Title: Relation of phenolic compounds to germination of peach seeds
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Title: Relation of phenolic compounds to germination of peach seeds
Physical Description: 91 leaves : ill. ; 28 cm.
Language: English
Creator: Aitken, James Bruce, 1938-
Publication Date: 1967
Copyright Date: 1967
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Subject: Peach   ( lcsh )
Fruit Crops thesis Ph. D
Dissertations, Academic -- Fruit Crops -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by James Bruce Aitken.
Thesis: Thesis (Ph. D.)--University of Florida, 1967.
Bibliography: Includes bibliographical references (leaves 81-89).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00097819
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 - 000415137
oclc - 37545966
notis - ACG2373

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











UNIVERSITY OF FLORIDA
August, 1967


RELATION OF PH;ENOLIC COM~dPOUNDS

TO GERMINATION OF PEACH SEEDS










By
JAMES BRUCE AITKEN














ACKNOWLEDGEMENT


The author wishes to express his sincere appreciation and gratitude

to Dr. R. H. Biggs, Associate Biochemist, Department of Fruit Crops, and

Chairman of the student's Supervisory Committee, for his most valuable

assistance and guidance during the course of research and the prepara-

tion of this manuscript.

Appreciation is extended to Dr. A. H. Krezdorn, Chairman, Depart-

ment of Fruit Crops; Dr. T. E. Humphreys, Associate Biochemist, Depart-

ment of Botany; Dr. C. H. Hendershott, Associate Professor of Fruit

Crops; and Dr. D. O. Spinks, Professor of Soils, Department of Soils,

for their constructive criticism and invaluable assistance in the pres-

entation of this manuscript.

The author also wishes to express his gratitude to Mr. J. K. Peter,

laboratory technician, for his assistance in conducting portions of the

research.

For her help in the preparation of this manuscript and also for her

interest and encouragement during the course of this study, the author

wishes to express his sincere appreciation to his wife, Patricia.



















TABLE OF CONTENTS





ACK(NOWLEDGEMIENT .. ... .. .. ... .. ... .. . .. ii


LIST OF TABLES. .. .. ... .. .. ... .. .. .. .. iv


LIST OF FIGURES .. .. ... .. .. .. .. .. .. .. .. vi


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


REVIEW OF LITERATURE. ... .. .. .. ... .. .. . .. 3
Germination Inhibitors ... .. ... .. .. .. .. 4
Physiology of Seed Germination ... .. . ... .. .. 12
Physiology of Peach Seed Germination . ... .. .. .. 17


MVATERIALS AND METHODS .. .. .. .. .. .. .. .. .. . .. 21


EXPERIM~ENTA L RESULTS. ....................... 31


DISCUSSION. .... .. ... .. .. .. .. . .... 67


SUMMl\ARY AND CONCLUSIONS ... .. .. .. .. . ... .. .. 73


APPENDIX: GAS CHROMATOGRAMS OF STANDARDS .. .. .. .. ... 75


LITERATURE CITED. .. .. .. .. .. ... .. .. . .. .. 81


BIOGRAPHICAL SKETCH .. .. .. .. .. .. .. ... .. .. 90
















LIST OF TABLES


Table

1. Influence of thiourea concentrations on germination and
per cent of production of abnormal seedlings from
'Okinawa' peach seeds ................

2. Effect of thiourea concentration and embryo excision on
germination of 'Okinawa' peach seeds 12 days after
start of imbibition and on abnormal seedling production
32 days after start of imbibition...........

3. Per cent germination of 'Okinawa' peach seeds 7 and 20
days after start of imbibition as influenced by cyanide

4. Per cent germination of 'Okinawa' peach seeds 7 and 12
days after start of imbibition as influenced by
mandelonitrile. . . . . . . . . . .


5. Per cent germination of 'Okinawa' peach seeds 7 and 12
days after start of imbibition as influenced by
benzaldehyde. . . . . . . . . . . .

6. Relative retention time and possible identity of com-
ponents separated by gas chromatography of the propyl
esters of the acidic fraction from an ethanolic
extract of peach seeds. ................

7. Paper chromatographic separation of the inhibitory
complex from dormant peach seeds. ...........

8. Influence of acids, bases and heat on the inhibitory
complex from peach seeds after paper chromatography .

9. Solubility of the inhibitor-complex in various organic
solvents as determined by the alfalfa bioassay. ....

10. M~ean per cent germination of dormant 'Okinawa' peach
seeds 30 days after start of imbibition as influenced
by benzaldehyde and mandelonitrile concentrations...

11. M~ean per cent germination of dormant 'Okinawa' peach
seeds 30 days after start of imbibition as influenced
by benzoic and p-hydroxybenzoic acid concentrations ..


