• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 Abstract
 Introduction
 Literature review
 Growth hormone involvement in germination...
 Respiratory control of Capsicum...
 Mechanical resistance of the seed...
 Gibberellic acid stimulation of...
 The appearance of cell wall degrading...
 Appendix: Germination rate of pepper...
 Bibliography
 Biographical sketch














Title: Germination of Capsicum annuum L. at low temperature
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Permanent Link: http://ufdc.ufl.edu/UF00099229/00001
 Material Information
Title: Germination of Capsicum annuum L. at low temperature
Alternate Title: Capsicum annuum
Physical Description: vii, 156 leaves : ill. ; 28 cm.
Language: English
Creator: Watkins, James Thomas, 1953-
Copyright Date: 1982
 Subjects
Subject: Sweet peppers -- Development   ( lcsh )
Horticultural Science thesis Ph. D
Dissertations, Academic -- Horticultural Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1982.
Bibliography: Bibliography: leaves 147-155.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by James Thomas Watkins.
 Record Information
Bibliographic ID: UF00099229
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000297774
oclc - 08512395
notis - ABS4149

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
        Page v
    Abstract
        Page vi
        Page vii
    Introduction
        Page 1
        Page 2
    Literature review
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
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    Growth hormone involvement in germination of Capsicum annuum L. at low temperature
        Page 21
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    Respiratory control of Capsicum annuum L. germination at low temperature
        Page 60
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    Mechanical resistance of the seed coat and endosperm in the control of Capsicum annuum L. germination at low temperature
        Page 79
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    Gibberellic acid stimulation of early endosperm degradation in Capsicum annuum L.
        Page 106
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    The appearance of cell wall degrading enzymes during germination of Capsicum annuum L.
        Page 132
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    Appendix: Germination rate of pepper seeds at various temperatures
        Page 145
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    Bibliography
        Page 147
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    Biographical sketch
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Full Text
















GERMINATION OF Capsicum annuum L. AT
LOW TEMPERATURE














BY

JAMES THOMAS WATKINS


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

UNIVERSITY OF FLORIDA
1982













ACKNOWLEDGEMENTS


The author wishes to express his sincere appreciation to

Dr. D.J. Cantliffe (Dan), for his friendship, guidance, support

and assistance in the preparation and completion of this dis-

sertation and degree. Special appreciation to Drs. H.H. Bryan,

C.B. Hall, T.E. Humphreys and T.A. Nell for their advice, re-

commendations and special talents so freely given. The author

is also grateful to Drs. M. Sachs (visiting professor), D.J.

Huber and R.C. Fluck (Agricultural Engineering) for their time

and instruction.

Thank you to Art, for his expertise and assistance in pre-

paration of the graphic materials.

The author wishes to thank the faculty, staff and students,

past and present, in the Horticultural Science Department for

their encouragement and help during this graduate study. Special

thanks go to those "Seed Physiologists" who served, worked, traveled

and supported each other.

The author is grateful to his parents for their devotion,

support and unwaivering belief in their son who appeared to be

a professional student.

The author is especially grateful to Carol, Jimmy, Kristy

and Charles. His wife, Carol, gave unendingly of herself for

the completion of this work and the author is very thankful for

her understanding, patience, assistance and love. To Jimmy,

ii








thank you for the happiness and love you have given. To Kristy

and Charlie who always gave love and friendship, thank you.














TABLE OF CONTENTS


PAGE
ACKNOWLEDGEMENTS . . . . . . . ... .. ii

ABSTRACT . . . . . . . . ... . . . vi

INTRODUCTION . . . . . ... . . . . 1


CHAPTER


I. LITERATURE REVIEW . . . . . . . . 3
Low Temperature Imbibition and Membrane
Reorganization . . . . . . . . 7
Growth Regulator Activity as a Function
of Low Temperature Germination . . . ... 13
Low Temperature Germination as it
Relates to Genetic Variability . . . ... .15
Seed Treatments for Enhancing Low
Temperature Germinability . . . . .... 16

II. GROWTH HORMONE INVOLVEMENT IN GERMINATION
OF Capsicum annuum L. AT LOW TEMPERATURE ..... 21

Materials and Methods . . . . . . 23
Results . . . . . . . .... . .. 26
Discussion . . . . . . . . ... . 54
Summary . . . . . .. . . . . . 58

III. RESPIRATORY CONTROL OF Capsicum annuum L.
GERMINATION AT LOW TEMPERATURE . . . ... 60
Materials and Methods . . . . . .... 62
Results . . . . . . . . . . . 65
Discussion . . .... .. .. .. . . ... 72
Summary . . . . . . . . . . . 77

IV. MECHANICAL RESISTANCE OF THE SEED COAT AND
ENDOSPERM IN THE CONTROL OF Capsicum annuum L.
GERMINATION AT LOW TEMPERATURE .... . . . 79
Materials and Methods . . . . . .... 81
Results . . . . . . ..... . . 85
Discussion ............. * * 97
Summary . . . . ..................... 104









V. GIBBERELLIC ACID STIMULATION OF EARLY
ENDOSPERM DEGRADATION IN Capsicum
annuum L. . . . . . . . ... . 106

Materials and Methods . . . . . . . 108
Results . . . . . . . . ... . . 110
Discussion . . . . . . . . ... . 122
Summary . . . . . . . . . . . 131

VI. THE APPEARANCE OF CELL WALL DEGRADING
ENZYMES DURING GERMINATION OF
Capsicum annuum L. . . . . . . . ... 132
Materials and Methods . . . . . . ... 133
Results . . . . . . . .... .. .. 135
Discussion . . . . . . . .... . 142
Summary . . . . . . . .... ... .. 144
APPENDIX GERMINATION RATE OF PEPPER SEEDS AT
VARIOUS TEMPERATURES . . . . .... 145
LITERATURE CITED . . . . . . . . ... . 147

BIOGRAPHICAL SKETCH . . . . . . . .... 156













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

GERMINATION OF Capsicum annuum L. AT
LOW TEMPERATURE

BY

James Thomas Watkins
May 1982

Chairman: Daniel J. Cantliffe
Major Department: Vegetable Crops

The cause of slow and nonuniform germination of pepper

(Capsicum annuum L.) seeds at low temperature was investigated.

There was no evidence of a leachable or extractable germination

inhibitor being activated or formed due to low temperature ex-

posure. Auxin and kinetin applications did not effect germina-

tion rate. However, gibberellic acid (GA) treatments effec-

tively increased germination rates at 150 and 250C. Gibberellic

acid4+7 was more effective than GA3. Gibberellic acid stimu-

lated germination rate under osmotic stress and the presence of

a GA synthesis inhibitor reduced germination rate.

High oxygen (02) concentrations stimulated pepper seed

germination rates at 250C but inhibited germination rates at 150

Gibberellic acid applications could not overcome the inhibitory

effect of 100% 02 at 150. Seed respiratory activity was greater

at 250 in 100% 02 than in air, but the additional 02 did not

alter respiratory pathways. At 150, total respiration, respira-








tory activities in the cytochrome and alternate pathways were

unaltered by additional 02.

The endosperm surrounding the embryo appeared to be

a mechanical barrier to radicle emergence. The amount of resis-

tance the endosperm imposed on radicle protrusion was measured

with the Instron Universal Testing Machine. Endosperm strength

decreased as imbibition time increased and the rate of reduction

was affected by imbibition temperature. Gibberellic acid treat-

ments and 100% 02 at 250 increased the rate at which the endosperm

strength decreased while 100% 02 at 150 inhibited the rate at

which the endosperm strength decreased.

The endosperm in front of the radicle was visually protruding

one day before radicle emergence regardless of imbibition tempera-

ture or seed treatment. This protrusion was correlated to a

loss of cellular integrity and endosperm thickness in front of

the radicle. Gibberellic acid treatments stimulated endosperm

breakdown.

Pepper seed cell wall degrading enzyme activity increased

with increased imbibition time, with temperature, and GA treat-

ment. Pepper seed enzyme extracts preferentially degraded a

galactomannan substrate but did not degrade a carboxymethyl-

cellulose substrate indicating that manno-polysaccharides may

be components of pepper seed endosperms.














INTRODUCTION


Florida's third most valuable vegetable crop is sweet pepper

(Capsicum annuum L.). It accounts for 6.5% of the total vegeta-

ble crop value for this state, the leading pepper producing state

in the United States. Florida harvested 17,300 acres in 1979

which was approximately one-third of the total United States acre-

age of peppers. The total value of the pepper crop in the United

States in 1979 was 109 million dollars and Florida production

accounted for 51 million dollars of that amount.

Germination of pepper seed at low temperatures is very erratic

and nonuniform and emergence may be spread out for several weeks

when the seeds are sown in cool soils. This problem is not only

encountered during direct field seeding in cool soils, but also

during seeding for transplants in cold frames and inadequately

heated greenhouses during Florida's winter months. This erratic

and nonuniform emergence often leads to nonuniform transplants,

poor crop stands, and may necessitate crop replanting. Often,

reduced yields and delayed harvest (losing early high market

prices) can be caused by slow stand establishment.

Emergence data from 80 cultivars of C. annuum suggested

that altered germination rates at low temperatures could be

controlled genetically (Gerson and Honma, 1978). However, they

reported the best mean emergence rate from the 80 cultivars

tested at 160C was 20.6 days. This long period of exposure in a

soil environment could lead to pathogenic attack and death of the seed.




2


The studies contained herein explore the cause or contri-

buting causes of slow and nonuniform germination of C. annuum

at low temperature. Investigations for inherent germination

inhibitors and applications of growth promoters were made to

determine their effects, if any, on pepper seed germination at

low temperature. Respiratory control of germination at low

temperature and the effects of the seed coat and endosperm on

germination were investigated. Additionally, the manner in which

the radicle emerges through the endosperm was determined in

relation to germination temperature.













CHAPTER I
LITERATURE REVIEW


Germination of seeds at low temperature has been corre-

lated with erratic and nonuniform germination, inhibition of

germination, abnormal seedling development and embryo death

(Gerson and Honma, 1978; Christiansen, 1968; Pollock and

Toole, 1966). The response of seeds to germination at low

temperatures has been grouped into three general categories:

1) seeds which will germinate at temperatures near freezing

2) seeds which fail to germinate at low temperatures but if

moved to a more favorable temperature will germinate normally,

and 3) seeds which when imbibed at low temperatures showed

injury symptoms when subsequently grown at warmer temperatures.

This injury is known as chilling injury (Simon, 1979).

Mustard seed germinated within a day at temperatures be-

tween 15 and 250C (Simon et al., 1976). As the temperature

was lowered, seed germination became slower, although mustard

seeds were able to germinate at temperatures of 3 to 40

Thompson (1973) demonstrated that seeds from various populations

of 'Grand Rapids' lettuce and Agrostemma githago L., a weed

of cereals, could germinate slowly at temperatures near 0.

Mustard, lettuce and Agrostemma githago all germinated at

low temperatures without injury to the subsequent seedlings.

Watermelon and tomato seeds do not germinate at constant

low temperatures. Sachs (1977) found that no germination of








watermelon seeds occurred at constant temperatures below 15C

while the optimal germination temperature was 300. However,

when watermelon seeds were imbibed at suboptimal temperatures,

as low as 00 for 10 days, no effect on subsequent germination

at optimal temperatures was found. Tomato seed exposed to

80 for 24 hours at the start of imbibition or just before

radicle emergence did not affect the final percent germination

(Bussell and Gray, 1976). The cold treatments, however, caused

the time to 50% germination to be increased but reduced the time

to 95% germination.

