen 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 aule to penetrate these cells as they lost their impermeability.

PAGE 95

84 Tetrazolium Staining Studies The dead areas en deteriorated seeds say be related to microflora. The relationship or fungus to dead tissue is not understood at this time. For instance, does aging kill parts of tae 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 cot 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 tne 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

PAGE 96

do scalpel. For the stain to penetrate the entire seed, it is suggested that the seeds be split between the cotyledons tnen iabibed directly in a tetrazolium solution. Cytology of the Plasma Membrane The cause of seed leakage was the guestion addressed by several preliminary experiments, suggested by current concepts, that aging causes deterioration of the membranes whicn 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 memnrane 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 oecause 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 twodimensional electrophoresis following ultracentrif uge purification. Aging was expected to produce an alteration in the protein composition. Also since cultivars differ in their aging rates, peraaps protein composition could account for tais difference.

PAGE 97

86 Membrane enrichment was accomplished by use of the discontinuous density gradient ultracentrif ugation techniyue. Determination of purity was based on electron microscopy. Plasma membrane was distinguished from other membrane material by use of the Ecland stain (142). Memnrane preparations were successfully extracted first from embryos and then from bean sprouts (Figure 11). Even thougn sufficient membrane quantity was obtained, the 70 percent purity was insufficient for futher 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 concept of homeostasis or anabolic repair in pregerminated seeds.

PAGE 98

t*% * $'*% 4
PAGE 99

a 6 P rim ing E xper i men ts At least five points can be Bade which supplement ar. understanding of the priming effects based on physiology experiments. Tnese 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] Bather 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 priming effect. The relationship between maximum seed moisture used tor 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

PAGE 100

89 first question is wnat determines the maximum priming effect onca 60 percent moisture is attained? For instance, if 60 percent moisture produced the Maximum priming effect then the line would be asyiatotic 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 propcrtionly less effective. Two separate experiments failed to ontain sufficiently consistent data to answer these questions with certainty. The limitation was finding a technique which gave the desired results. The first technigue 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 tae seeds had germinated, it was possible to maintain vitality after redrying the seeds. The sprouted seeds survived, grew secondary roots, and demonstrated geotropic grcwth.

PAGE 101

90 70 65 60 M • 55 c ^ R ft 50 P s 45 40 35 ACCELERATED AGED R=-0.77 Y= 750.49 X N NATURAL AGED R=-0.89 Y = 55-0.35X N=ll 10 20 30 40 50 % MOISTURE 60 Figure 12: Seed Moisture Effect on Priming. Seed analyzer results (Y axis) are a function of the moisture used to prime the seeds. Each point represents average test results of 20 seeds, replicated twice.

PAGE 102

9 1 Test of p riming hypotheses, Eriming reduced the leakage of seeds in experiments which have been repeated regularly. Tne explanation consistent with contemporary thought is that the plasaa membrane is repaired, probably by metabolic processes. This explanation is satisfactory for all experiments which have been conducted to test this concept, It may not ultimately be true but it is a useful construct tor conducting future experiments and does explain the results obtained thus far. Other experiments tested the alternative hypotheses that if priming followed by redrying reduced the rate or water uptake, then the reduction in electrolyte leakage may result not from repair of the plasma membrane but from reduced injury. Table 12 contains the data from one ox these experiments and is the basis for concluding that priming did not affect the rate of uptake (imbibition time) in this test. In order to determine if electrolyte leakage was reduced by reducing the rate of water uptake, two sources of primed seeds (Prime 1, Prime 2) were compared to three sources of unprimed seeds (Check 1, Check 2 and Check 3). The samples were imbibed on one layer of germination paper saturated with 20 milliliters of water. The weights of each sample were recorded four times during the eight hours of uptake. Priming did not affect the rate of uptake in tais test. Another hypothesis concerning the mechanism cf priming was tnat residual moisture after priming protected the seeds

PAGE 103

92 TABLE 12 Effect of Priming on the Bate of Imbibition Uptake Prime 1 Prime 2 Check 1 Check 2 Check 3 rh a 3 a a ^ 4.95 5.00 4.95 5.07 4.97 1 5.76 5-55 5.70 6.22 5.73 4 8.72 7.77 8.05 8.33 8.15 8 10.69 10.26 10.62 10.66 10.59 r Uptake imbibition interval (hours) from soakinj (and chilling) injury. The first experiment was designed to measure the leakage of seeds soaked in cold water as previously described. The second approach was to measure the residual moisture of the seeds after redrying. The thinking behind this experiment was that if even a small residual amount or moisture remained, then leakage of redried seeds may leak less than that of controls. Careful moisture measurements, made on oven drying control seeds and redried seeds at 106 C for 16 hours, showed no difference in moisture, even though replicates of these samples were very different on the ASA test. The oven method was used in the previous experiment to compare the moisture content of two seed treatments. Another method used routinely was to start with 5.0 grams of seeds, prime, tnen redry to 5.0 grams. One concern with

PAGE 104

93 this method was that respiration ot the hydrated seeds used stored reserves. It was found that seeds redry to iess than their original weignt. About three percent of the seed dry weight was consumed during priming. Another experiment compared seeds primed at 15 C and 25 C. Although both priming temperatures reduced ASA values to an equal extent, germination and growth was better after the 25 C priming treatment. This again suggests the importance of priming to the entire seed, not just to the plasma memcrane. Pregermination events evidently not only restore the membranes but an increase in seed vigor becomes apparent when primed seeds are compared to unprimed controls. P£iS±iiii_2sLS_i3££S§se_3ermination_rate. When primed and unprimed seeds were slowly nydrated tor two days on five layers of germination paper, neither group had started to germinate until an additional 20 milliliters of water was added to the 20 milliliters already in each petri dish. Germination (broken seedcoat was the criterion) began the same day for the previously primed group of seeds. The following day primed seeds had an average of 21 percent germination whereas unprimed seeds had none. The second day almost all of the primed seeds had germinated whereas the average for tae unprimed group was 16 percent. This experiment indicated that priming did increase the rate of germination even though the seeds had been redried. Priming and redrying were considered as separate components of the

PAGE 105

94 partial priming technique. Page 31 contains descriptions of tnese procedures. Similar experiments have indicated that germination and growth rates of seedlings increase if they are primed without redrying. This event may, however, depend on the moisture content of the seeds. For instance, seeds imbibed to 50 percent moisture may not grow more rapidly when the moisture is allowed to increase to the 60 percent required for germination. It may be possible to prevent cell expansion with moisture less than 60 percent. Cell division may have another limiting value. Metabolism would take place at still lower moisture. Therefore, it may be possible to use seed moisture as a means to separate each of these three processes. Deterioration_of_Perf or mane e_is_ Permanent The reduction in growth rate of aged seeds was a permanent condition. When aged seeds were grown in a screen-house, most failed to germinate and those which did remained dwarfed. These stunted plants were grown along with unaged controls. Plants from aged seeds flowered with the control plants but they were smaller and had fewer and smaller root nodules. Part of the explanation for this stunting may be related to chromosome or genetic damage (145). However, if mechanical damage causes a similar growtn reduction, then genetic damage does not offer a complete explanation (45).