Pap~e


. . 36




. . 37





. . 40



. . 42



. . 43



. . 44


.58









12. Comparison of retention times of p-hydroxybenzoic acid
and L-mandelic acid as influenced by various acetylation
procedures. ..........*************** 64

13. Comparison of peak areas of p-hydroxybenzoic acid and
L-mandelic acid as influenced by various acetylation
procedures. ........****************. 66
















LIST OF FIGURES


Figure

1. Influence of benzaldehyde and mandelonitrile on peach
seed germination. ... . .... ... ...

2. Gas chromatogram of the propyl enters of the acidic
fraction from an ethanol extract of peach seeds.
Time is in minutes. .................

3. Gas chromatograms of a known composite sample of an
etheral solution of benzaldehyde-mandelonitrile:
(a) initial solution; (b) after addition of a
solution of sodium bisulfite; and (c) after addition
of potassium cyanide.................

4. Changes in benzaldehyde-mandelonitrile content of
peach seeds as measured at various intervals after
start of imbibition under the designated treatments.

5. Change in the content of mandelonitrile in peach
seeds at various intervals after start of imbibition
under the designated treatments...........

6. Change in the content of benzaldehyde in peach seeds
at various intervals after start of imbibition under
the designated treatments..............

7. Germination of peach seed as influenced by embryo
excision, and thiourea treatments as determined
periodically after the start of imbibition. .....

8. Relative inhibitory activity, as measured by the
alfalfa bioassay, of the inhibitory complex in an
ethanolic extract of peach seeds chromatographing
between Rf's 0.6 to 0.8........... ...

9. Germination of peach seeds as influenced by 50 C
of varying durations.................

10. Gas chromatogram of diazomethane-solvent control.
Retention time is in minutes.............

11. Gas chromatograms of L-mandelic acid(a) and
p-hydroxybenzoic acid(b) treated for 30 minutes
with diazomethane. Retention time is in minutes. ..


Page


. . 39


. . 52


. . 61









12. Gas chromatogram of an ethanol extract from peach
seeds treated for 30 minutes with diazomethane.
Retention time is in minutes. .. ... ... .. .. 63

APPENDIX: Gas chromatograms of standards.

13. Gas chromatograms of the propyl esters of benzoic
acid(a) and mandelic acid(b). Retention time in
minutes ...,. .. . .. .. .. .. .. ... 76

14. Gas chromatograms of the propyl esters of o-hydroxy-
beenzoic acid(a) and p-hydroxybenzoic acid(b). Reten-
tion time in minutes. .. ... .. .. .. ... .. 77


15. Gas chromatogarams of the propyl ester of 2,6-dihydroxy-
benzoic acid. Retention time in minutes. ... .. .. 78

16. Gas chromatograms of the propy, ester of 2,4-dimethoxy-
benzoic acid. Retention time in minutes. . .. .. .. .. 79


17. Gas chromatograms of the propyl ester of o-hydroxy-
cinnamic acid. Retention time in minutes .. ... .. 80














INTRODUCTION


The phenomena of seed dormancy have interested researchers for many

years. Little by little the details are being unfolded in various plant

species. Seed dormancy may result from such sources as mechanical re-

striction, immature embryo, or chemical inhibition.

Various chemicals, e.g., thiourea, have been found which will termi-

nate seed donnancy, but in many cases this results in the production of

abnormal seedlings (36, 37, 76, 97). However, reports in the literature

show that abnormalities may be due to temperature (80).

The breakdown of amygdalin in germinating peach seeds possibly

presents a fruitful area for investigating seed dormancy. Amygdalin is

hydrolyzed to mandelonitrile and glucose by prunsin (104). Mandelo-

nitrile is further hydrolyzed by emulsin to cyanide and benzaldehyde

(104). It has been shown previously that benzaldehyde strongly affects

growth (46). Phenolic compounds have been isolated from many plant

tissues (39, 82, 92, 93, 94) and their influence on certain biochemical

systems within the plant has been investigated (38, 74, 78, 79, 83, 95,

105). Recent evidence would indicate that they are involved in plant

growth and development (78, 105). Therefore, phenolic compounds could

play a prominent role in controlling dormancy of peach seed.

With this knowledge at hand, research was undertaken to determine

the role of phenolic compounds in peach seed germination. It was recog-

nized that this role could be stimulatory, inhibitory, both, or neither.









Also various means of terminating dormancy were compared with regard to

their influence upon certain of the phenolic compounds. In order to

conduct this investigation, several new techniques were established for

isolating and aiding in the identification of certain of the phenolic

compounds.