In an earlier report, Goodall and Bolas (1942) chilled

imbibed tomato seeds at temperatures of 0 to 11 C for periods

of 10 to 20 days, then germinated them with untreated seeds at

more favorable temperatures. The plants grown from the chilled

seeds produced more fruit and had a concentrated yield at the

beginning of the cropping season.

Pepper seeds germinate slowly at less than optimal

temperatures. Cochran (1935) reported that no peppers ger-

minated after 45 days in a greenhouse with a temperature range

between 4 and 100C. However, with each 60 increase in temp-

erature range, the speed of germination increased until ger-

mination was obtained in five to six days in a greenhouse with

a temperature range between 32 and 380. Heydecker (1974)

reported that when imbibed pepper seeds were held for three

weeks at 3, 7 and 100, they failed to germinate. But, when they

were transferredto 300 they germinated faster than untreated

seeds placed at 300 from the outset, with no apparent injury.




5


This was contrary to the work of Harrington and Kihara (1960).

They reported chilling injury symptoms on pepper seedlings

imbibed at temperatures of 0 to 100 for one-half to four weeks

then subsequently germinated at 250. The greatest percentage

of chilling injury, 33%, occurred at the 100 imbibitional

temperature. Chilling injury symptoms were described as the

collapse of cells 1 to 2 mm behind the root tip, necrosis of

this area and decaying of the seedlings.

Imbibition and germination at low temperatures may affect

subsequent plant growth and development. Highkin and Lang

(1966) reported that pea plants obtained from seeds germinated

at 30C compared to 270, grew slower, had fewer flowers, pods

and seeds and had shorter final heights. Similarly, cotton-

seed imbibed at low temperatures, produced plants with reduced

heights, delayed fruiting, and adveresely affected fiber qual-

ity (Christiansen and Thomas, 1969). The degree of damage was

in direct proportion to the amount of time the seeds were ex-

posed to the low temperature. Imbibition of soybeans at 50

for 12, 24, or 36 hours before planting and germination in a

greenhouse at 20 to 300 reduced survival, dry matter accumula-

tion and seedling heights after two weeks compared to soybeans

imbibed at 250 (Obendorf and Hobbs, 1970). In a later investi-

gation, Hobbs and Obendorf (1972) reported that surviving plants

from soybeans imbibed at 50 for 36 hours were shorter, produced

fewer fruits and seeds compared to plants from soybeans imbibed

at 250 for 12 hours.







The sensitivity to low temperature during germination

appears to occur early during seed imbibition. Pollock and

Toole (1966) noted that lima bean seeds and excised embryonic

axes had reduced survival rates and seedling size when imbibed

at 5 or 15C then allowed to grow at 250. However, if the

seeds were initially imbibed at 250 for two or six hours before

low temperature exposure, seed and seedling injury was much

less or completely avoided. Exposure of cottonseed to 50

during imbibition, for as little as 30 minutes, resulted in

a reduction in germination rate and an increase in seedling

abnormalities (Christiansen, 1968). The sensitivity to low

temperatures in cottonseed persisted for the first two to four

hours of imbibition. If the seeds were initially imbibed for

four hours at 310, the optimum temperature for germination, they

were less sensitive to subsequent chilling temperatures.

Likewise, Bramlage et al. (1978) determined that soybean em-

bryos had reduced axis elongation if imbibed for 30 minutes

at 140 then placed at 280 for three days. Additionally,

injury resulted to soybean embryos which were imbibed at 20

for as little as five minutes. Bramlage et al. (1978) pro-

posed that chilling injury may occur during the initial hy-

dration of the embryo and the time to injury depends on seed

protection structures and the rate of imbibition.







Low Temperature Imbibition and Membrane Reorganization


Chilling injury due to low temperature imbibition of seeds

has largely been attributed to the affect water and tempera-

ture has on the cellular membranes (Simon, 1974; Bramlage et

al., 1978; Leopold, 1980). Using the fluid mosaic model of

cellular membranes, Simon (1974) described the effect water

content has on membrane architecture. In animal tissues using

X-ray diffraction, it was noted that as water content of the

membrane fell below 20%, the phospholipids in the cell membrane

rearranged from a lamellar phase to a hexagonal phase. The

hexagonal state would be more porous, permeable to solutes and

would initially allow substances to rapidly diffuse out when

cells were rehydrated. Lyons (1973) determined that a similar

phase change occurred in plant mitochondrial membranes as

temperatures were lowered. The phase change from the liquid-

crystalline lamellarr) to the solid-gel (hexagonal) state

occurred at 10 to 120C. This correlated with the tempera-

tures at which injury occurred in chilling sensitive tissues.

Ilker et al. (1979) found that ultrastructural chilling symp-

toms, as examined from electron micrographs, of tomato coty-

ledons held at 50 for two to 24 hours, exhibited themselves

primarily as a progression of membrane deterioration. Mito-

chondrial and nuclear membranes became less definite and more

disorganized in advance of the plasmalemma which appeared more

resistant to chilling.

Lyons et al. (1964) compared several plant species that

differed in sensitivity to chilling. Mitochondrial membranes







from chilling sensitive plants exhibited inflexibility when

compared to chilling resistant plants. Membrane lipid com-

ponents also differed between chilling sensitive and resis-

tant species. Mitochondria of chilling resistant species con-

tained a greater content of unsaturated fatty acids than did

mitochondria from chilling sensitive species. In seeds, phos-

pholipid composition and synthesis in cell membranes may be

critical to low temperature sensitivity (Dogras et al., 1977).

During imbibition of a chilling sensitive species, lima bean,

more labelled 14C-glycerol was incorporated into phosphatidyl-

ethanolamine and phosphatidylglycerol than occurred in broad

beans and peas, chilling resistant species. The latter species

incorporated a higher proportion of the label into phosphatidyl-

choline. Broad beans and peas had a greater unsaturated to

saturated fatty acid ratio compared to lima beans. The authors

speculated, due to the fact that phosphatidylcholine (P.C.) had

a lower temperature requirement for phase change than phospha-

tidylethanolamine (P.E.), that membranes which contained greater

amounts of P.C. than P.E. could have lower temperature phase

changes and greater chilling resistance.

The ability of germinated pepper seed to withstand low

storage temperatures was correlated with the degree of unsatura-

tion in total fatty acids in dry seed (Sosa-Coronel and Motes,

1981). Pregerminated seed from pepper cultivars most sensitive

to chilling injury during storage had a lower degree of fatty

acid unsaturation in dry seed extracts compared to less sensitive

cultivars.








Injury to seeds during low temperature imbibition has been

attributed to the inability of cell membrane phospholipids to

rearrange from the dry hexagonal state to the hydrated lamellar

architecture, because they were gelled in a rigid molecular

shape (Simon, 1974). This inability of cellular membranes to

reorganize to the semipermeable condition results in the chil-

ling injury demonstrated by the embryo.

Further support for this theory came in work which de-

termined cellular leakage during imbibition. The majority of

carbohydrate leakage was restricted to the first 18 hours of

imbibition at temperatures of 22 to 300C in pea seeds (Short and

Lacy, 1976). However, at an imbibition temperature of 10,

significant leakage occurred for 48 hours after the addition of

water. Pollock and Toole (1966) noticed that lima bean axes

imbibed at 5 or 150 lost organic material directly and to a

greater extent than occurred at 250. Bramlage et al. (1978)

found that soybean embryos imbibed at 250 had a short period of

rapid leakage that leveled off and became fairly constant.

Imbibition of embryos at 100 or lower prolonged that period of

rapid leakage. These authors suggested that the prolonged period

of rapid leakage at the low temperatures was evidence that low

temperature imbibition interfered with normal membrane reorgan-

ization by altering the physical state of the membrane phospho-

lipids.

Additional support to this theory came from evidence

that seed leakage can be reduced if seed moisture was increased

beforelow temperature imbibition. A reduced cold sensitivity








of soybean embryos occurred when seed moisture was increased

to 35 to 50% by exposure to high humidity before low temperature

imbibition (Bramlage et al., 1978). This increased seed mois-

ture was presumed to allow partial membrane reorganization

before exposure to the cold stress. Protection against chil-

ling injury has also been achieved by raising seed moisture

contents in other cold sensitive species. Chilling resistance

has been imparted to lima beans by an increased seed moisture

content of 20% (Pollock, 1969), peas by an increase to 30%

(Simon and Wiebe, 1975), snap beans by increased moisture con-

tents of 12% or greater (Roos and Manalo, 1976) and corn to

13% or higher (Obendorf et al., 1971).

Contrary to the presumption that seed membranes are in

the hexagonal configuration at low moisture contents and be-

fore imbibition, McKersie and Stinson (1980) reported that no

change in membrane architecture occurred during seed dehydra-

tion and reimbibition. Birdsfoot trefoil seeds at moisture

contents from 5 to 40% were investigated by low and wide angle

X-ray diffraction. No phase change was noted in any sample

and all membrane phospholipids were found exclusively in the

lamellar state. However, dehydration did appear to induce a

subtle reorganization of cellular membranes which altered per-

meability properties but did not involve a phase change or

total loss of selective permeability.

The disorganization of membranes and the increased per-

meability associated with it during low temperature imbibition

can influence other subcellular reactions which can have direct








affects on chilling injury (Lyons, 1973). The phase transition

or the inability of membranes to reorganize completely not only

increased permeability but also increased the activation energy

of membrane-bound enzyme systems. This could lead to a sup-

pressedreaction rate of the mitochondrial krebs cycle and an

imbalance with the nonmembrane-bound glycolysis system allowing

accumulation of metabolites (ie. pyruvate, acetaldehyde and

ethanol). Accumulation of these metabolites can cause injury

or death depending on the ability of the species to withstand

or metabolize these compounds.

Mitochondrial respiration was suppressed as temperature

was reduced below the temperature associated with membrane

phase changes in chilling sensitive tissues (Lyons and Rai-

son, 1970). Arrhenius plots of mitochondrial respiration

isolated from soybean embryonic axes held at 23 and 100C dif-

fered (Duke et al., 1977). The cold treated axes had an inflec-

tion in the Arrhenius plot at 80, whereas control axes held at

230 had an inflection temperature at 12.50. Duke et al. addi-

tionally determined that the slopes of the plots for axes NADP-iso-

citrate dehydrogenase were very similar to those for mitochon-

drial respiration isolated from the control axes. They suggested

that this enzyme may limit mitochondrial respiration at low

temperatures in soybean tissue.

Other subcellular systems are affected by low temperature

imbibition. DNA synthesis in cottonseed was reduced drasti-

cally by exposure to 20C for five hours during a sensitive








period of imbibition between 30 and 36 hours (Clay et al.,

1976). The reduced DNA synthesis was correlated with reduced

DNA polymerase activity. When a less chilling sensitive geno-

type of cotton was compared to more sensitive varieties, the

resistant type's nuclear DNA polymerase activity was more

efficient. Chilling resistance was also correlated with a higher

degree of unsaturation in the nuclear membranes of the resistant

genotype. Clay et al. (1977) concluded that even though many

physiological parameters were probably affected adversely by

chilling stress, the capacity to synthesize DNA following chil-

ling was closely associated with seedling performance under

unfavorable temperatures.