PAGE 106

95 E ffects of Monovalent and Divalent Cati ons Several tests were conducted to determine if salts, such as calcium chloride or potassium phosphate, affected the growta of seeds in otherwise standard germination tests. Concentrations were in the millimolar range for the salts and deionized water was the control. £ach root length was measured after four days of growth. Root lengths were ranked and then plotted in ascending order. The growth curve was sigmoid for each seed treatment which reflected the expected normal distribution in growth rates among the seeds. Overlays or these plots provided a sensitive means of visualizing small differences among the treatments. Water aloue was the least favorable medium for germination, but txiese differences were not large enough to justify routine use of these salts in germination tests. A parenthetical comment is related to the distribution of growth among seedlings. Both germination and growth are described by sigmoid curves. Statisticians transform germination data to linearize the relationship between points of comparison. Growth data probably should be linearized before statistical analysis although this is not the practice. Before routine transformation, however, it should be shown that enough curvilinear relationsnip exists to justify the conversion. Effect of calcium on priming. Further improvement in priming was sought tnrough which fortified the water with 2

PAGE 107

9n X 10 -3 H calciua chloride solution as the iBediuB. No difficulties were encountered due to the additional electrolyte but no benefit of this treatment could be detected. The calciua concentration tested was one based on the literature and since the effect was so negligible, no additional experiments were conducted. Vacjium_Drxin^_to_Prevent_Seed_peteri oration Seed moisture on a fresh weight basis could be reduced from eleven percent to three percent by reducing the air pressure to one-third atmosphere for two days and the low moisture level retained by hermetically sealing. This approach may have economic advantages over continuous refrigeration as a means of maintaining seed performance. No adverse effect was detected from vacuum treatment. A very strong vacuum was also used to dry seeds without loss of vitality. Freeze drying seeds has been reported by Woodstock (199), but vacuum drying without freezing would have obvious advantages. Stimulation_of _^o wth_B_y_Slightlx_&gifig_Seeds According to Woodstock and Tao (200), slightly aged seeds demonstrated improved performance. Also Perl in 1981 (122) reported a similar observation. The author conducted at least two experiments in an attempt to confirm these two reports.

PAGE 108

9 7 In the first experiment, "dry" seeds seeds were accelerated aged. Seeds, equilibrated to 11 percent moisture, were incubated at 4 1 C in a sealed jar. Samples of 20 seeds, aged 11 days in this manner (5 replicates), were compared to unaged seeds (4 replicates) . After four days of growth at 25 C, no differences could be detected in total root-hypocotyl weights of the aged and unaged seeds by Student's "t" test: t = [X1-X2]/ SQET [ (S1VN1)* (S22/N2) J The symbols XI, X2 are the means, 31, S2 are the standard deviations. The number of replicates are N1 and N2. In the second experiment, seeds were aged in the conventional manner on a wire screen over water at 41 C. Aging times in hours were 0, 6,5, 8.5, 22.0, and 28.5. The seeds were not redried before the germination test. Again growth was not improved by aging in any of the treatments. There have been occasions in this study when slight aging did appear to stimulate the growth of seeds. This occurred in one instance when seeds were added to petri dishes containing two layers of germination paper and various amounts of water (1 through 16 milliliters). During 20 hours of aging at 4 1 C, growth was improved by the addition of some water. Up to two milliliters of water improved subsequent growth. Greater amounts were detrimental. An explanation for taese apparent contradictious can te advanced. In Perl's (122) work., the conditions were in

PAGE 109

9 8 effect priming the seeds with water vapor which resulted in an improvement in vigor. Aging does not improve performance, but since priming does, and may occur as a result of moisture at the beginning of aging, the two effects may have become confounded. R ates of Uptake and Leakage Compared Three considerations were evident from specific experiments directed toward the following questions: (1) For a given sample of seeds, does the loss of electrolytes depend directly on the rate of water uptake? (2) What is the optimum soaking interval for the automatic seed analyzer? (3) When does the plasma membrane depend on respiration for active transport of ions across the plasma membrane? W ate r Dptak e Ra te. The rate of uptake was described in the results chapter of this dissertation as a log-linear function between seed moisture and the log of time. Leopold recently described the rate of seed volumetric changes with a log-linear function (87). -In [ (m-a)/m] = kt The maximum seed volume is (m) , and (a) is the actual volume at any point in time. The proportionality constant, (k) , indicates that seed volume increases at a constant rate which is proportional to the remaining possible expansion. Both Leopold's equation for seed volume and the equation

PAGE 110

99 used in this study are applicable to water uptake data. Moreover, electrolyte loss conforms to similar profiles (99, 149) . Tcis suggests that deformation due to unequal volume changes throughout the seed may cause leakage from tissue injury. Figure 13 shows that the rate of water uptake is paralleled oy the accumulation of electrolytes in the steep solution. Leakage and uptake rate was correlated during the first six hours. One of the factors influencing the rate of water uptake is viscosity (184). Ihe viscosity of PEG, not its osmotic potential, was likely responsible for Woodstock and Tao's findings (200). Probably, the viscosity of cold water reduces the leakage of soaking seeds (Figure 8). Optimized Soaking Interval for the ASA. T he manufacturer's recommended 24 hour soaking interval was not sensitive to differences in seed leakage between samples. This was apparent at the eight to twelve hour interval. For this reason, the shorter period was used for much of tnis work. Effect of respiration on seed_ieaka_ge. After reading Simon's review (158), it became apparent that lack of oxygen (anaerobiosis) could result in an increase in aembrane permeability due to insufficient active transport. Consideration of this possibility along with another report by Hullett and Considine (99) , who showed that electrolyte

PAGE 111

100 HOURS OF SOAKING Figure 13: Hates of Hydration versus Leakage. Both the rate of nydration (W) and electrolyte leakage (E) conform to a log-linear relationship with time. This holds true wnen the seeds are imbibed on paper as well as when they are soaked in water.

PAGE 112

101 loss increased with the depth of the seeds in water, it would explain these results. This would influence the ASA so seed leakage was partitioned into a dependence on rate of water uptake and air dependence. An experiaent (Figure 14) did give evidence for anaerobiosis in the ASA test, but this type of leakage does not contribute greatly to the ASA test results in less than 24 hours of soaking.