REVIEW OF LITERATURE


In many plants the phenomena of seed dormancy, regardless of cause,

have a survival benefit. The term I'dormancylt as applied to viable seeds

is generally restricted to those which fail to germinate in a reasonable

length of time when subjected to an adequate moisture supply, a temper-

ature within the range of 18-300 C and the normal gaseous composition of

the atmosphere. Dormancy can be due to various causes. It may be due

to the immaturity of the embryo, impermeability of the seed coat to

water and/or gases, prevention of embryo growth by mechanical restric-

tions, special requirements for temperature or light, endogenous factors

which inhibit germination, age of seed, and, in certain cases, immaturity

of the embryo. These factors have been discussed in several classic

reviews on seed germination (16, 17, 24) and in some excellent reviews

in the last several years (64, 96, 102, 108). This review will be par-

ticularly concerned with endogenous factors which control seed dormancy

since this is the type of dormancy we are dealing with in the case of

peach seeds (11, 14, 36).

Viable seed that fail to germinate when exposed to conditions

generally considered favorable for germination can be induced to germi-

nate in most cases by the correct exposure to certain environmental

factors. Very often the environmental cue for the resumption of growth

is attained from climatic components, e.g., low temperature of a given

duration, alternating periods of moisture stress, daylength, etc. These








environmzental components precondition t~he seeds, so; thant i:`rmi~nat~itn\

occurs when they are supplied with adequate moisture, a warm temperature

and atmospheric gases of the normal composition. After-ripening may be

defined as physiological changes occurring in any part of the seed which

enable the seed to germinate and the seedling to grow normally (64).

The necessity for a period of after-ripening may be due to several

factors. In the case of the immature embryo, further developmental

changes may be required before germination (115). In other seeds,

chemical changes must occur in the embryo before they germinate (64).

In still others, chemical changes must occur in the integuments and/or

other tissues associated with the embryo (96).

In contrast to seeds which will not germinate until subjected to

certain environmental factors before being placed under favorable Germi-

nation conditions of moisture, warm temperature and atmospheric gases,

some seeds will germiinate readily without preconditioning. However, it

is interesting to note that the latter will also lose their readiness to

germinate if subjected to stress conditions much the same as seeds that

require factors to terminate dormancy (65, 98). This phenomenon is

referred to as secondary dormancy. Secondary dormancy can be induced

in certain seeds by subjection to high or low temperatures, high CO2

levels or continuous light.


Germination Inhibitors

A large number of substances are capable of inhibiting germination.

Those compounds which are generally toxic to living organisms will also,

at toxic concentrations, prevent germination simply by killing the seed.

However, these compounds have been of little value in determining the








underlying causes of dormancy. Compounds which prevent germination

without killing the seeds are by far the more valuable in determining

the mechanism of dormancy.

The simplest type of inhibition is caused by non-toxic chemicals

in high concentration and this has been shown to be due to high osmotic

pressures (16). These high osmotic conditions may be obtained by in-

organic salts, sugars, or other substances. An example of such inhibi-

tion is the inability of seed within some mature fruit to germinate.

The large quantities of soluble solids present in the flesh create high

osmotic conditions around the seed and prevent germination. The thresh-

old of osmotic pressure which prevents germination differs with the

species. As soon as the seeds are removed from the high osmotic environ-

ment and placed in water, they will germinate (64).

A more complex type of inhibition is that caused by substances

which are known to interfere with certain metabolic pathways. Since

germination cannot occur without active metabolism, any substance that

would alter normal metabolism, would probably alter the germination

pattern of seed. Compounds such as cyanide (4), dinitrophenol (68),

azide (68), fluoride (24), hydroxylamine (24), and others (4, 24) which

are respiratory inhibitors, have inhibited germination at concentrations

approximating those which inhibit metabolic processes. Therefore, it

seems that inhibition of germination by this class of compounds is a

result of their effect on metabolism (64), but only in the case of

cyanide (4) have these chemicals been implicated in natural seed

dormancy.

Another class of compounds that inhibited germination are auxins

(54). An example of such a case would be the use of low concentrations









of 2,4-dichlorophenoxyacetic acid (2,4-D) to inhibit germination. Al-

though auxins have been shown to be necessary for growth of isolated

embryonic tissues and to increase at the time of germination or shortly

before (32, 51); however, there has been no convincing evidence that

they are directly involved in the dormancy mechanism (25, 42, 55). Only

in a few instances (14, 25, 37) have auxins been shown to stimulate

germination and these instances were cases where the dormant state of

the seed was altered by pretreatments. This is in contrast to the in-

fluence of auxins on fruit growth and development (59, 60, 61).

On the other hand, growth inhibitors are of general occurrence in

dormant seeds, and there is abundant evidence for their involvement in

the physiological mechanisms of dormancy.

Evidence for the involvement of growth inhibitors in seed dormancy

is the demonstration that they are often present in dormant seeds and

that the application of such materials can impose dormancy on seeds in

certain cases. Nutrile (70) was the first to show this. He applied

coumarin to lettuce seeds and showed they required preconditioning again

before they would germinate. These experiments were substantiated by

Evenari (25).