Protein synthesis, glutamine synthetase, acid phosphatase

and fumarase levels decreased in crimson clover seeds when

germinated at 100C compared to 200 (Ching, 1975). However,

no differences were found in soluble protein, protease, a-

amylase, ATPase and RNase levels between the two germinating

temperatures.

The effect of imbibitional chilling injury on energy

metabolism in corn was examined by Cohn and Obendorf (1976).

Corn kernels at 5 and 13% moisture were imbibed at 50C for

12, 24 or 48 hours. Radicle growth was reduced in the low

moisture seeds exposed to the 50 temperature, indicating

chilling damage. However seed respiration, ATP content and

energy charge of the embryos were the same regardless of initial

moisture content. ADP/0 ratios and respiratory control ratios

of isolated mitochondria were not altered by initial kernel








moisture. The authors concluded that disruption of energy me-

tabolism was not the primary cause of seed moisture-mediated

imbibitional chilling injury.

Contrary to previously mentioned work, Simon et al. (1976)

found that failure of cucumber seeds to germinate in the cold

was not due to imbibition injury, loss of membrane integrity,

or failure of mitosis in the cold. Cucumber seed failed to

germinate at cold temperatures when first imbibed for 12 hours

at 20 C and there was no evidence for loss of membrane integrity

in the cold. Mitosis was eliminated as a restriction to low

temperature germination when cucumber seeds germinated in the

presence of colchicine or after y-irradiation at 200. The

authors proposed, after reviewing high Q10 figures during low

temperature germination, that failure to germinate may result

from protein denaturation. Denaturation of proteins may have

inhibited low temperature germination by inactivating enzymes

or preventing the orderly association of proteins into organelles.

Growth Regulator Activity as a Function of Low Temperature
Germination

Growth regulator formation or activation has been indicated

to affect the rate, in part, in which tomato seeds could ger-

minate at low temperatures (Abdul-Baki and Stoner, 1978; Maluf

and Tigchelaar, 1980). Abdul-Baki and Stoner (1978) reported

the presence of a growth promoter and an inhibitor in leachates

from tomato seed which were possibly responsible for the ability








of tomato seeds to germinate at low temperatures. Leachate

from seeds of PI 341984, a line which germinated well at low

temperatures, promoted germination of that line and other

tomato cultivars at 1000C. The leachate from 'Red Rock' seeds,

a cultivar which germinated poorly at low temperatures, in-

hibited germination of that and other tomato cultivars at

100. The growth regulator activity was highest in fresh seed,

declined with age and responses were best exhibited at low

temperatures. The researchers did not identify the promoting

or inhibiting substances nor did they determine the exact role

or mode of action these substances had with respect to each

other or tomato germination. However, they did find the pro-

motive and inhibitory effects of the leachates were highly

specific and restricted to tomato seeds. In support of Abdul-

Baki and Stoner's work, Maluf and Tigchelaar (1980) found ger-

mination of noncold-germinating lines of tomato could be

enhanced by the addition of activated carbon to the germination

media. No such enhancement of germination was found when cold-

germinating lines of tomato were tested. These researchers

determined that inhibition or delay in germination of noncold-

germinating lines in tomato was due, in part, to a low temp-

erature activation or formation of an activated carbon ad-

sorbable inhibitor of seed germination.








Low Temperature Germination as it Relates to Genetic
Variability

The ability of seeds to germinate at low temperatures, in-

dependent of physiological considerations, appears to be geneti-

cally controlled within individual species. Gerson and Honma

(1978) in reviewing 105 cultivars from five species of Cap-

sicum for emergence response from low soil temperatures found

great variability between species and within species. Mean

emergence indices of seven pepper species and subspecies at 16C

ranged from 22.9 for C. baccatum v. pendulum to 49.0 for C.

baccatum v. microcarpum. Within C. annuum at 160, mean emer-

gence indices varied from 20.6 to 46.5 for the 80 cultivars

tested. From these results the authors suggested the ability

to germinate at low temperatures may be heritable in peppers.

Differences in the degree of chilling sensitivity within

species, among cultivars, has also been reported for the chilling

sensitive crops of cotton (Buxton and Sprenger, 1976), soybean

(Hopper et al., 1979), corn (Pinnell, 1949; Cal and Obendorf,

1972 a and b) and carrot (Hegarty, 1973).

The inheritance of low temperature germination ability in

tomato has been widely reviewed (Cannon et al., 1973; DeVos et

al., 1981; El Sayed and John, 1973; Gatherum et al., 1970;

Maluf and Tigchelaar, 1980; Ng and Tigchelaar, 1973). However,

the genetic nature and mode of inheritance for low temperature

germinability is still unclear and debated. Reported claims

from single gene control (Cannon et al., 1973) to polygenic








and primarily additive with dominance for inability to germi-

nate at suboptimal temperatures (Ng and Tigchelaar, 1973) are

found within the literature. Cold germinating accessions

have been identified in tomato with the ability to reach 50%

germination in eight days at 100C, whereas noncold-germinating

accessions may take 22 days or longer to reach 50% germination

(Maluf and Tigchelaar, 1980). The emphasis on studying the

inheritance of low temperature germination in tomato seed was

an attempt to incorporate low temperature sprouting ability

into commercially acceptable cultivars to allow early spring

direct seeding of tomatoes.

Seed Treatments for Enhancing Low Temperature
Germinability

A number of seed treatments have had favorable results on

the subsequent performance of seeds under conditions of low

temperature stress. Ells (1963) described a seed treatment in

which tomato seeds were placed on filter paper moistened with

1% potassium phosphate and 1% potassium nitrate solution for

six days. The seeds were dried at room temperature to approxi-

mately the original moisture content. This treatment proved

beneficial in advancing seedling emergence by up to five days

over untreated seed when planted in a greenhouse with a night

temperature of 100C. Ells attributed the effect of this seed

treatment not primarily to the salts but to the osmotic pressure

maintained by the salt solution. The solution prevented seed

germination but permitted enough moisture to enter the seed to

"foster enzymatic activities."








Similarly, other researchers increased germination rates

of tomato seed at low temperatures by pretreating seeds with

potassium salt soaks (Oyer and Koehler, 1966; Wickham and Nicols,

1976; Dimov et al., 1977). The seed treatments increased the

speed of germination at low temperatures and increased the

uniformity of germination.

Pregermination treatments with potassium salts have had

favorable results on other crops. Watermelon (Sachs, 1977),

pepper (Sachs et al., 1980), carrot, celery, cucumber, egg-

plant, muskmelon and Kentucky blue grass (Malnassy, 1971) ex-

hibited increased germination rates at low temperatures when

pretreated in aerated potassium salt solutions for several

days then redried.

Polyethylene glycol (PEG) has proved favorable as an os-

moticum for the pretreatment of seeds for low temperature ger-

mination (Heydecker, 1974). Heydecker and associates demon-

strated the ability of several species of seeds, including

onion, carrot, french bean and geranium, to germinate more

rapidly and uniformly after osmotic treatment with PEG (Hey-

decker et al., 1973; Heydecker, 1974). The benefit gained

from osmotic pretreatment before low temperature germination

was retained when onion seeds were dried and held in storage

for eight weeks. Knypl and Khan (1981) improved the perfor-

mance of soybean seeds at low temperatures with osmotic pre-

treatment. The seeds were treated by placing them on filter

paper moistened with a PEG-6000 solution (-8.6 to -11.0 bars)








for four to eight days at 150C in a sealed container. The seeds

were then removed from the container, washed and dried. Similar

pretreatment of pepper seeds allowed for faster, more uniform

germination at 250 in the laboratory and greenhouse, but did

not speed germination in the field with cool soil temperatures

(Yaklich and Orzolek, 1977).

These hydration-rehydration treatments performed on seeds

with salt or PEG solutions have generally been referred to as

priming (Malnassy, 1971; Heydecker, 1974). That is, the tech-

nique of starting germination processes, bringing them to the

brink of cell elongation and radicle emergence then stopping

all processes by dehydration. This process apparently advan-

ced the onset of germination by increasing the permeability of

the seed coat, and initiating metabolic events which would

withstand dehydration, such as increased protease activity

(Berrie and Drennan, 1971). Increased protein levels and

activation and/or synthesis of a number of enzymes, including

acid phosphatases and esterases, have been reported during

priming of seeds (Koehler, 1967; Khan et al., 1978). Hanson

(1973) noted that oxygen uptake and leucine incorporation in

protein was enhanced in aleurone layers, more than in embryos,

isolated from primed wheat seed. In addition, amylase ac-

tivity developed faster in primed seed than in non-primed seed

during germination. Sen and Osborne (1974) found that when rye

embryos were hydrated for three to six hours then dehydrated,

the embryos when rehydrated had a rate of protein synthesis

equal to the pretreatment time plus the duration of the second








imbibition time. Additionally, hydration-dehydration treatments

enhanced subsequent RNA synthesis and if the hydration time was

increased to nine hours before dehydration, DNA synthesis began

immediately upon rehydration. Sen and Osborne (1974) concluded

that many changes that occurred during the period of hydration

pretreatment must be stable to subsequent dehydration.

Priming, however, can be detrimental if during the hy-

dration step embryo growth was apparent or radicle emergence

occurred (Berrie and Drennan, 1971). Dehydration usually then

resulted in some embryo or radicle damage.

The use of a gibberellic acid (GA) as a pretreatment to

seed has proved beneficial in speeding germination of some

species at suboptimal temperatures. Nelson and Sharples (1980)

reported the rate and total germination of cucumber seeds ger-

minated at 120C was increased by acetone infusion of fusicoccin,

GA4/7 or GA3. Gibberellic acid4/7 treatment was less effective

than fusicoccin, but more effective than the GA3. Gibberellic

acid4/7 treatments to muskmelon seeds increased total germina-

tion at 160, bud did not increase germination rate over untreated

seeds. However, no fusicoccin or gibberellic acid treatment

was effective in promoting watermelon germination at low temp-

eratures. Cole and Wheeler (1974) noted that chilling injury

to cottonseed was reduced at suboptimal temperatures by a pre-

conditioning in water or gibberellic acid. These authors

suggested that elevated seed moisture in combination with

growth regulator treatments may be a satisfactory treatment for

obtaining cotton stands at lower than optimal germination
temperatures.





20


In the study which follows, germination of sweet pepper

(Capsicum annuum) seed was investigated to determine the cause

or contributing causes of slow germination rate at a low

temperature.













CHAPTER II
GROWTH HORMONE INVOLVEMENT IN GERMINATION OF Capsicum
annuum L. AT LOW TEMPERATURE


The control of germination at lower than optimal temperatures

may be governed by the presence of growth inhibitors or promoters.

Abdul-Baki and Stoner (1978) described the presence of a growth

promoter in the leachate from the seed of a tomato accession,

PI341984, which germinated well at low temperatures. The leach-

ate promoted germination of seeds at 100C of the same and other

tomato cultivars. Leachates from the seed of the tomato cul-

tivar, Red Rock, which germinated poorly at low temperatures

inhibited or slowed germination of tomato seeds. The promotive

and inhibitory components of the leachates were not isolated or

identified, but they were found to be highly specific and

restricted in nature to tomato seeds. The presence of a low

temperature germination promoter and inhibitor was further

supported by work done by Maluf and Tigchelaar (1980). They

found that germination of noncold-germinating lines of tomato

was improved by adding activated carbon to the germination

media. They felt that inhibited or slowed germination at 100

by noncold-germinating lines was, in part, due to a low tempera-

ture triggered activation or formation of an activated carbon

adsorbable inhibitor.

