REVERSAL OF THE EFFECTS OF DETERIORATION
IN AGED SOYBEAN SEEDS
(L.) MERR. CV. VICCJA]
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
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
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
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.
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
SUPPLEMENTARY EXPERIMENTS . . . . . .. . 81
Microflora and Seed Studies . . . - 81
Fungal Survival .... .. . . . 81
_______ ______ __
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
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
LIST OF FIGURES
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. . .
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
(L.) MERR. CV. VICOJA]
Robert Luther Tilden
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
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.
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
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
Statement of Objectives
Seed detetioration was analyzed as several contributing
causes including microflora, nonuniform tissue aging and
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
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.
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
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
1 Homeostasis a biological tendency to balance anabolism
and catabolism (metabolic turnover and refurbishment)
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
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
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
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
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
areas which degenerate first under natural or accelerated
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
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
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
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.
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
(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
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
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
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
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
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
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
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
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
Literature on Membrane Activity of Growth Regulators
System, Activity and References
5, 61, 123, 151
23, 25, 91
5, 67, 70, 71, 90,
62, 65, 114, 167
115, 116, 172, 176,
Adsorption of Ions
33, 117, 162
33 34, 117, 135,
165, 166, 174
135, 174, 182
8, 12, 13, 39, 83,
84, 92, 93, 103,
123, 135, 147
to this idea, inhibitors of ethylene synthesis such as
amino-oxyacetic acid, AOA, should produce effects similar
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.
Literature on Regulatory Activity of Plant Hormones
System, Activity and References
De Novo Synthesis
8, 60, 70, 129, 175,
34, 36, 58, 124,
136, 172, 191
11, 52, 151, 193
52, 54, 59, 150, 202
10, 36, 114, 124,
84, 92, 93,
r----- ---- -- --- ----------~
MATERIALS AND METHODS
Microtlora of Seed Deterioration
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.
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.
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
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
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 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
glutaraldehyde (4 percent) at 4 C. The low temperature was
used to slow temperature dependent processes within the
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.
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
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.
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).
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-
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
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
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.
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.
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
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
r ----- ----- -- -------- -- -
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.
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.
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).
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.
^^^*^**^ *\?y- '*1t* *
^ -* **- *A So T*.*" *' '?
'r ^ -* *- *. i 4'r' -i "'
***''** ~:. t^ .*** .* '*', -
*<. 't .f^ . ~*: 'i1, f 1,- *-
t -'- >a k
"~' "," '. '
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
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
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-
10. HOURS O
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.
of 11 percent
Each plate also
ve layers of
~- I -- ------------.
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.
Effect of Aging on Rate of Water Uptake
Age Layers of Paper
r 1 -------------
1 2 3 4 5
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
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
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
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.
Vigor as a Function of Age and Bate or Hydration
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
40- PAPER LAYERS
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
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
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,
r-------- ------------ __--^----_----__^--------
50 ;./ 5^/
rated agi 988(0, 20, 301ours) ith
Z f862 251
/ 786 262
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
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.
Effect of Accelerated Aging on Primed Seeds
C------- ------- -----
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.
Priming-Dependence on Temperature
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
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.
(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
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 ---------- ------------------------
Interaction of Age and Abscisic Acid
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
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
T total weight; RH root hypocotyl weight; MEAN of two
replications; CV coefficient of variation
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
Effect of ABA on the Vigor of Aged Seeds
r --- --
GROWTH RATIO RATIO
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
r________~__l~~ __ I
After five days of growth, seeds were weighed before and
carter detaching the cotyledons and Table 9 contains the
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-
r -- ---------------~
~----------- --- ------------------
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).
Growth Response to Fusicoccin and Ethephon
r 1--- 1--- -
%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
Since the data in Table 9 contains a missing value, the
generalized linear model was used for statistical analysis
(Table 11). No significant interaction was attributed to
Lusicoccin, with age and ethylene. All other interactions
and main effects were significant or highly significant.
Generalized Linear Regression Model
SUiM O S
TYPE I SS
F VALUE PR > F
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 ----- -----
0 0 S 25 30
0 0 .75-
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
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
.istoloiogof Seed Deterioration
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.
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.
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.
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.
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
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
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
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
ABA treatment of aged seeds. Ethylene, on the other hand,
did have a positive age interaction.
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
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.
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
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
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
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
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
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
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
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
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
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
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).
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.
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
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.
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
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
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.
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
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
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
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
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
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
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
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
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
4--- -- V-- 142
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.
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
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:  What is the relationship between
the maximum seed moisture used for priming and the leakage
of redried seeds?  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).  What is the
"point of no return" for redrying seeds without loss of
vitality?  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?  If seeds are redried to their
original weight, do they return to their original moisture
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
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.