PAGE 113

102 p E fl c uoE s T C 30H R N G E 20-201 LEGEND: COVERED 22.5 27.5 32.5

PAGE 114

103 ML2ch§mistrx_of_Growth_R emulators Work which eventually evolved to partial priming, began with the use of abscisic acid to induce dormancy in seeds. The idea behind this work was that dormancy would alter the effects ex aging on seeds by allowing homeostatic processes to repair age-related damage. Another impetus to use growth regulators to study the effects of aging on seeds came from the work of Petruzzelli et al. (123) . Fusicoccin as well as monovalent cations were siiowu to improve the germination of low vigor wheat. Dr. west was able to obtain a 25 milligram sample of fusicoccin from Dr. Marre* so we had an opportunity to incorporate the use of this chemical into studies concerned with stimulation of soybean seeds. Considered in the subsections below are respiration studies, ABA dormancy, and the stimulation studies involving fusicoccin and ethylene. Respiration of dormant seeds was measured with an oxygen specific electrode (Yellow Springs Instrument Co., Inc.). This polarographic oxygen sensor responds to the oxygen dissolved in water. Respiration of seed samples were measured during a 10 minute interval as submerged seeds removed oxygen from the medium. Between measurements, seeds were not submerged so respiration could resume normally. i Dr. E.E. Marre', Milan, Italy

PAGE 115

104 Growth regulator experiments involved inducing dormancy and reversing dormancy with other growth regulators such as fusicoccin and ethylene. The effects of biosynthetic inhibitors, sucrose, and selected salts were also tested in an effort to enhance the mode of action for growth regulators and to gain insight into their mechanisms of action, I§§£i£§tioji_ExEe£i^ents Respiration was monitored during imbicition and early germination (YSI oxygen electrode). The effects of accelerated ajiny on seeds was also measured with the same methods. Seeds which were dormant after abscisic acid treatment were tested to determine if their respiration was sufficient to support active metabolic repair processes. Respiration versus imbibition. The increase in seed moisture corresponded tc an increase in seed respiration until the seed reached 60 percent moisture. Cxygeu uptake was 0.2 1 microliters per minute per seed, after six hours of imbibition. Twenty nours later, just prior to radicle emergence, respiration was doubled to 0.46 microliters per minute per seed. These results confirmed other studies (26,98) and served to validate this methodology. In summary, respiration increased as a result of seed hydration until saturation. Afterwards, there was a plateau in water uptake and

PAGE 116

105 respiration for almost 20 hours. As the radicle began to emerge, there was an increase in respiration, probably supplying energy for growth. is §£i£§:iioS._of _i£S d_se eds . Respiration was reduced by aging. This was not a new observation (197), but the work provided experience and confidence in the methods. Respira tion of pri med se eds. There was no improvement attributable tc priming within the first 24 hours of hydration. This may signify that dry seeds which had been primed had a similar oxygen uptake versus time profile as unprimed seeds. It does seem likely that, at some later stage during this development, the respiration would exceed the unprimed controls it there is a concomitant increase in germination rate or growth rate due to priming. ifi§cisic_Acid_Dormanc^_InJuctio^ A new model fcr the dose response was established with the log-logit linear transform (page 56). Cnce the optimum range was established for inducing dormancy, several methods were used which would reverse the effects of abscisic acid. Other growth regulators which were effective were fusicoccin, ethylene (as etnepnon) and thiourea. Non-growth regulators explored included hydrogen peroxide, water eluticn, sucrose and selected nitrogen, phosphorous and potassium (NPK) salts. Other treatments tried to remove

PAGE 117

106 abscisic acid ay leaching seeds with water and by oxidation of ABA with hydrogen peroxide. Several of these treatments demonstrated a positive effect. No single treatment was as dramatic as the use of hormones, however. Reversal of ABA dormancy was spontaneous when the dose was limited. Low doses of abscisic acid delayed germination and slowed growth. Restriction of dose rates was a practical way to avoid the necessity of dormancy reversal. Another finding in the abscisic acid experiments which may be new, was its influence on seed respiration. Seeds which had been held dormant tor a week had maintained respiration at the "plateau rate." Prevention of germination may have prevented increased respiration or vice versa . A a£§.22Bi§ tic_ Experiments £a§i£2£cin_antagcnism. Factorial experiments used three concentrations of A3A versus three concentrations of fusicoccin. This design was used to obtain data for a statistical (SAS) procedure which optimizes the data on the basis of a three dimensional plot. The program supplies dummy variables, therefore, only nine treatments are reguired. Fusicoccin in tne 10~* M concentration range did antagonize the effects of ABA in tne 10~* M range. This was confirmed by several related experiments.

PAGE 118

107 II*i2U£§§:_§:S£i:12I*isfi' Experiments with the same 3 2 design, demonstrated reversal of abscisic acid dormancy (1.3 X 102 «) by thiourea in the (3.5 X 10" 2 M) range. Ethylene_antag_onism. The design used to optimize the ethylene concentration (ethephon) was different from the previous optimization procedures. The maximum concentration of ethylene which reversed the effects of ABA without inducing dormancy was determined and used in subsequent experiments. Serial dilutions of ethephon added to AEA dormant seeds promoted germination. Antagonism of 0.25 X 10~ 3 H abscisic acid was accomplished with (1.7 X 10~ 6 H) ethephon. Mechanisms of ABA activity. Reversal of ABA dormancy by fusicoccin suggested that the mechanism of action tor ABA was linked to the active transport by the plasma memnrane. The basis of this suggestion was that fusicoccin is thought to have this single mechanism of action (93). Fusicoccin_Stimulation_of_Seeds An overall objective of experiments with fusicoccin was to find seed treatments which would improve the performance of low vigor seeds. Also fusicoccin was combined with sucrose and/or salts with the purpose of finding a synergistic response, Other combinations included fusicoccin and growth regulators such as ethylene, as previously described in the results chapter (page 60).

PAGE 119

108 Water potential. Germination paper was used to control the water availability to seeds germinating on a solution of fusicoccin. Three levels of water potential were combined with three concentrations of fusicoccin to determine if fusicoccin was able to promote germination under conditions of restricted water availability. Fusicoccin in the 10~ 5 B range did stimulate germination or seeds growing in petri dishes when the water potential was reduced witu germination paper. These results indicated that fusicoccin can increase the turgor of germinating seeds. A similar experiment attempted to evaluate the effect of fusicoccin on turgor by stressing the seeds with 30 percent polyetnylene glycol. This single concentration of osmcticum was too concentrated to permit germination even when the seeds were stimulated with fusicoccin. Several concentrations of osmoticum should be used to bracket the degree of stimulation due to the growth regulator. Mode of action. Fusicoccin did alter seedling morphology. Concentrations in the 10~ 5 M range tended to produce short hypocotyls and stimulated secondary root formation. There was also a stimulation of chlorophyll development of seedlings in a manner characteristic of an auxin. But, unlike auxins, it is doubtful that fusicoccin acted through stimulation of ethylene biosynthesis.

PAGE 120

109 Ethylene Biosyuthesis_apd_Stifflulation Experiments were conducted with aiaino-oxyacetic (AOA) acid and cobalt chloride to determine if fusicoccin stimulated endogenous ethylene production. The ACA (10~ 3 M) stunted seedling growth whereas the cobalt chloride (10 -3 H) showed little effect. Exogeneous ethylene in the form of ethephon did not reverse the ACA retardation. This concentration of AOA was probably excessive. It was not determined whether fusicoccin stimulated ethylene synthesis by use of AOA or cobalt ions. This was probably because the concentrations suggested by a review of the literature were inappropriate for this application. Amino-oxyacetic acid stunted growth so severely that nc meaningful results were obtained. In contrast, cobalt in millimolar concentrations was ineffective in altering a growth response. Optimization of concentrations might have solved these problems. Another approach, based on interaction of ethylene and fusicoccin, described in Chapter III on results, indicated tnat fusicoccin does not stimulate ethylene biosynthesis. Hecnanism of .action. Both ethylene and fusicoccin break ABA dormancy. If fusicoccin stimulated endogenous ethylene syntaesis, then this would indicate that ethylene and not fusicoccin antagonizes ABA. Dormancy induction/reversal may be related to membrane permeability. The energy economy of the cell is dependent

PAGE 121

110 on the semipermeability of the membranes. Perturbation of this ability to produce chemical concentration gradients across aembranes would reduce the rate of energy production to meet the demands for cell expansion and growth. Use of cell-free systems may avoid the inherent problem in isolating cause and effect relationships. ftode o f action . Both sucrose and ethylene stimulated the growth of root-hairs which suggest part of the ethylene response may be associated with nobilization of sugar with in tne seedling. Ihis was observed in isolated embryos as well as whole seeds.