The addition of growth regulators to seeds or to the

germination media has proved beneficial in stimulating germination







under adverse temperature conditions. Cytokinins have been

used to overcome thermodormancy and the inhibitory effect of

abscissic acid on lettuce seed germination (McCoy and Harring-

ton, 1970; Reynolds and Thompson, 1973). Cytokinins have also

been suggested as having the permissive role in seed germina-

tion because of their ability to oppose the effect of an in-

hibitor and their inability to trigger seed germination per se

(Khan, 1971). The primary role in the control of seed germination

can be attributed to gibberellins. The ability of gibberellic

acid (GA) to control many germination processes has been well

documented (Mayer and Shain, 1974) including the production of

a-amylase by de novo synthesis in barley endosperm (Varner,

1964). Gibberellins, GA3 and GA4+7, have been beneficial in

stimulating germination of several species at unfavorable

temperatures. Cucumber, cotton, and lilac (Syringa reflexa)

have all been stimulated to germinate faster and more uni-

formly at low temperatures by GA application (Nelson and

Sharples, 1980; Cole and Wheeler, 1974; Junttila, 1973).

Auxin, indoleacetic acid (IAA), is an important plant

growth hormone which is active in many growth processes.

However, its role in seed germination is relatively unknown

(Tillberg, 1977). Exogenously applied auxin generally has

little or no effect on seed germination. Tillberg (1977)

felt that seeds may already contain adequate levels of auxin

for germination and thus extra quantities of the hormone

would be without effect.








The experiments which follow investigated the effect of

growth hormones on Capsicum annuum seed germination at opti-

mal and low temperature (150C). Determinations were made

to find if low temperature stimulated the formation or acti-

vation of a germination inhibitor.


Materials and Methods


Seed source. Seeds used in all experiments were from

the same seed lot of Capsicum annuum 'Early CalWonder' (Lot

number 0031019) obtained untreated from Petoseed Company,

Saticoy, California. The seeds were stored in plastic bags

at 100C and 50% relative humidity until used.

Germination procedure and data. Seeds were germinated

at 15 and 250C on moistened Whatman No.1 and No.3 filter papers

in 5.5 or 9 cm Petri dishes. Germination data for all of the

experiments were taken at 24 hour intervals and all treatments

were replicated 4 times with 25 seeds per replicate. Seeds

with visible radicles were counted as germinated. Data taken

for each treatment included total percent germination and mean

number of days to germination. The mean number of days to

germination (MDG) was calculated by the formula adapted from

an emergence index used by Gerson and Honma (1978):


MDG= z(days to germination) (number of seeds germinated)
total number of seeds germinated


Imbibition rate of pepper seeds. Seeds were imbibed at

15 and 25 C in distilled water. After the appropriate treat-

ment time, the seeds were removed, blotted dry, then weighed.








The total number of seeds which germinated were counted, the

seeds were dried at 700 for 48 hours, and dry weights were

recorded.

Determination of inhibitor activity after low temperature

imbibition. To determine if there was a water soluble leachable

low temperature germination inhibitor present in pepper seeds,

5 g of air dry seed were leached in 40 ml of cold distilled

water, on a reciprocating shaker for 0, 2, 4, or 8 days at

40 0.50C. The water was changed midway during each leaching

period and saved. Moist, fully imbibed, and dry seeds were also

subjected to the 40 temperature as controls. At the end of each

leaching period, seeds were taken for germination tests.

Samples were prepared by combining the leachate from each

of the time periods, freezing at -200C and then concentrating

the leachate by freeze-drying. The freeze-dried residue was

taken up in 10 ml of distilled water then filtered through a

double thickness of Miracloth. Pepper seeds were germinated

in 1 ml of the filtered, concentrated sample, representing

leachate from 0.5 g of seed. The osmotic potential of the

sample was determined by a vapor pressure osmometer (Wescor Inc.

Logan, Utah) and suitable osmotic comparison treatments were

prepared using a polyethylene glycol (PEG-6000) (Michel and

Kaufmann, 1973) or a 0.2M sodium phosphate buffer (ph 6.5)

(Ross, 1974) diluted to the appropriate osmotic potential.

Seeds leached for 0, 2, 4 or 8 days, representing approxi-

mately 4.5 g of air dry seed, were homogenized in distilled

water by a Sorvall Omni-mixer for 5 minutes. The homogenate








was filtered and washed through a double thickness of Miracloth,

centrifuged for 10 minutes at 3600 X g, and concentrated in a

freeze dryer. The residue was taken up in 9 ml of distilled

water and 1 ml was used for germination tests.

Organically extractable inhibitor activity after low

temperature imbibition. Ethanol and acetone extractions were

performed on 5 g of either a) air dry pepper seed b) seeds

imbibed 2 days at 25C, or c) seeds imbibed 4 days at 150

The seeds were homogenized in 10 ml of absolute ethanol or

redistilled acetone in a Virtis homogenizer for 5 minutes then

filtered through a double thickness of Miracloth. Seeds were

treated in Petri dishes containing 1 ml of the filtrate and

4 ml of distilled water or the appropriate organic solvent.

The Petri dishes were sealed and placed in a constant tempera-

ture chamber (250) for 2 hours. Immediately after treatment, the

solutions were decanted and the seeds were vacuum dried in a

desiccator for 2 hours. After drying, germination tests were

performed.

Application of growth hormones to pepper seeds germinated

at low temperatures. IAA and gibberellin A3 or A4+7 were

applied in concentrations from 0 to 1000 ppm to pepper seeds

to determine if they would stimulate germination at low

temperature. Kinetin was similarly tested at concentrations

of 0 to 100 ppm. The IAA, GA3 and kinetin were obtained from

Sigma Chemical Company, St. Louis, Missouri, and GA4+7 was

obtained from Imperial Chemical Industries, Plant Protection

Limited, Surrey, England. The IAA was dissolved in 2 ml of








acetone whereas the GA's and kinetin were dissolved in 10%

KOH. The solutions were corrected to pH 6.5 with 10% HC1

and used to moisten the filter papers for germination tests.

Redistilled acetone was also used to apply GA4+7 at

100 ppm to pepper seeds. Seeds were soaked in 5 ml of

acetone or acetone containing GA4+7, 100 ppm, for 0, 2, 4,

8, 12, or 24 hours at 250C. After the prescribed durations,

the solutions were decanted and the seeds were dried in a

vacuum desiccator for 2 hours. Germination tests were then

performed.

Effect of osmotic potential on pepper seed germination.

Pepper seeds were germinated in PEG-6000 solutions from -1 to

-8 bars with and without GA4+7, 100 ppm. The osmotic poten-

tials of the aqueous solutions of PEG-6000 were determined

from Michel and Kaufmann's (1973) paper.

Pepper seed germination with a GA synthesis inhibitor.

At 250C, 1.5 ml of GA4+7 at 0, 10, 100 and 1000 ppm in

combination with 2'isoproply-4'-(trimethylammonium chloride)-

5' methylphenyl piperidine carboxylate, (AMO-1618), a GA syn-

thesis inhibitor, at 0, 100 and 1000 ppm was used as a germina-

tion medium for pepper seeds.

Results

Total germination of pepper seeds does not differ at 15

and 250C (Figure 2-1). However, germination at 150 becomes

less uniform and the MDG is 2 times that at 250 (Figure 2-2).




























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Water uptake at 15 and 250C was similar and increased

with imbibition time until 12 hours (Figure 2-3). After 12

hours, the moisture content of pepper seeds remained constant

until germination exceeded 50%, then the moisture content

increased.

Leaching pepper seeds in cold distilled water for 2, 4 or

8 days had no effect on the mean number of days to germina-

tion at 250C when compared to unleached seed (Table 2-1).

Seeds which were kept moist for 8 days germinated slower than

unleached seed. A similar treatment for 4 days germinated

slower than 4 and 8 day unleached seeds. There was no diff-

erence noted in the total germination of leached, moist or

unleached seeds when germinated at 250 (Table 2-2).

Pepper seeds leached in cold distilled water for 8 days and

germinated at 150C, germinated at the same rate as the unleached

and moist seeds after the same treatment time (Table 2-3). Leach-

ing pepper seeds for 2 or 4 days speeded the germination rate of

these seeds when compared to unleached or moist seeds after the

same treatment times. However, pepper seeds leached 2 days, ger-

minated at a faster rate than any other treatment. Total germina-

tion of pepper seeds was unaffected by leaching when germinated

at 150 (Table 2-4).

Seeds germinated slower in the leachate extract than the

untreated control at 250C (Table 2-5). Germination in the -2

bar sodium phosphate buffer was also slower than the untreated

control. There was no difference in the total germination with

any treatment at 250. When germinated at 150, untreated seeds
























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Table 2-1. The effect of leaching seed at 40C for various times on the
mean number of days to germination (MDG) when germinated
at 250C



Days Treated
0 2 4 8

(MDG)

Leached Seed -- 6.4 c 7.1 abc 7.2 abc

Moist Seed -- 6.6 c 7.8 ab 8.2 a

Unleached Seed 6.7 bcz 6.7 bc 6.5 c 6.6 c


ZMean separation by Duncan's multiple range test, 5% level.






Table 2-2. The effect of leaching seed at 40C for various times on the
total germination when germinated at 250C



Days Treated
0 2 4 8



Leached Seed -- 79 a 73 a 75 a

Moist Seed -- 83 a 75 a 70 a

Unleached Seed 76 az 74 a 81 a 75 a


ZMean separation by Duncan's multiple range test, 5% level.











Table 2-3. The effect of leaching seed at 400 for various times on the
mean number of days to germination (MDG) when germinated
at 150C



Days Treated
0 2 4 8

(MDG)

Leached Seed -- 8.0 c 9.4 b 9.2 b

Moist Seed -- 9.2 b 10.7 a 10.2 ab

Unleached Seed 10.1 abz 10.6 a 11.0 a 10.1 ab


ZMean separation by Duncan's multiple range test, 5% level.








Table 2-4. The effect of leaching seed at 40C for various times on the
total germination when germinated at 150C



Days Treated
0 2 4 8

(%)
Leached Seed -- 76 a 74 a 78 a

Moist Seed -- 80 a 67 a 75 a

Unleached Seed 72 az 68 a 78 a 77 a


ZMean separation by Duncan's multiple range test, 5% level.
















Table 2-5. The effect of leachate, from 0.5 g of seed, on the
germination of pepper seed at 25 and 150C



250 150
Treatments Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.6 bz 78 a 8.5 d 79 ab
2 day leachate 5.3 a 80 a 10.8 b 82 ab

4 day leachate 5.4 a 80 a 10.1 bc 86 ab

8 day leachate 5.6 a 85 a 10.6 b 86 ab

-1 bar PEG-6000 4.6 b 82 a 8.8 d 91 a

-2 bar PEG-6000 4.4 b 84 a 9.7 c 84 ab

-2 bar sodium
phosphate buffer 5.4 a 79 a 11.6 a 77 b


ZMean separation within
5% level.


columns by Duncan's multiple range test,








and seeds soaked in -1 bar PEG-6000 germinated more rapidly than

all other treatments. Seeds soaked in -2 bar sodium phosphate

buffer germinated slower than all other treatments. Total ger-

mination in all treatments was similar to the untreated control

at 150

Filtrate from ground seeds which had been previously leached

slowed the rate of germination compared with the untreated control

at 250C (Table 2-6). Total germination of treated seeds at 250

was similar to the untreated control. After 2 or more days of

leaching, the extract of ground leached seeds reduced germination

rate at 150 compared to the untreated control. When these leached

seed treatments were compared to the -3 bar PEG or -2 bar sodium

phosphate treatments, germination rate was unaffected. Total

germination was again unaffected by germinating the seeds in

a ground seed extract compared to the untreated control.

Soaking pepper seeds in 20% ethanol extract had no affect

on germination at 250C (Table 2-7). Adding ethanol seed ex-

tracts to seeds germinated at 150 slowed germination rates

compared to the untreated control. The ethanol extracts did not

affect total germination at 150

When seeds were soaked in similar extracts containing

absolute ethanol, total germination at 25 or 150C was unaffected

(Table 2-8). However, ethanol extracts slowed germination rate

over both controls at 250. All ethanol treatments increased

the MDG of pepper seeds germinated when compared to the untreated

control at 150.

Similar 20% acetone extracts had no affect on germina-

tion rate at 250C or total germination at 25 or 150 (Table 2-9).

















Table 2-6. The effect of an aqueous solution from ground leached seed,
0.5 g/treatment, on pepper seed germinated at 25 and 150C



250 150
Treatments Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.8 bz 79 ab 9.3 c 84 ab

0 day leached seed 7.2 a 77 ab 10.9 abc 77 b

2 day leached seed 6.6 a 71 b 12.2 a 80 ab

4 day leached seed 7.5 a 77 ab 11.2 ab 78 ab

8 day leached seed 7.2 a 82 ab 11.2 ab 77 b

-2 bar PEG-6000 4.4 b 84 a 9.7 bc 84 ab

-3 bar PEG-6000 4.5 b 85 a 12.3 a 88 a

-2 bar Sodium
phosphate buffer 5.4 b 79 ab 11.6 a 77 b


ZMean separation within columns by Duncan's multiple range test,
5% level.









Table 2-7. Germination of pepper seeds at 25 and 150C when treated
with 1 ml of ethanol seed extract diluted with 4 ml of
distilled water



250 150
Treatments. Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.8 az 84 a 10.4 c 78 a

Dry seed-extract 5.9 a 82 a 13.1 b 81 a

4 day imbibed seed
at 150-extract 5.3 a 87 a 15.9 a 87 a


ZMean separation within columns by Duncan's multiple range test,
5% level.


Table 2-8. Germination of pepper seeds at 25 and 15C when treated
with 1 ml of ethanol seed extract diluted with 4 ml of
ethanol



250 150
Treatments Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.8 bz 84 a 10.4 b 78 a

Ethanol soak
control 6.4 b 88 a 14.1 a 84 a

Dry seed-extract 7.6 a 85 a 14.3 a 76 a

2 day imbibed seed
at 250-extract 8.6 a 80 a 14.8 a 73 a

4 day imbibed seed
at 150-extract 8.6 a 80 a 13.8 a 81 a


ZMean separation within
5% level.


columns by Duncan's multiple range test,









Table 2-9. Germination of pepper seeds at 25 and 150C when treated
with 1 ml of acetone seed extract diluted with 4 ml of
distilled water



250 150
Treatments Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.8 az 84 a 10.4 b 78 a

Dry seed-extract 5.1 a 77 a 12.3 a 86 a

4 day imbibed seed
at 150-extract 4.6 a 86 a 11.9 a 74 a


ZMean separation within columns by Duncan's multiple range test,
5% level.




Table 2-10. Germination of pepper seeds at 25 and 150C when treated
with 1 ml of acetone seed extract diluted with 4 ml of
acetone



250 150
Treatments Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)

Untreated control 4.8 bz 84 a 10.4 b 78 a

Acetone soak
control 10.9 a 79 a 17.7 a 78 a

Dry seed-extract 13.8 a 75 a 17.3 a 78 a

4 day imbibed seed
at 150-extract 10.8 a 76 a 16.4 a 84 a


ZMean separation within
5% level.


columns by Duncan's multiple range test,








Germination rate at 150 was slower in the acetone treatments

when compared to the untreated control.

Extracts in 100% acetone slowed the rate of germination

when compared to the untreated control at both 25 and 150C

(Table 2-10). The treatments did not alter total germination

percentage at either temperature.

IAA concentrations from 0 to 500 ppm did not effect the

speed of germination at 250C (Table 2-11). At 1000 ppm, IAA

reduced the germination rate at both 25 and 150. Concentra-

tions of IAA from 0 to 250 ppm had no effect on germination

rate at 150; however, higher concentrations slowed germination.

IAA treatments had no effect on the total germination of pepper

seeds at 25 or 150

Kinetin, 1 to 100 ppm, did not alter germination rate or

total germination of pepper seeds germinated at 25C (Table

2-12). At 150, 0 ppm treated seeds germinated faster than the

50 or 100 ppm kinetin treatments. Total germination was un-

affected by kinetin at 15.

At 250C, seeds germinated in GA3 had faster germination

rates than the 0 ppm treatment (Table 2-13). At 150, only the

three highest concentrations of GA3 (100, 500 and 1000 ppm) sig-

nificantly increased the germination rate when compared to the

0 ppm treatment. The GA3 applications had no effect on the total

germination of pepper seeds at 25 or 150

All GA4+7 applications at 25 or 15C stimulated germina-

tion rates when compared to the 0 ppm (Table 2-14). Applications

of GA4+7 did not affect the total germination.

















Table 2-11. The effect of indole acetic acid (IAA) concentration on
germination of pepper seeds at 15 and 250C



250 150
IAA Concentration Germ. Total Germ. Total
Rate Germ. Rate Germ.

(ppm) (MDG) (%) (MDG) (%)

0 4.0 bcz 82 a 8.8 c 85 a

10 4.2 bc 86 a 8.7 c 86 a

25 4.1 bc 83 a 8.6 c 82 a

50 4.0 bc 85 a 8.6 c 83 a

100 4.0 bc 87 a 8.6 c 83 a

250 4.2 bc 77 a 8.7 c 82 a

500 4.4 b 85 a 9.3 b 87 a

1000 5.3 a 78 a 9.7 a 81 a

Acetone control 3.8 c 84 a 9.3 b 85 a


ZMean separation within
5% level.


columns by Duncan's multiple range test,


















Table 2-12. The effect of kinetin (Kn) concentration on germination
of pepper seeds at 15 and 250C



250 150
Kn Concentration Germ. Total Germ. Total
Rate Germ. Rate Germ.

(ppm) (MDG) (%) (MDG) (%)
0 3.8 az 77 a 7.8 b 86 a

1 4.0 a 86 a 8.1 ab 78 a

5 4.2 a 85 a 8.3 ab 80 a

10 4.0 a 84 a 8.2 ab 76 a

25 3.9 a 87 a 8.1 ab 85 a

50 4.2 a 86 a 8.4 a 74 a

100 3.8 a 79 a 8.6 a 82 a


ZMean separation
5% level.


within columns by Duncan's multiple range test,




















Table 2-13. The effect of gibberellic acid3 (GA ) concentration on
germination of pepper seeds at 15 and 250C


GA3 Concentrationz


(ppm)

0

10

50

100

500

1000


250
Germ. Total
Rate Germ.

(MDG) (%)

4.7 ay 79 a

4.5 b 91 a

4.3 bc 83 a

4.4 bc 78 a

4.2 c 84 a

4.4 bc 85 a


150
Germ. Total
Rate Germ.

(MDG) (%)

12.9 a 81 a

12.2 ab 79 a

12.3 ab 80 a

11.6 b 84 a

11.7 b 76 a

11.7 b 86 a


ZGibberellic acid was in 0.05 M Sodium phosphate buffer.

YMean separation within columns by Duncan's multiple range test,
5% level.




















Table 2-14. The effect of gibberellic acid4+ (GA4 .) concentration
on germination of pepper seeds at 15 and 25C


GA4+7 Concentrationz


(ppm)

0

10

50

100

500

1000


250
Germ. Total
Rate Germ.

(MDG) (%)

4.7 ay 79 a

4.2 b 82 a

3.9 bc 78 a

3.8 c 75 a

3.7 c 80 a

3.7 c 82 a


150
Germ. Total
Rate Germ.

(MDG) (%)

12.9 a 81 a

11.5 b 79 a

10.7 c 79 a

10.8 c 86 a

10.7 c 82 a

10.9 c 85 a


ZGibberellic acid was in 0.05 M Sodium phosphate buffer.

YMean separation within columns by Duncan's multiple range test,
5% level.








All GA4+7 applications in acetone generally increased the

germinaton rate of pepper seeds compared to the untreated control

or acetone treated seeds at 250C (Table 2-15). Seeds soaked

in acetone for 2 or 24 hours and GA4+7 in acetone for 8 or

24 hours, reduced total germination at 250 (Table 2-16). All

GA4+7 applications in acetone increased the germination rate

compared to acetone treatments or untreated control at 150

(Table 2-17). Acetone treatments with or without GA4+7 had no

affect on the total germination compared to the untreated con-

trol (Table 2-18).

Increasing the osmotic concentrations of the germination

medium over -4 bars greatly reduced germination rate at 250C

(Table 2-19). Gibberellic acid4+7 applications increased ger-

mination rate at 250 regardless of osmotic concentration.

Total germination was reduced at -8 bars when compared to the

untreated control. Similar results were observed with GA4+7 appli-

cation at 150 when the osmotic concentration of the germination

medium was varied (Table 2-20). The -4 bar treatment germinated

more rapidly than the -6 bar treatment and -8 bar treatment

did not germinate. Gibberellic acid4+7 applications reduced

the time to germination at the -4 and -6 bar osmotic concentra-

tions. Total germination was not affected by osmoticum, except

that no seeds germinated at the -8 bar treatments.

The application of AMO-1618 at 100 or 1000 ppm in the absence

of GA4+7 reduced the rate of germination when compared to the

untreated control (Table 2-21). Application of GA4+7 overcame the

inhibition of AMO-1618 on germination rate. There was no






47
























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Table 2-19. The effect of GA+7, 100 ppm, on
in osmotic solut ons at 250C


pepper seed germination


Germ. Rate
+GA -GA


Total Germ.
+GA -GA


(MDG)


Untreated control

-1 bar PEG

-2 bar PEG

-4 bar PEG

-6 bar PEG

-8 bar PEG


ZMean separation within main observations by
test, 5% level.


Duncan's multiple range


Treatment


6.0 efa

6.3 def

7.0 de

7.8 d

10.7 c

17.8 a


80 ab

80 ab

78 ab

86 ab

75 b

6d


















Table 2-20. The effect of GA4+7 100 ppm, on
in osmotic solutions at 150C


Treatment


Germ. Rate
+GA -GA


pepper seed germination


Total Germ.
+GA -GA


(MDG) (%)

Untreated control 7.6 fz 9.6 def 84 a 82 a

-1 bar PEG 7.4 f 9.7 def 85 a 76 a

-2 bar PEG 8.6 ef 10.3 de 79 a 82 a

-4 bar PEG 11.3 d 13.9 c 86 a 83 a

-6 bar PEG 22.3 b 26.6 a 83 a 84 a

-8 bar PEG -- -- 0 0


ZMean separation within main observations by
test, 5% level.