PAGE 122

LITER A TUBE CITED 1. Abdul-Baki AA 1980 Biochemical aspects of seed vigor. HortScience 15: 765-771 2. Abdul-Baki AA, JD Anderson 1972 Physiological and biochemical aeterioraticn of seeds. In TT Kozlowski, ed, Seed Biology, Vol 2, Academic Press, New Ycrk pp 238-315 3. Abdul-Baki AA, GB Chandra 1977 Effects of rapid aging on nucleic acid and protein synthesis by soybean embryonic axis during germination. Seed Sci Technol 5:689-698 4. Adegbuyi E, SB Cooper, B Don 1981 Osmotic priming of some herbage grass seed using polyethylene glycol (PEG). Seed Sci Technol 9: 867-878 5. Adkins Si, JD Boss 1981 Studies in wild oat seed dormancy. Plant Physiol 67:353-362 6. Aducci P, G Crcsetti, B Federico A Ballio 1980 Fusicoccin receptors. Evidence for endogenous ligand. Planta 148:208-210 7. Akalehiywot I, Bewley JD 1977 Promotion and syncronization of cereal grain germination by osmotic pretreatment with polyethylene glycol. J Agric Sci 89:503-506 8. Aldasoro JJ, A Jiatilla, G Nicola 1980 Effect of ABA, Fusicoccin, K+, and glucose uptake in chickfea seeds at different temperatures. Physiol Plant 53:139-145 9. Anderson JD, JE Baker, K Horthington 1970 Ultrastructural changes of embryos of wheat infected witu storage fungi. Plant Physiol 46:857-859 10. Apelbaum A, SF Yang 1981 Biosynthesis of stress ethylene induction by water deficit. Plant Physiol 68:594-596 11. Baker JE, JD Anderson, DC Adams, A Apelbaum 1982 Biosynthesis of ethylene from methionine in aminoethoxyvinylglycineresistant avacado tissue. Plant Physiol 69: 93-97 111

PAGE 123

112 12. Ballarin-Denti A, M Cocucci 1979 Effects of absicisic acid and fusicoccin on the transmembrane potential during the early phases of germination on radish (Rapaanus sativus L) seeds. Planta 146: 19-23 13. Ballio A 1977 Fusicoccin: structure activity relationships. In E Marre» and CCiferri, eds, Regulation of Cell Membrane Activities in Plants, Nortn-Holand, Amsterdam, pp 217-223 14. Bass LN 1977 Paysiolcg ical and other aspects of seed preservation. In I Rubenstein, RL Phillips, CE Green, and GB Gengenbach, eds. The Plant Seed: Development, Preservation, and Germination, Academic Press, New York, pp 145-170 15. Bass LN, DC Clark, E James 1962 Vacuum and inert-gas storage of safflower and sesame seeds. Crop Sci 3: 237-240 16. Bass Ll», DC Clark, E James 1963 Vacuum and inert-gas storage of crimson clover and sorghum seeds. Crop Sci 3: U25-428 17. Basu EM, N Dhar 1979 Seed treatment for maintaining vigor, viability and productivity of sugar beet (Beta vulgaris) . Seed Sci Technol 7: 225-233 18. Becwac Hfi, PC Stanwood, EE Boos 1982 Dehydration effects on imbibitional leakage from desiccationsensitive seeds. Plant Physiol 69: 1132-1135 19. Berjak P, TA Villiers 1970 Ageing in plant embryos. I. The establishment of tne seguence or development and senescence in the root cap during germination. New Phytol 69: 929-938 20. Berjak P, TA Villiers 1972 Ageing in plant embryos. II. Age-induced damage and its repair during early germination. New Phytol 7 1: 135-144 21. Berjak P, TA Villiers 1972 Ageing in plant embryos. III. Acceleration of senescence following artificial ageing treatment. New Phytol 71: 513-518 22. Berjak P, TA Villiers 1972 Ageing in plant embryos. IV. Loss of regulatory control in aged embryos. New Phytol 71: 1069-1074 23. Bewley JD 1979 Dormancy breaking by hormones and other chemicals — action at the molecular level. In I Rubenstein, RL Phillips, C£ Green, and BG Grengenbach, eds. The Plant Seed: Development, Preservation, and Germination, Academic Press, New York, pp 219-239

PAGE 124

113 24. Bewley JD 1979 Physiological aspects of desiccation tolerance, Ann fiev Plant Physiol 30: 195-238 25. Bewley JD , 8 Black 1978 Physiology and biochemistry of seeds in relation tc germination. Vol I. Developmant, Germination, and Growth. SpringerVerlag, Berlin 26. Bewley JD, M Black 1982 Physiology and Biochemistry of Seeds in Relation tc Germination. Vol 2. Viability, Dormancy, and Environmental Control. Springer-Verlag, Berlin 27. Bittar EE 1980 Membrane Structure and Function. Vol 1, 2, and 3. John Wiley and Sons, New York 28. Bramlage wJ, AC Leopold, DJ Parrish 1978 Chilling stress to soybeans during imbibition. Plant Physiol 6 1: 525-529 29. Brandts JF 1967 Heat effects on proteins and enzymes, In AA Bose, ed, ThermoBiolog y. Academic Press, London, pp 25-70 30. Buttrose MS 1973 Bapid water uptake and structural changes in imbibing seed tissues, Protoplasraa 77: 111-122 3 1. Cartledge JL , LV Barton, AF Blakeslee 1936 Heat and moisture as factors in the increased mutation rate from Datu ra seeds. Proc Am Philos Soc 76: 663-685 32. Chabot JF , AC Leopold 1982 Ultrastruct ural changes in membranes with hydration in soybean seeds. Amer J Bot 69: 623-633 33. Chastain CJ, Jfi Hanson 1981 Proton efflux from corn roots induced by t riprcpyltin. Plant Physiol 68: 981-982 34. Chastain CJ, JB Hanson 1982 Control of proton efflux from corn root tissue by an injury-sensing mechanism. Plant Science Letters 24: 97-104 35. Chippindale HG 1934 The effect of soaking in water on the 'seeds' of some Graminae. Annals of Applied Biology 23: 225-232 36. Chrispeels MJ, JE Varner 1967 Hormonal control of enzyme synthesis: on the mode of abscision on aleurone layers of barley. Plant Physiol 42: 1008-1016