Duncan's multiple range


YNo seeds germinated and this data was not included in the statistical
analysis.











Table 2-21. The effect of AMO-1618 on the germination rate of pepper
seeds at 250C when GA4+7 is applied



GA
GA4+7 AMO-1618 Concentration (ppm)
Concentration 0 100 1000

(ppm) (MDG)
0 5.6 bz 6.7 a 6.7 a

10 4.4 cd 4.5 cd 4.8 c

100 3.8 d 4.2 cd 4.2 cd

1000 4.0 d 4.0 d 4.2 cd


ZMean separation by Duncan's multiple range test, 5% level.





Table 2-22. The effect of AMO-1618 application on the total
germination of pepper seeds at 250C when GA4+7 is applied



GA
G4+7 AMO-1618 Concentration (ppm)
Concentration 0 100 1000

(ppm) (%)

0 84.0 az 87.0 a 85.0 a

10 82.5 a 77.0 a 83.0 a

100 83.0 a 85.0 a 84.5 a

1000 80.0 a 80.0 a 85.5 a


ZMean separation by Duncan's multiple range test, 5% level.








effect of varying the concentration of GA on this enhancement.

AMO-1618 did not affect the total germination (Table 2-22).


Discussion


Pepper seed germination under optimal conditions was uniform

with most radicle emergence occurring after 4 days of imbibi-

tion. However, at 150C, germination became less uniform with

radicle emergence occurring after 7, 8 and 9 days of imbibi-

tion. Similarly, Gerson and Honma (1978) screened 80 culti-

vars of Capsicum annuum for emergence rates at low temperatures.

The average emergence indices for all C. annuum cultivars in-

creased from 17.8 at 180 to 26.5 at 160 and further increased

to 41.5 at 130

Water uptake at 15 and 250C was similar and did not differ

until 50% or more of the seeds germinated. Sachs et al. (1980)

also found little difference in the rate of water uptake in

pepper seeds imbibed at 15 and 250. Water content increased

appreciably only after germination. The authors reported that

any differences in water uptake due to temperature did not

lead to a delay in germination.

There does not appear to be any evidence for a leachable

inhibitor to low temperature germination of pepper seed as was

found for tomato seed (Abdul-Baki and Stoner, 1978; Maluf and

Tigchelaar, 1980). No increase in the rate of germination

was found at 250C when seeds were leached at 40 for up to 8

days. However, the germination rate generally increased at

150 when seeds were leached compared with the unleached seeds.








This may provide evidence for a leachable low temperature

inhibitor, but leaching only for 2 days proved effective

in speeding germination rate when compared to unleached

seeds. Increasing leaching times to 4 or 8 days was not

beneficial in speeding germination. Heydecker (1974) reported

that pepper seeds imbibed at 3, 7 and 100 failed to germinate

but when transferred to 300 germinated faster than seeds placed

at 300 from the outset. In similar experiments, Sachs et al.

(1980) observed that pepper seeds imbibed at 300 for 2 days

enhanced germination rates at 150 when the seeds were not re-

dried after treatment. The benefit of the 2 day leaching time

on pepper seed germination rates may be similar to what Hey-

decker (1974) and Sachs et al. (1980) found.

When seed leachate was applied back to unleached seed,

it slowed germination at 25 and 150C. The PEG-6000 treatments,

osmotic concentrations equal to the leachates, did not account

for the reduction in the speed of germination, but a -2 bar

sodium phosphate buffer treatment reduced the speed of germina-

tion to the same rate or greater than the leachates. Heydecker

(1974) found similar results with pepper seed extracts. The

delay in germination rate imposed by pepper extracts could not

be fully explained by the osmotic potential of the extract,

duplicated by PEG-6000. He felt that one of the components of

the extract imposed a delay in germination chemically. However,

in this study, germination rate was not reduced further by in-

creasing leaching time. Furthermore, germination temperature

had no effect on these trends. Thus, there does not appear to








be a leachable inhibitor which was formed or activated during

low temperature exposure, but merely a salt-osmotic effect

on germination rate due to the addition of the leachate it-

self.

Pepper seeds were extracted in distilled water, ethanol

or acetone to determine if there was a non-leachable germina-

tion inhibitor formed or activated during low temperature

imbibition. Aqueous extracts of leached pepper seeds delayed

germination regardless of leaching time. The delay in ger-

mination at 150 was similar to the osmotic treatments. No

"build-up" or activation of germination inhibitors at low

temperatures was observed in aqueously extracted pepper seeds.

Ethanol extracts inhibited germination rates at 150 when di-

luted in water and at 250 in ethanol. Acetone extracts did

not affect germination rate at 15 or 250 regardless of how

they were applied. Singh and Khattra (1977) found levels of

an abscissic acid-like compound, equivalent to 2 ppm, present

in 80% methanol extractions of Capsicum annuum seeds. If an

abscissic acid-like compound was present in the ethanol ex-

tracts of the present experiments, it did not appear to be

formed or activated in response to low temperature.

Auxin or kinetin applications had no effect on increasing

the germination rate of pepper seeds. However, GA3 and GA4+7

did effectively increase the germination rate at both 15 and

25 C. Gibberellic acid4+7 stimulated the germination rate to

a greater extent than GA3. Gibberellic acid stimulated ger-

mination in cucumber (Nelson and Sharples, 1980), cotton (Cole

and Wheeler, 1974), and Syringa reflexa (Junttila, 1973) at








suboptimal temperatures. Gibberellic acid4+7 was more effec-

tive than GA3 in stimulating germination in cucumber (Nelson

and Sharples, 1980) and Labiatae species (Thompson, 1969).

Gibberellic acid applications were effective in increasing

germination rates whether the seeds were in continuous contact

with the growth regulator or whether it was applied with an

organic solvent before imbibition was initiated.

Gibberellic acid4+7 treatments also had the effect of

increasing germination rate under osmotic stress at 15 and

250C. Conditions of high osmoticum have been associated with

inhibition of cellular expansion while not affecting cell

division (Haber and Luippold, 1960). Gibberellic acid treat-

ments have also been correlated with increasing the water po-

tential of embryos (Junttila, 1973). This suggests that GA

may promote germination rate in pepper by increasing the water

potential (rate of radicle elongation) of the embryo or by

stimulating processes other than cell division. This was

supported by the fact that GA4+7 increased germination rates

of pepper seeds at high (35 and 400) and low (10 and 120)

temperatures (Appendix). High temperatures inhibit germina-

tion in lettuce but not cell division (Haber and Luippold, 1960).

At low temperatures, cell division was delayed in lettuce until

after radicle emergence occurred.

AMO-1618, an inhibitor to GA synthesis, delayed germina-

tion of pepper seeds at 100 and 1000 ppm. However, the delay

in germination was overcome with GA applications. Harvey and

Oaks (1974) found AMO-1618 at 200 pM concentrations inhibited

protease and amylase production in excised corn endosperms.








Additions of GA3 did not overcome this inhibition. Moore (1967)

found that 1 pg of GA3 could overcome the inhibitory effect

that 10 ug of AMO-1618 had on hypocotyl elongation of cucumber

seedlings. It appears from this study that low levels of GA

may be synthesized preceding germination of pepper seeds.

Gibberellic acid synthesis may not be a prerequisite of ger-

mination but in effect could be necessary to allow germina-

tion to proceed at a rapid pace. Added GA stimulated germina-

tion at 15 and 250C by possibly triggering or enhancing some

germination process, such as the formation or activation of

enzymes as seen in monocots or by increasing embryo growth

potential allowing germination to proceed at a more rapid

rate.


Summary


Germination of pepper seeds was reduced by 1/2 and became

less uniform as the temperature was decreased from 25 to 15 C.

Water uptake at 15 and 250 was similar and increased appre-

ciably only after radicle emergence occurred.

There was no evidence for a leachable or extractable

germination inhibitor being activated or formed due to low

temperature exposure. Leachates and aqueous extracts of pepper

seeds did slow germination but the effect appeared to be a

salt-osmotic effect due to the addition of the leachates or

extracts to the germination medium.

Auxin and kinetin applications had no effect on increasing

germination rates of pepper seeds at 15 or 250C. However, GA3








and GA4+7 effectively increased germination rates at both temp-

eratures. Gibberellic acid4+7 was more effective than GA3.

Gibberellic acid applications stimulated germination whether

the seeds were in continuous contact with the growth regulator or

whether it was applied with an organic solvent and redried before

imbibition was initiated.

AMO-1618 applications inhibited pepper seed germination rate

indicating GA synthesis may be necessary for rapid germination

but is not a prerequisite for germination. Gibberellic acid

applications were effective in increasing germination rates at

15 and 250C when seeds were under osmotic stress, a condition

associated with inhibition of cellular expansion but not cellular

division.













CHAPTER III
RESPIRATORY CONTROL OF Capsicum annuum L. GERMINATION AT
LOW TEMPERATURE

The availability of oxygen for germination is critical for

most species. With the exception of certain water plants,

most seeds will not germinate in its absence (Come and Tissaoui,

1973). A 2% oxygen environment inhibited germination of all

dicots tested except Cucumis sativa, Celosia plumosa and Celosia

cristata (Heichel and Day, 1972). However, these three species

exhibited severely suppressed growth in the low oxygen environ-

ment. Embryos may receive low oxygen concentrations when seeds

are first imbibed (Come and Tissaoui, 1973). The only oxygen

that can pass to the embryo must be dissolved in water that

is covering the structures of the seed during imbibition.

Under optimum conditions, increased germination of several

species has been achieved by increasing the oxygen concentra-

tion above normal (Edwards, 1973). Sachs et al. (1981) found

that clay-coated pepper seeds germinated faster when placed in

100% oxygen. They felt the clay coating limited oxygen supply

to the embryo and thus inhibited germination. In addition,

the authors felt that water was also a barrier to oxygen diffusion

in clay-coated pepper seeds leading to a reduction in germination

rate.

The supply of oxygen may regulate respiratory pathways

during seed germination (Roberts, 1973). Roberts (1973) found

60








that in dormant seeds, any treatment that increased alternate

respiration tended to decrease or alleviate dormancy. Such

treatments included the removal of covering structures, in-

creased oxygen pressure, or application of respiratory inhibi-

tors to the cytochrome mediated-cyanide sensitive pathway.

Higher oxygen pressure may have stimulated alternate respiration

by reducing the competition between the high affinity cytochrome

oxidase and the lower affinity oxidase of the alternate respira-

tory pathway. Yentur and Leopold (1976) concluded that the

alternate respiratory pathway, was needed during the early stages

of soybean germination. In addition, they found that the early

stages of soybean germination required a normal tension of oxy-

gen but later when alternate respiration was lower, this was

no longer required. Burgullio and Nicolas (1977) reported that

the alternate pathway of respiration was not the major respiratory

pathway early in germination of chick peas, but reached its

maximum between 72 and 96 hours. Oxygen stimulated the appear-

ance of the alternate respiratory pathway and the stimulation

was dependent on cytoplasmic protein synthesis.

In the following study, the effect of oxygen on germina-

tion of Capsicum annuum seeds was investigated to determine the

ability of oxygen to control cyanide sensitive and resistant

respiratory pathways at 15 and 250C. Gibberellic acid has been

shown to stimulate germination of pepper seeds at 15 and 250

(Chapter II). The effect of GA on respiratory activity at

15 and 250 was determined to find out if it correlated with

increased germination rate. Effectiveness of GA and 02 on

stimulating germination rate were compared.








Materials and Methods


Effect of 0, on germination. Twenty-five pepper seeds were

placed on a moistened No.3 Whatman filter paper in an open 5.5

cm Petri dish. The Petri dishes were placed into 450 ml glass

jars and sealed. The lid to each jar had 2 rubber septums.

Jars receiving the same gas treatments were connected via need-

les and rubber tubing. The jars were placed into constant

temperature chambers, 15 or 250C, and attached to gas streams

containing the desired atmospheric concentrations of 02 and

N2. The gas streams were humidified by bubbling them through

distilled water at the desired germination temperature. Oxy-

gen mixtures of 10, 21, 40, 60 and 100 percent, were achieved

by mixing 100% 02 and 100% N2 in a 1000 ml erlenmeyer flask,

after passing the gases through distilled water. Flow rates

for individual gases were monitored by the use of flowmeters

(Airco Industrial Gases, Scientific Products, McGaw Park, Il-

linois) and the gas mixture before and after passing through

the sealed jars, was monitored with a Beckman oxygen analy-

zer (Beckman Instrument Inc., Fullterton, California).

Effect of 0, and GA combinations on termination. Twenty-

five pepper seeds were placed on a No.3 Whatman filter paper

in an open 5.5 cm Petri dish. Three milliliters of distilled

water or GA4+7 at 100 or 1000 ppm was used to moisten the

filter paper. The Petri dishes were placed into 450 ml glass

jars, sealed and treated with 21 or 100% 02 gas streams at

15 and 25C as described above.








Germination data for all the experiments in this section

were taken at 24 hour intervals and all treatments were re-

plicated 4 times. Seeds with visible radicles were counted

as germinated. Data taken for each treatment included total

percent germination and mean number of days to germination

(MDG) as described in Chapter II.

Respiratory measurements. One hundred pepper seeds per

treatment were imbibed or germinated in the manner described

above in 21 or 100% 02 at 15 or 250C for various time

durations. The percent germination was recorded and all the

seeds were quickly transferred to 15 ml Warburg respirometer

flasks. One milliliter of distilled water was added to each

flask with or without the respiratory inhibitors, KCN (Fisher

Scientific Company, Fair Lawn, New Jersey) at 10 mM or salicyl-

hydroxamic acid (SHAM) (Sigma Chemical Company, St. Louis,

Missouri) at 1 or 10 mM. The center wells contained 0.4 ml

of 10% KOH (weight/volume) and a filter paper wick. The 10%

KOH was used to absorb the CO2 during the 02 uptake determina-

tions. The KOH may have absorbed some CN from the respirometer

flask environment (Purvis, 1980) but the concentrations of

CN in the reaction medium remained sufficient to maximally

inhibit 02 uptake. This was apparent from the observation

that any concentration of CN above 5 mM inhibited 02 uptake

to the same extent at both 15 and 250. The flasks were

mounted onto a Gilson differential respirometer (Gilson

Medical Electronics, Middleton, Wisconsin), submerged into

the water bath and equilibrated at the appropriate treatment








temperature, 15 or 250. The flasks for the high 02 treatments

were flushed through a sidearm on the respirometer flask with

100% 02 as previously described (Umbreit et al., 1959). The

flasks were equilibrated for one hour before commencing 02

uptake measurements. Oxygen uptake was measured three times

for 15 or 30 minutes with a 15 minute equilibration between

readings for each treatment. Upon the termination of each

experiment, the seeds were removed from the flasks, rinsed,

dried at 700 and weighed. The results were expressed on a

dry weight basis. Each treatment was replicated 4 times.

Determination of total respiratory activity in germinating

pepper seeds treated with GA4+7 at 100 ppm was performed as

described above, but without the use of any respiratory inhibi-

tors. One hundred pepper seeds were imbibed or germinated in

9 cm Petri dishes in a constant temperature chamber at 15 or

250C. Whatman No.1 and No.3 filter papers were placed in the

bottom of the Petri dish and moistened with 0.05 M pH 6.5 sodium

phosphate buffer or 100 ppm GA4+7 in buffer solution. Seeds

were removed at the appropriate time intervals from the Petri

dishes, total percent germination was determined, and the seeds

were placed into the Warburg respirometer flasks. One milliliter

of the treatment solution was added to each flask. Oxygen

determinations were taken as described above. Treatments were

replicated 4 times.








Results

Oxygen concentrations above 21% increased the rate of pepper

seed germination at 250C (Table 3-1). A 10% 02 concentration

reduced the speed of germination when compared to the 21%

level. No apparent trends in altering total germination were

observed by increasing 02 concentrations above 21% 02, although

there was a significant increase at 60% 02. Reducing the 02

concentrations to 10% at 250 led to a reduction in total

germination. At 150, the 02 concentrations above or below

21% generally reduced the rate of germination. Total germina-

tion was unaffected by 02 treatments at 150

At 250C, GA4+7 application or 100% 02 increased germination

rate above 21% 02 alone; however, combining both materials had no

further effect on germination rate (Figure 3-1). Total germina-

tion was unaffected by these treatments at 250. All treatments

at 150C exhibited different germination rates (Figure 3-2).

Oxygen concentrations of 100% delayed germination, while GA+7

and 21% 02 increased the germination rate above 21% 02 alone.

When GA4+7 was added to seeds treated in 100% 02, it increased

the germination rate but did not totally overcome the inhibitory

effect of 100% 02. No treatment affected total germination of

pepper seeds at 150. Increasing the GA4+7 concentrations to

1000 ppm did not decrease the inhibition effect of high 02 on

the germination rate of pepper seeds at 15C (Table 3-2).

Total respiratory activity was greater at 250C when pepper

seeds were germinated in 100% 02 compared to 21% 02 (Figure 3-3).




66


Table 3-1. The effect of O concentration on germination of pepper
seed at 25 and f50C



250 150
Oxygen concentration Germ. Total Germ. Total
Rate Germ. Rate Germ.

(%) (MDG) (%) (MDG) (%)

10 12.0 az 70.0 c 12.3 a 80.0 a
21 (air) 4.2 b 80.3 b 8.0 d 82.7 a

40 3.4 c 86.0 ab 9.0 c 85.0 a

60 3.2 c 88.0 a 8.6 cd 85.0 a
100 3.4 c 80.0 b 11.2 b 81.3 a


ZMean separation within columns by Duncan's multiple range test,
5% level.





Table 3-2. The effect of high 02 concentration and various GA4+7
levels on germination of pepper seeds at 15 C


Oxygen concentrations (%)
Germ. Rate Total Germ.
GA4+7 Concentration 21 100 21 100

(ppm) (MDG) (%)

0 9.4 cz 14.6 a 80 a 73 a
100 8.5 d 12.7 b 82 a 85 a
1000 8.3 d 12.2 b 75 a 77 a


ZMean separation with main observations by Duncan's multiple range
test, 5% level.




































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0-0 i
a 0





o o
0 0
U --
" 0 /


o^ / ,, < _\Ih


0 0 0 0
co qMN

AVG / NOIIVNIlAdi39 IN303d






71








2646
2200-

-oo

1800- -80-

/ 60
rI-
z 40-
1400-
xr 20-
0.
24 68
1000- TIME (DAYS)



600 a 21% OXY (AIR)
S21% OXY.+ KCNIOmM
o 21% OXY +KCNIOmM +SHAM OmM
L- oA 100% OXY.
S 500- m 100% OXY +KCN IOmM

/ IOO%OXY +KCN IOmM+SHAM 10 mM

. 40C?-




300-



200



100



0
2 4 6 8 10 12
TIME(DAYS)
Figure 3-3. Respiration (Cyanide sensitive and resistant
components) of pepper seeds ggrminated in 21
or 100% 02 environments at 25 C








Cyanide sensitive respiration was more active than cyanide

resistant respiration (indicated by the hatched areas in Figure

3-3) throughout germination. The alternate respiratory pathway

generally accounted for 5 to 8% of the total respiration.

At 150C, respiratory activity was similar at the 21% and

100% 02 (Figure 3-4). However, total respiration was higher in

21% 02 at 6 and 12 days. Cyanide resistant respiration was

low and no differences were observed between 02 treatments

throughout germination.

Respiratory activity was similar during the first two

days of germination at 25C between GA4+7 treated and untreated

seed (Figure 3-5). However, after day 4, GA4+7 treated seeds

had higher respiratory activity than untreated seeds. At

150, respiration was similar until day 6 when respiration

rates were higher in GA treated seeds. At both temperatures,

GA treated seeds had higher respiration rates than untreated

seeds after radicle emergence occurred.


Discussion


Oxygen concentrations above that found in air stimulated

pepper seed germination rate at 250C. When seeds imbibe water,

02 becomes less soluble and less available to the embryo as

germination temperatures increase (Come and Tissaoui, 1973).

At 250, diffusion of 02 to the pepper embryo may be limited by

solubility or covering structures. Increasing the 02 concen-

tration, 40% or greater, may allow the embryo to proceed with

metabolic processes near optimal rates. This is supported




















1800-



1400



1000


z
100
Z 80-
60
40
20



2 4 6 8 10 12
TIME(DAYS)
a 21%OXY.(AIR)
o 21% OXY.+KCN IOmM
o 21%OXY. + KCNIOmM+SHAM IOmM


500 a100% OXY.
S100% OXY.+KCN IOmM
*100% OXY.+KCN IO mM +SHAM lOmM


300



200


2 4 6 8 10 12
TIME (DAYS)
Figure 3-4. Respiration (cyanide sensitive and resistant com-
ponents) of pepper seeds germinated in 21 or 100%
02 environments at 150C















2200 -
2100
so-
1800-
60
40
1400- 20
So0
2 4 6 8 0O [2 14
TIME (DAYS)
1000-



600: NCONTROL 250C

A 100 ppm GA4/7 25C

500- CONTROL 15C
S/ / 100 ppm GA4/7 15C


400-



300-



200



100



0 -1-- ----7- ---- [-- i-i ---
2 4 6 8 10 12 14

TIME (DAYS)
Figure 3-5. Respiration of pepper seeds germinated at 15 and 250C
with or without GA4+7, 100 ppm








by the increased total respiratory activity in 100% 02 and

that GA4+7 did not further stimulate germination rate in 100%

02. Ohmura and Howell (1960) found that when corn, soybean

and barley tissues were soaked in water, a marked decrease in

02 uptake occurred at 290. This was overcome by substituting 100%

02 for air. They felt that 02 solubility and diffusion through

the tissues was reduced by the water.