PAGE 125

114 37. Caristen CM 1972 Microflora and seed deterioration. In EH Roberts, ed, Viability of Seeds, Chapman and Hall, London, pp 59-93 38. Clowes FAL 1963 The guiescent centre in aeristeas and its behavior after irradiation. Brookhaven Syap Biol 16; 46 39. Cocucci 5, « Cocucci 1977 Effect of ABA, GA, and FC on the development of potassium uptake in germinating radish seeds. Plant Science Letters 10: 85-95 40. Copeland LO 1976 Principles c£ seed science and technology. Burgess Publishing Company, Minneapolis 41. Crevecoeur H, B Deitour, fi Bronchar 1976 Cytological study on Mater stress during germination of Zea mays. Planta 132: 31-41 42. Delouche JC, CC Baskin 1973 Accelerated aging techniques for predicting the relative storability of seed lots. Seed Science Technol 1: 427-452 43. Delouche JC 1980 Environmental effects on seed development and seed guality. HortScience 15; 775-780 44. Delouche JC, KT Nouyen 1964 Methods for overcoming seed doraancy in rice, Proc Assoc Offic Seed Anal 54:41-49 45. Dickson BH, MA Boettger 1976 Factors associated with resistance to mechanical daaage in snap beans (Phaseglus vulgaris L) 101:541-544 46. Dohraana 0, k Hertel, P Pesci, SM Coccucci, E Marre 1 , G Handazzo, A Ballio 1977 Localization of in vitro binding of the fungal toxin fusicoccin to plasaaaembrane-rich fractions from corn coleoptiles. Plant Sci Lett 9: 291-299 47. Duff as C, C Slaughter 1980 Seeds and Their Uses. John Wiley and Sons, New York 48. Duke SH, G Kakefuda 1981 Bole of the testa in preventing cellular rupture during imbibition of legume seeds. Plant Physiol 67: 449-456 49. Duke SH, G Kakefuda, TM Harvey 1983 Differential leakage of intra-cellular substance from imbibing soybean seeds. Plant Physiol 72: 919-924 50. Dunn BL, BL Obendorf, DJ Paclillo Jr 1980 Imbibitional surface damage in isolated hypocotyl-root axes of soybean. Plant Physiol 65: S-139

PAGE 126

115 51. Elliott DC 1982 Inhibition of cytokinin action and of heat aging induced potential for cytokinin action by inhibitors of membrane synthesis and function, Plant Physiol 69: 1169-1172 52. Franklin D, P« Morgan 1978 fiapid production of auxin induced ethylene. Plant Physiol 62: 161-162 53. Gicaner T, J Veleminsky, V Pokorny 1977 Changes in the yield of genetic effects and DNA single-stranded breaks during storage of barley seeds after treatment vita diethyl sulrate. Environ Expt Bot 17: 63-67 54. Goeschl JD, HK Pratt, 8A Bonner 1967 An effect of light on the production of ethylene and growth of the plumular portion of etiolated pea seedlings. Plant Physiol 42: 1077-1080 55. Grabe DF 1970 Tetrazolium Testing Handbook, AOSA Handbook on Seed Testing, No. 29 56. Gracen VE Jr 1970 Physiological and Ultrastructural Studies of Oat Membranes Treated with Helminthos^orium XiSISti^ toxin. PhD Diss, University of Florida, Gainesville 57. Haber AH, HJ Luippold 1960 Separation of mechanisms initiating cell division and cell expansion in lettuce seed germination. Plant Paysiol 35: 168-173 58. Halloin JM 1976 Inhibition of cottonseed germination with abscisic acid and its reversal. Plant Physiol 57: 454-455 59. Hendricks SB, HB Taylorson 1978 Dependence of phytochrome action in seeds on membrane organization. Plant Physiol 61: 17-19 60. Hanson AD, H Kende 1975 Ethy iene-ennanced ion and sucrose efflux in morning glory tissue. Plant Physiol 55: 663-669 6 1. Banson J3, A J Trewavas 1982 Eegulation of plant cell growtn: the changing perspective. New Phytol 90: 1-18 62. aelleburst JA 1976 Osmoregulation. Ann Rev Plant Physiol 27: 485-505 63. Heydecker W 1977 Symposium on seed problems in horticulture: the search tor practical solutions. Acta Horticulturae

PAGE 127

116 64. Heydecker W # P Coolbear 1977 Seed treatment for improved performance: survey and attempted progress, Seed Sci Technol 1: 427-452 65. Heyser JW, HI Nabors 1981 Growth, water content, and solute accumulation of two tobacco cell lines cultured on sodium chloride, dextran, and polyethylene glycol, riant Physiol 68: 1454-1459 66. Hockling IJ, J Clapham, KJ Cattele 1978 Abscisic acid binding to subcellular fractions from leaves of Vicia faba. Planta 138: 303-304 67. Holfman HE, JF Fu, and SF Yang 1982 Identification and metabolism or 1-malcnylaminocyclopropane-1-carboxylic acid (MACC) in germinating peanut seeds. Plant Physiol 69: S-136 68. Jensen MA 1981 Bioiogy of the Cell. Wadsworth Publishing Co. lac. Belmont, Calif. 69. Justice OL, LN Bass 1978 Principles and Practices of Seed Storage. USDA Handbook No. 506 71. Keen AB, J Van Staden 1982 The effect of ethylene on_ dormant Sicinodendron rautanentii seeds. Plant Physiol 69: S-3 72. Keenan TH, El Leonard, TK Hodges 1973 Lipid composition of plasma membranes from hvena. sativa roots. Cytofcios 7: 103-112 73. Keith B, LM Srivastava 1980 In Vitro binding of gibberellin A in dwarf pea epicotyls. Plant Physiol bo: 962-967 74. Khan AA 1977 The Physiology and Eiochemistry of Seed Dormancy and Germination. North-Holland Publishing Company, Amsterdam 75. Khan AA 1982 The Physiology and Biochemistry of Seed Development, Dormancy and Germination. Elsevier Biochemical Press, Amsterdam 74. Khan SA, MA Humayun, TM Jacob 1977 A sensitive radioimmunoassay for isopentenyladenosine. Anal Biochem 83: 632-635 76. Kidd F, C West 1918 Physiological predetermination: the influence of physiological condition of the seed upon the course of subseguent growth and upon the yield. I. The effects of soaking seeds in water. Annals of Appied Biology 5: 1-10

PAGE 128

117 77. Klein S, B Pollock. 1968 Cell fine structure of developing lima bean seeds related to seed desiccation. Amer J Bot 55; 658-672 78. Kozlowski TT 1972 Seed Biology. Vol, I. Importance, Development, and Germination, Academic Press, New York 79. Kozlowski TT 1972 Seed Biology. Vol. II. Germination Control, Metabolism, and Pathology. Academic Press, New York 80. Kozlowski TT 1972 Seed Biology. Vol. III. Insects and Seed Collection, Storage, Testing, and Certification. Academic Press, New York 81. Kubowicz BD, LN Vanderhoef, JB Hanson 1982 ATPDependent calcium transport in plasmalemma preparations from soybean hypocotyls. Plant Pyhsiol b9: 187-191 82. Kulik MM, HW Yaklich 1982 Evaluation of vigor tests in soybean seeds: relationship of accelerated aging, cold, sand bench, and speed of germination tests to field performance. Crop Science 22: 766-770 83. Kurkdjian A, JJ Leguay, J Guern 1979 Influence of fusicoccin on the control of cell division by auxins. Plant Physiol 64:1053-1057 84. Kuzmanoff KM, ML Evans 1982 Calmodulin-like protein in corn roots: evidence for its existance and potential involvemant in root growth. Plant Physiol 69: S-56 85. Larson LA 1968 The effect of soaking peas without seed coats on seedling growth. Plant Physiol 43: 255-259 86. Leopold AC 1980 Aging and senescence in plant development. In KV Thimann, ed. Senescence in Plants, CfiC Press, Boca Raton pp 2-12 87. Leopold AC 1983 Volumetric components of seed imnibition. Plant Physiol 73: 677-680 88. Leopold AC, PE Kriedemann 1975 Plant Growth and Development. 2nd Edition, McGraw-Hill, New York 89. Lewin B 1980 Gene Expression 2. Vol. 2Eucaryotic Chromosomes. John iiley and Sons, New York 90. Lieberman M 1979 Biosynthesis and action of ethylene. Ann Bev Plant Physiol 30: 533-591