At 150C, high 02 concentrations inhibited the rate of

pepper seed germination. Gibberellic acid4+7, 100 ppm,

applications could partially relieve the inhibitory effect that

high 02 concentrations had on germination rate. Increasing

GA4+7 concentration to 1000 ppm in high 02 did not further increase

germination rate. At 150, respiration rates were similar in

21 and 100% 02. Apparently, at 150 pepper seed respiration

cannot utilize higher 02 concentrations. Siegel and Gerschman

(1959) found that excised bean embryos which received pure

02 at 250 exhibited reductions in growth and enzymatic activity.

Wheat root growth was inhibited by exposure to pure 02 for longer

than 2 days (Eliasson, 1958). The effect of pure 02 on root

growth was an inhibition of cellular elongation. Albaum et al.

(1942) found that when oat seeds were soaked in oxygenated water,

subsequent growth was inhibited when compared to aerated controls.

Enzyme assays on the extracts of the oat seedlings indicated little

or no change in cytochrome oxidase due to oxygenation, but, catalase

and dehydrogenase activity was lower. The authors suggested

that high 02 levels may interfere with proteolytic breakdown








of the endosperm, preventing nitrogen transport from the endo-

sperm to embryo, development of enzyme activity and growth.

Thus, in the present study, at 150 lack of 02 utilization may be

related to altered metabolic processes at the low temperature.

It should be noted that total germination was unaffected by

increasing 02 concentrations at 150

At 25 cyanide sensitive and resistant respiration were

at higher levels with 100% 02. However on a percentage basis,

total respiratory activity was the same as in air. At 150

respiratory activity was similar, regardless of the concentra-

tion of the 02 in the environment. In any event, cyanide

resistant respiration regardless of 02 treatment made up a

small percentage of the total respiratory activity at both

15 and 250

Yentur and Leopold (1976) found that respiration in

soybean seeds was dominated by cyanide resistant respiration

during the first 4 to 5 hours of germination. After this time,

the cytochrome pathway gradually took over and after 9 hours

nearly all respiration was cyanide sensitive. Burguillo and

Nicolas (1977) reported that in chick pea, respiration was

dominated by the cytochrome pathway for the first 12 hours of

germination shifting to more cyanide resistant respiration,

reaching maximal amounts at 72 to 96 hours. They also found

that high 02 concentrations could initiate the appearance of

the alternate respiratory pathway. Pepper seeds did not ex-

hibit an increase in respiration in the alternate pathway in

this study. These differences could be due to variations among








species and the time periods that data were recorded. However,

McCaig and Hill (1977) found that 100% 02 did not stimulate

the appearance of the alternate pathway in wheat mitochondria

isolated from etiolated coleoptiles. In fact, germination in

100% 02 seemed to have little effect on either the cytochrome

or cyanide resistant pathways.

The addition of GA to pepper seeds stimulated germination

and respiration rates. However, respiration rates of GA treated

seeds did not increase over untreated seeds until radicle

emergence had already taken place. Therefore, GA does not

appear to stimulate respiratory controlled metabolic activities

prior to radicle emergence.


Summary


Higher than normal 02 concentrations stimulated germination

of pepper seeds at 25C and inhibited germination rates at 150

Low 02 concentration, 10%, slowed germination rates at both

temperatures. Gibberellic acid4+7 increased germination rate

at 15 and 250 in air. At 250 in 100% 02, GA did not further

increase the germination rate. Gibberellic acid treatments

did not overcome the inhibition 100% 02 treatments had on the

germination rate at 150

Total respiratory activity of pepper seeds germinated at

25C was higher in 100% 02 treated seeds compared to air

treated seeds. However, high 02 treatments did not affect

the proportion of respiration which was in the cyanide sensi-

tive and resistant pathways. Cyanide resistant respiration





78


comprised only a small percentage of total respiratory activity.

At 150, total respiration and the cyanide sensitive and resis-

tant components were similar regardless of 02 treatment.

The addition of GA to pepper seeds increased respiratory

activity only after radicle emergence occurred at 15 and 250C.











CHAPTER IV
MECHANICAL RESISTANCE OF THE SEED COAT AND ENDOSPERM
IN THE CONTROL OF Capsicum annuum L.GERMINATION
AT LOW TEMPERATURE

Seed coats and surrounding structures may have a profound

influence on the ability of a seed to germinate (Mayer and

Shain, 1974). These structures have been attributed to inter-

fering with water uptake, gas exchange and diffusion of endo-

genous inhibitors. In addition, seed coats and surrounding

endosperms may offer mechanical restriction to embryo growth

(Mayer and Shain, 1974; Ikuma and Thimann, 1963).

In seeds that do not have hard seed coats or require

scarification for water uptake, the endosperm in endospermic

dicotyledonous seeds, may mechanically restrict embryo expan-

sion preventing radicle emergence (Pavlista and Haber, 1970).

Junttila (1973) described the mechanism of low temperature

dormancy in Syringa species. Low temperature dormancy ( 9 to

150) was imposed by an endosperm embedding the radicle and

could be alleviated by removal of endosperm from around the

radicle. The endosperm did not appear to contain significant

quantities of germination inhibitors, and his results indicated

that dormancy was mainly due to mechanical restriction of

radicle emergence through the endosperm.

In lettuce, a 2-celled layer of endosperm, characterized

by thick cell walls and dense cytoplasm, encases the lettuce

79








embryo (Jones, 1974). The ability of this endosperm to control

or to mechanically restrain lettuce radicle emergence has been

greatly debated. Ikuma and Thimann (1963) proposed that red

light relieved endosperm restriction on the embryo in photo-

sensitive lettuce varieties. They proposed that the red light

triggered cellulolytic enzyme production in the embryo weak-

ening the endosperm layer. Nabors and Lang (1971) also felt

the lettuce endosperm restricted radicle emergence; however,

this could be overcome by increasing the growth rate of the

embryo. Red light could induce greater growth rate in the

lettuce embryo enabling it to grow through the endosperm.

Pavlista and Haber (1970) proposed that mechanical force of

the growing embryo pushing against the endosperm and chemical,

enzymatic, weakening of the endosperm were necessary for let-

tuce seed germination. This was suggested from their observation

that when chemical weakening of the endosperm was inhibited,

the embryos grew and buckled in the endosperm encasing but did

not germinate.

Jones (1974) in microscopic and electron microscope in-

vestigations determined that the cell walls of lettuce endosperm

were increasingly degraded with longer imbibition times and at

the time of germination the walls were extensively broken down.

In a study using the Instron Universal Testing Macnine to test

puncture forces of endosperm in lettuce seed, Tao and Khan

(1979) determined that endosperm strength did not appear to be

directly related to radicle protrusion. Halmer et al. (1976)

reported that an enzymatic degradation of a mannose containing








polysaccharide, a major component of lettuce endosperm cell

walls, was stimulated by red light or gibberellin. The appear-

ance of this process did not correspond with radicle emergence

and enzymatic activity increased markedly only after radicle

emergence.

Pepper embryos are totally surrounded by endosperm materials

making up the bulk of food reserves for the embryo and young

seedling (Cochran, 1938). The experiments which follow inves-

tigate the possible mechanical control that the seed coat and

endosperm have on germination of Capsicum annuum at optimum

and suboptimal temperatures.

Materials and Methods

Decoated seeds. Air dry pepper seeds were decoated by

inserting a scalpel or probe into the seed cavity and peeling

back the seed coat material until a coatless seed was obtained.

Twenty-five decoated or raw (untreated) pepper seeds were placed

on a moistened No.1 and No.3 Whatman filter paper in a 9 cm

Petri dish. The Petri dishes were placed in 15 or 250C con-

stant temperature chambers for germination.

Scarified endosperm test. Decoated air dry seeds were

scarified by removing 0.5 mm of endosperm material directly in

front of the radicle. Scarified seed and decoated control

seeds were then germinated as described above.

Germination data for both experiments were taken at 24

hour intervals and all treatments were replicated 4 times.








Seeds with visible radicles were counted as germinated. Data

taken for each treatment included total percent germination

and mean number of days to germination (MDG) as described in

Chapter II.

Endosperm strength measurements. The Instron Universal

Testing Machine (Instron Engineering Corporation, Canton, Massa-

chusetts) was used to determine resistance to radicle emergence.

Seeds were previously treated with distilled water, 100 ppm

GA4+7 or 100% 02 when imbibed at 15 and 250C. Fifty pepper

seeds per treatment time were imbibed in 5 ml of distilled

water or GA4+7, on Whatman No.1 and No.3 filter paper in 9 cm

Petri dishes at 15 or 250. Seeds treated with 100% 02 were

gassedin a continuous flow system at 15 and 250 as described

in Chapter III. After the appropriate pretreatment time, the

seeds were immediately subjected to testing with the Instron.

Each of the seeds was prepared immediately before measurement

on the Instron. The seeds were decoated and approximately

1/4 of the seed, the area of radicle emergence, was excised.

The radicle was teased out of the surrounding endosperm material

with a microprobe, leaving a clean undamaged radicle cavity in

the endosperm. The endosperm section was placed onto a

No. 78 drill blank, 0.4 mm in diameter, attached to a basal

load cell (Figure 4-1). The crosshead had a bolt placed in it

with a 0.8 mm diameter counter hole drilled into it. The load

cell was set to 100 g (0.98 newton) full scale load. Cross-

head and chart speeds were 1.3 and 25.4 cm/minute respectively.

Each treatment consisted of 5 seeds and all treatments were

replicated 5 times.




























Figure 4-1. The endosperm section placed onto the drill
blank ready for puncture force measurement
with the Instron Universal Testing Machine.
The drill blank is held by a drill chuck
mounted on a platform attached to the
basal load cell. The crosshead of the
Instron has a bolt placed in it with a
counter hole drilled into it.

























































Figure 4-1









Results


Decoated seeds germinated more rapidly than control seeds

at 250C (Figure 4-2). There was no difference in total germina-

tion between the two treatments. At 150, germination rate

was unaffected by decoating (Figure 4-3). Again, decoating did

not affect the total germination of pepper seeds at 150

When decoated pepper seeds were scarified by removing

0.5 mm of endosperm directly in front of the radicle, germina-

tion rates increased dramatically at both 15 and 250 (Table

4-1). The scarification treatment did not affect the total

germination of pepper seeds at 15 or 250

Puncture force of the endosperm directly in front of

the radicle decreased with increasing imbibition time at both

15 and 250C (Figure 4-4). The endosperm resistance decreased

more rapidly when imbibed at 250 than at 150. Puncture force

decreased to 0.3 to 0.4 newtons at both 15 and 250 before the

majority of radicle protrusion occurred.

Gibberellic acid treatment at 250C reduced the endosperm

resistance and increased the germination rate compared to

untreated seeds (Figure 4-5). Gibberellic acid decreased

endosperm resistance rapidly after 24 hours of treatment whereas

in untreated seeds, endosperm resistance did not decrease

appreciably until 48 hours. At 150, GA treatments had a sim-

ilar effect on endosperm weakening and germination rate as

at 250, except that the effects were slower (Figure 4-6).












































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Table 4-1. The effect of removing 0.5
in front of the radicle 8n
germination at 15 and 25 C


mm of endosperm directly
decoated pepper seed


250 150
Treatment Germ. Total Germ. Total
Rate Germ. Rate Germ.

(MDG) (%) (MDG) (%)
Decoated control 3.1 az 72 a 9.1 a 94 a

Scarified Seed 1.7 b 78 a 3.4 b 90 a


ZMean separation between
5% level.


columns by Duncan's multiple range test,













































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