PAGE 129

118 91. Loveys BH 1977 The intracellular localization of abscisic acid in stressed and non-stressed leaf tissue. Plant Physiol 40: 6-10 92. Marre 1 E 1977 Effects of Fusicoccin and hormones on plant cell membrane activities: observations and hypothesis. In E Marre' and C Ciferri, ed. regulation or Cell Membrane Activities in Plants, North-Holland, Amsterdam, pp 185-202 93. Marre 1 E 1979 JFusicoccin: a tool in plant physiology, Ann fie? Plant Physiol 30: 273-288 94. McDonald Jr MB 1980 Assessment of seed guality. HortScience 15: 784-788 95. McDonald Jr MB, DO Wilson 1980 ASA-6 10 ability to detect changes in soybean seed quality. Journal of Seed Technology 5: 56-66 96. McKue E 1935 Vernalization experiments with forage crops. US Dept of Agriculture Circular 377 97. Moore TC 1979 Biocaemistry and Physiology of Plant Hormones, Sprinyer-Verlag, New York 98. Morohashi Y, JD Bewley 1980 Development of mitochondrial activities in pea cotyledons during and following germination of the axis. Plant Physiol 6t»: 7 0-73 99. Mullett JH, JA Considine 1980 Potassium release and uptake in germinating legume seeds in relation to seed condition and germination environment. J Exp Bot 31: 151-162 100. Murata M 1979 Genetic changes induced by artificial seed aging in barleyPhD Diss. , Colorado State University, Ft Collins 101. Murata M, T Tsuchiya, EE Soos 1982 Chromosome damage induced by artificial aging in barley. II. Types of chromosomal aberrations at first mitosis. Bot Gaz 143: 111-116 102. Murphy J3, TL Noland 1982 Temperature effects on seed imbibing and leakage mediated by viscosity and membranes. Plant Physiol 69: 428-431 103. Nakamura S 1981 Effect of Fusicoccin on the germination or agricultural seeds. Seed Sci technol 9: 879-884 104. Navashin MS 1933 Aging of seeds is a cause of chromosome mutations. Planta 20: 233-243

PAGE 130

119 105. Neergaard P 1977 Seed Pathology. Vols. I and II. , The Macaillan Press Ltd., London 106. Nickell LG 1983 Plant Growth Regulating Chemicals, CRC Press, Boca Raton 107. Nilsson NH 1931 Sind die induzierten autanten nur selective erscheicung? Hereditas 15: 320-328 108. Gbendorf RL, PR Hobbs 1970 Effect of seed moisture on temperature sensitivity imbibition of soybean. Crop Sci 10: 563-566 109. O'Neill SD, AC Leopold 1982 Assessment of phase transitions on soybean membranes. Plant Physiol 70: 1405-1409 110. Osborne DJ 1980 Senescence in seeds. In KV Thiiuann, ed. Senescence in Plants, CRC Press, Boca Raton, Fla pp 14-37 111. Osborne DJ 1982 Deoxyribonucleic acid integrity and repair in seed germination: the importance in viability and survival. In AA Khan, ed. The Physiology and Biochemistry of Seed Development, Dormancy and Germination, Elsevier Biochemical Press, Amsterdam, pp 435-463 112. Gvcharov KE 1977 Physiological basis of seed germination (translated frota Russian) Amerind Publishing Co Pvt Ltd, New Delhi 113. Pai K, BK Gaur 1981 Quantitive and qualitative changes in mitochondrial proteins induced by gamma irradiation of bean hypocctyl segments. Plant Physiol 69: S-552 114. Paleg LG 1965 Physiological effects of gibberellins. Ann Rev Plant Physiol 16: 291-322 115. Pallaghy CK, K Rasschke 1972 No stomatal response to ethylene. Plant Physiol 49: 275-276 116. Pallas JE Jr , SJ Kays 1982 Inhibition of photosynthesis by ethylene a stomatal etfect. Plant Physiol 70: 598-601 117. Pardi D, S Horgutti, S Cocucci 1980 K+ Uptake in the early phases of germination of the photoblastic and thermosensitive seed of Phacelia ta na cetifolia. Physiol Plant 46: 379-384 118. Parrish DJ, AC Leopold 1977 transient changes during soyDean imbibition. Plant Physiol 59: 1111-1115

PAGE 131

120 119. Parrish DJ, AC Leopold 1978 On the mechanism of aging in soybean seeds. Plant Physiol 61: 365-368 120. Parrish DJ, AC Leopold 1982 Turgor changes with accelerated aging or soybeans. Crop Science 22: 666-669 121. Pengelly WL, ES Bandurski, A Schulze 1981 Validation of a radioimmunoassay for indole-3-acetic acid using gas chromatography-selected ion moni toring-mass spectrometry. Plant Physiol 68: 96-98 122. Perl M, Z Feder 1981 Improved seedling development of pepper seeds (Capsicum annuum) by seed treatment for pregermination activities. Seed Sci Technol 9: 055-663 123. Petruzzelli, L Lioi, G Carello 1982 The effect of Fusicoccin and monovalent cations on the viability of wheat seeds. Journal of Experimental Botany 33: 118-124 124. Pilet PE, MC Elliot 1981 Some aspects of the control of root yrowth and geotropism: the involvement or indole acetic acid and abscisic acid. Plant Physiol 67: 1047-1050 125. Pollock BM 1969 Imbibition temperature sensitivity of lima bean seeds controlled by initial seed moisture. Plant Physiol 44: 907-911 126. Pollock BM, JS Manalc 1970 Simulated mechanical damage to garden beans during germination. J Amer Soc Hort Sci 95: 415-417 127. Pollock BM, EE Roos, Jfi Manalo 1969 Vigor of garden bean seeds and seedlings influenced by seed moisture, sunstrate oxygen and imbibition temperature. J Amer Soc Hort Sci 94: 557-584 128. Pollock BM, VK Toole 1966 Imbibition period as the critical temperature sensitive stage in germination of lima bean seeds. Plant Physiol 41: 221-229 129. Poole aJ 1978 Energy coupling for membrane transport. Ann Rev Plant Physiol 29: 437-460 130. Powell AA, S Matthews 1978 The damaging effect of water on dry pea embryos during imbibition. J Exp Bot 20: 1215-1220

PAGE 132

121 131. Pratt lit, fiA Coleman, JM MacKenzie Jr 1976 Immunological visualization of phytochrome. In Light and Plant Development, B Smith, ed, Butterworth £ Co, Ltd, London, pp 75-94 132. Priestley DA, AC Leopold 1979 Absence of lipid oxidation during accelerated aging of soybean seeds. Plant Physiol 63: 726-729 133. Priestley DA, SB McBride, AC Leopold 1980 Tocopherol and organic free radical levels in soybean seeds during natural and accelerated aging. Plant Physiol 715-719 134. Hay Pfl, U Dohrmann, B Hertel 1977 Chacterization of naphthalene-acetic acid binding to receptor sites on cellular membranes of maize coleoptile tissue. Plant Physiol 59: 357-364 135. Eayle DL, R Cleland 1977 Control of plant cell enlargement by hydrogen ions. In AA Hoscona, A aonroy, eds, Current Topics in Developmental Biology, Vol 2, Pattern Development, Academic Press, New York 136. Reid MS, Y Mor, AM Kofranek 1981 Epinasty of poinsettiasthe role of auxin and ethylene. Plant Physiol 67: 950-952 137. Roberts EH 1972 Storage environment and the control of viability. In Viability of Seeds, EH Roberts, ed, Chapman Hall, London, pp 14-58 138. Roberts EH 1973 Loss of viability. 01 trastructural and pnysiological aspects. Seed Sci Tecnnol 1: 529-545 139. Roberts EH 1973 Loss of seed viability: chromosomal and genetical aspects. Seed Sci Technol 1: 515-527 140. Roberts EH 1981 The guantif ica tion of aging and survival in orthodox seeds. Seed Sci Technol 9: 373-409 141. Roberts EH, RH Ellis 1982 Physiological, ultrastructural and metabolic aspects of seed viability. In AA Khan, ed, The Physiology and Biochemistry of Seed Development, Dormancy and Germination. Elsevier Biomedical Press, New York, pp 465-485 142. Roland JC , CA Lembi, DJ Morre 1972 Phosphotungstic acid-cnromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Technol 47: 195-200

PAGE 133

122 143. Soos £E 1980 Physiological, biochemical, and genetic changes in seed quality during storage, HortScience 15:781-78U 144. Soos EE 1982 Induced genetic changes in seed gemination during storage, In AA Khan, ed. The Physiol Biochemistry of Seed Development, Dormancy and Germination. Elsevier Biochemical Press, New York, 1982, pp 409-434 145. fioos EE, CM Eincker 1982 Genetic stability in •pennlate 1 orchardgrass seed following artificial aging. Crop Science 22: 6 11-613 146. Koyse DJ, MA Ellis, JB Sinclair 1975 Movement ot penicillin into soybean seeds using dichloromethane. Phytopathology 65: 1319-1320 147. fiubinstein B, EE Cleland 1981 Responses of Avena coleoptiles tc suboptimal fusicoccin: kinetic and comparisons with indolacetic acid, Plant Physiol 68: 543-547 148. Salisbury FB , CW Ross 1978 Plant Physiology, Wadsworth Publishing Company, Belmont, California 149. Samad MA, ES Pearce 1978 Leaching of ions, organic molecules, and enzymes from seeds of peanut (Arachis hyeqgea L) Imbibing without testas or with intact testas. J Exp Bot 29: 1471-1478 150. Samrmy C 1978 Effect of light on ethylene production and hypocotyl growth of soybean seedlings. Plant Physiol 61: 772-774 151. Samimy C 1978 Influence of cobalt on soybean hypocotyl growtn and its ethylene evolution. Plant Physiol 62: 1005-1006 152. Saunders MJ, PK Helper 1982 Calcium ionophore A23187 stimulates cytokinin-like mitosis in Fjinaria. Science ^17: 943-945 153. Schopmeyer C.S. (ed-) 1974 Seeds of Hoody Plants in the United States. Agriculture Handbook No. 450, Forest Service, USDA, Washington D.C. 154. Sen S 1975 Germination and viability of rye embryos. Nucleic acid and protein synthesis during early hours of germination. Ph.D. Diss.. Cambridge, OK 155. Sen S, DJ Osborne 1977 Decline in ribonucleic acid and protein synthesis during early hours of imbibition of rye (Secale cereale L) embryos. Biochem J 166: 33-38

PAGE 134

123 156. Senaratna T, BD HcKersie 1983 Dehydration injury in germinating soybean (Glycine max L Merr) seeds. Plant Physiol 72: 620-624 157. Senaratna T, BE HcKersie 1983 Characterization of solute efflux from dehydration injured soybean (Glycine max L Merc) seeds. Plant Physiol 72: 9 1 1-91 U 158. Simon Ew" 1974 Phospholipids and plant membrane permeability. New Phytol 73: 377-419 159Simon EH, BM Harum 1972 Leakage during seed imbibition. J Exp Bot 23: 1076-1085 160. Skadsen fi«, JH Cherry 1983 Quantitive changes in in vitro and in vivo protein synthesis in aging and rejuvenated soybean cotyledons. Plant Physiol 71: 861-868 16 1. Smith H, D Grierson 1982 The Molecular Biology of Plant Development. University of California Press, Berkeley 162. Smith FA, JA Baven 1979 Intracellular pH and its regulation. Ann Bev Plant Physiol 30: 289-311 163. Sterferud A, ed. Yearbook of Agriculture 1961 Seeds. USDA Publication, Washington, D.C. 164. Stewart EC, JD Derk 1980 Lipid peroxidation associated with accelerated aging of soybean axes. Plant Physiol 65: 245-248 165. Stevenson TT , BE Cleland 1981 Osmoregulation in the A ve na coleoptile in relation to auxin and growth. Plant Physiol 67: 749-753 166. Stevenson TT, BE Cleland 1982 Osmoregulation in the Avena coleoptile. Plant Physiol 69: 292-295 167. Stillwell H, P Hester 1983 Kiuetin increases water permeability of phosphatidylcholine lipid bilayers. Plant Physiol 71: 524-530 168. Stout BG, BE Cleland 1980 Partial characterization of fuscicoccin binding to receptor sites on oat membranes. Plant Physiol 66: 353-359 169. Tanford JB 1980 The Hydrophobic Effect: Formation of Micelles and Biological Membranes. Edition 2, John Wiley and Sons, New York

PAGE 135

124 170. Tao KL, AA Khan, GE Harman, CJ Eckenrode 1974 Practical significance of the application of chemicals in organic solvents to dry seeds. J Am Soc Hort Sci 99: 217-220 171. Tao KL, MB McDonald, and AA Khan 1974 Life Sci 15: 1925-1933 172. Taylor JS, DM Raid, RP Pharis 1981 Mutual antagonism of sulfur dioxide and abscisic acid in their effect on stomatal aperture in broad bean (Vicia f aba L) Plant Physiol 68: 1504-1507 173. Taylorson RB, SB Hendricks 1977 Dormancy in seeds. Ann Rev Plant Physiol 28: 331-354 174. Terry ME, RL Jones 198 1 Effect of salt on auxin-induced acidification and growth by pea internode sections. Elant Physiol 68: 59-64 175. Terry ME, RL Jones, and EA Bcnner 1981 Soluble cell vali polysaccharides released from pea stems by centrif ugation. I. Effect of auxin. Plant Physiol 68:531-537 176. Terry ME, B Rubinstein, and RL Jones 1981 Soluble cell wall polysaccharides released from pea stems by centrif ugaticn. II. Effect of ethylene. Plant Physiol 68: 538-542 177. Thompson JR 1979 An Introduction to Seed Technology. Parts 1 to 6. John wiley and Sons, New York 178. Tolbert NE (ed) 1980 The Biochemistry of Plants. A Comprehensive Treatise. Vol. 1. The Plant Cell. Academic Press, New York 179. Toole EH, VK Toole 1946 Relation of temperature and seed moisture to the viability of stored soybean seed. OS Dept Agr Cir 753 180. Toole VK, EH Toole 1953 Seed dormancy in relation to longevity. Proc Int Seed Test Assoc 18: 325-328 181. Toole VK, L« Woodstock 1973 Seed Quality Research and Technology. Vol I. Norway: Internat Seed Test Assoc 182. Vanderhoef LN, RR Dute 1981 Auxin-regulated wall loosening and sustained growth in elongaton. Plant Physiol 67: 146-149 183. Vegis A 1964 Dormancy in higher plants. Ann Rev Plant Physiol 15: 185-224

PAGE 136

125 184. Vertucci CW , AC Leopold 1983 Dynamics of imbibition by soybean embryos. Plant Physiol 72; 190-193 185. Villiers TA 1971 Cytological studies in dormancy. I. Embryo maturation during dormancy. New Phytol 70: 751-760 18b, Villiers TA 1972 Cytological studies in dormancy. II. Pathological ageing changes and recovery upon release. New Phytol 71: 145-152 187. Villiers TA 1972 Cytological studies in dormancy. III. Changes during low-temperature dormancy release. New Phytol 71: 153-160 188. Villiers TA 1980 Oltrastructural changes in seed dormancy and senescence. In KV Thimann, ed, Senescence in Plants, CBC Press, Boca Baton, pp 40-66 189. Villiers TA 1974 Seed ageing: chromosome stability and extended viability of seeds stored fully imbibed. Plant Physiol 53: 875-878 190. Villiers TA, DJ Edgecumbe 1975 On the cause of seed deterioration in dry storage. Seed Sci Technol 3: 761-764 191. Vitagliano C, GV Hoad 1978 Leaf stomatal resistance, etnylene evolution and ABA levels as influenced by (2-chloroethyl) phosphonic acid. Sci Hortic 8: 101-106 192. Wardrop AJ, GM Polya 1980 Ligand specificity of bean leaf soluble auxin-binding protein. Plant Physiol 66: 112-118 193. Walton DC 1980 Biochemistry and physiology of abscisic acid. Ann Rev Plant Physiol 31: 453-489 194. Wareing PF 1982 Plant Growth Sunstances. Academic Press, New York 195. Widall S, T Lundbor, and C Larsson 1982 Plasma membranes from oats prepared by partition in an aqueous polymer two-phase. Plant Physiol 70: 1429-1435 196. Wclk WD, RC Herner 1982 Chilling injury of germinating seeds and seedlings. HortScience 17: 169-173 197. Woodstock LW, DF Grabe 1967 Relationships between seed respiration during imbibition and subseguent seedling growth in Zea ma_ys L. Plant Physiol 42: 1071-1076

PAGE 137

126 198. Woodstock LW, BM Pollock 1965 Physiological predetermination: imbibition, respiration, and growth of lima bean seeds. Science 150: 1031-1032 199. Woodstock Li 1975 Freeze-drying as an alternative method for lowering seed miosture, Proc, Association of Official Seed Analysists 65: 159-163 200. tfoodstock La", KLJ lao 1981 Prevention of imbifcitional injury in low vigor soybean embryonic axes by osmotic control of water uptake. Physiol Plant 51: 133-139 201. Yaklich RW, MD Orzolek 1977 Effect of polyethylene glycol 6000 on pepper seed, Hortic Sci 12: 263-264 202. Yu YB, SF Yang 1979 Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiol 64: 1074-1077 203. Yves H, Y Chartier 1981 Hormonal control of mitotic development on tobacco protoplast. Plant Physiol 68: 1273-1278 204. Zimmermann U 1978 Physics of turgor and osmoregulation. Ann Rev Plant Physiol 29: 121-148 205. Zimmermann 0, E Steudle, PI Lelkes 1976 Turgor pressure regulation in Valgnia utricular is: effect of cell wall elasticity and auxin. Plant Physiol 58: 608-613

PAGE 138

BIOGRAPHICAL SKETCH Three generations of Tildens have pioneered in Florida agriculture. Partly because of this precident, it has been very satisfying for me to engage in agricultural research. Education began for me in Winter Garden, Florida. High school graduation, however, was from Darlington School, Home, Georgia, in 1958. This education continued at Stetson University in DeLand, Florida, where a B.S. degree in chemistry was earned. An opportunity to use chemistry in a practical manner was provided during military service. This was at the Eocket Propulsion Laboratory, Edwards Flight Test Center, in California. After returning to Florida in 1967, I was employed io pesticide research with the University of Florida to develop new, sensitive analytical metnods. Several presentations and a publication resulted from this experience. In 1970 my career continued as a research chemist in nuclear medicine at the Veterans Hospital, Gainesville. Developments in hormone analysis were communicated in a number of pun licaticns. Khile employed by the VA, my association continued with the University. On a part-time basis, graduate credits were accrued and a Master of Education degree was awarded. 127

PAGE 139

123 A desire to apply research findings to available products led to employment in private industry as a product development cnemist (1977). Several research findings were manifest as commercial products tor hormone analysis. In 1980 my education resumed with the goal of applying uy education and experience in chemistry to research problems in agriculture. The final goal was to earn the doctorate degree and to contribute to agricultural research. Atter earning a master's degree in horticultural science, I began work toward the doctorate in agronomy. Fortunately, my wire Martha has understood the importance of this effort toward "self actualization. " She has been very patient in this regard. This experience has also served as an example to our daughters, Beth and Becky, who take their education seriously.

PAGE 140

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scnolarly presentation and is fully adequate, in scope and quality, a: a dissertation for the degree of Doctor of Philosophy. ^&fe^*^_ S. H. west, Chairman Professor, Agronomy I certify that I have read this study and that in ay opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for tne degree of Doctor of Philosophy. >ert fi. "Hxgg; Professor, Horticultural Science I certify that 1 have read this study and that in ay opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, a; a dissertation for the degree of Doctor of Philosophy. Daniel J. CaJntliffe Professor, Horticultural Science I certify that I have read this study and taat in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. iy H./Gaskins Professor, Agronomy

PAGE 141

I certify that I have read this study and that in ay opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy, v^^AK >i^M jU-. Donald J.' fiuber Assistant Professor, Horticultural Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. April 1984 Dean, College of Agriculture Dean for Graduate Studies and Research

PAGE 142

UNIVERSITY OF FLORIDA 3 1262 08285 166 7