Many phenolic compounds have been found to inhibit germination.

These have a widespread occurrence and distribution in plants and

fruits and thus it is thought that they may occur as natural germination

inhibitors (92). It was suggested by van Sumere (92, 93) that the

phenolic compounds may be classified along with coumarins as dormancy

inducing agents. Coumarin, ferulic acid and other phenolic compounds

have been found to occur in the skin as well as the cortical tissue of








potato and Hemberg (42, 43) suggested that the rest period of the

potato may be due to an abundance of growth inhibiting substances in the

periderm. Koves and Varga (53) surveyed the dry fruits of several

species with reference to inhibitory substances. Inhibitors were found

in all fruits and those that have been chemically identified were

phenolic acids or their depsides and polydepsides. Numerous benzoic and

cinnamic acid derivatives such as high molecular weight tannic acids,

protocatechuic, caffeic and chlorogenic, ferulic, p-coumaric and p-

oxybenzoic acids had a lesser activity (53). Salicylic acid, and in

some cases unidentified cinnamic acid derivatives, had strong activity.

Miost of these inhibitors were washed out or destroyed as the fruit re-

mained on the tree for a prolonged period of time.

Although the phenolic substances range in structure from simple

phenols to complex compounds, such as lignin, it seems that the most

important phenols, insofar as growth regulation is concerned, are the

monocyclic aromatic compounds (82). In recent years attention has been

given to the role of hydroxycinnamic and hydroxybenzoic acids in plant

growth and development. The biosynthesis of these acids in higher

plants has received renewed attention recently (22, 82, 94). The major

pathway for the formation of these compounds undoubtedly involves

phenylalanine via shikimic acid. The inter-conversion of the hydroxy-

benzoic acids gave rise to many derivatives (39, 45, 50). p-Hydroxy-

benzoic acid and caffeic acid have been isolated from plants and shown

to be active as growth regulators (103, 105). Other phenolic compounds

that have shown lesser activity include salicylic, gallic, ferulic,

caffeic, vanillic, protocatechuic, chlorogenic, p-oxybenzoic, and p.-

coumaric acids (53, 64, 92).







Another possible function of the phenolic acids in seed germ~ina-

tion may be their role in the synthesis and degradation of indoleacetic

acid (IAA) (74, 83). Pilet (78, 79) reported that the mono-hydroxyben-

zoic acids increased the in vitro destruction of IAA. Of these, p-hy-

droxybenzoic acid had the greatest effects, causing stimulatory growth

of stem sections at low concentrations and inhibiting elongation at higher

concentrations. Many other naturally occurring phenolic acids were

studied by Zenk and Muller (116) as to their influence on the destruc-

tion of exogenously applied IAA. By growth experiments with IAA-1_14C

and determination of the 14CO2 evolved, it was shown that monophenols

stimulate the decarboxylati~on of IAA under conditions where growth was

suppressed (95). WVhen Mn++ was present, this decarboxylation was enhanced.

To add to the complexity of the relation of phenols to growth, Gordon and

Paleg (38) have shown that phenols, under conditions leading to their

oxidation, reacted with tryptophan to form1 IAA.

Probably the most active and most widely used germination inhibitor

is coumarin. Coumarin is characterized by an aromatic ring and an un-

saturated lactone structure. No single group in the coumarin molecule

has been shown to be the cause of its inhibitory action. Reduction of

the unsaturated lactone ring or substitution by hydroxyl, methyl, nitro,

chloro and other groups in the ring system reduced the inhibitory activi-

ty (63, 70).

The flavonoid, naringenin, which has been isolated from peach buds

by Hendershott and Walker (44), has ain action similar to coumarin on

lettuce seeds. Phillips (77) demonstrated that it will impose dormancy

on lettuce seeds that can be reversed by light or by application of

gibberellins.







Recently, several new compounds have been isolated which exhibited

growth regulatory properties. One group of compounds which show a

marked elongation effect on rice and lettuce is related to helmintho-

sporol (84). ':Dormin', a terpenoid compound has shown a marked influence

on the regulation of bud growth in some woody plants. It appears that

the structure of 'dormiin' and 'abscisin II' are the same (15, 71).

Eagles and WVareing showed that an inhibitor \'dormin') concentrated from

an ex-tract of birch leaves could completely arrest apical growth when

applied to the leaves of seedlings. Evidence was also found for high

levels of 'dormin' in birch leaves under short days, with the emergence

from dormancy presumably resulting from an interaction between 'dormin'

and growth-promoting substances (20, 21). A recent finding in the study

of dormancy regulation in peach seeds was that an inh;ibitor isolated

from the seed integuments chromatographed identical to 'dormin' (57, 58).

However, Daley (18) has shown that several inhibitors are present in

peach seed cotyledons and that several chromatographed in the zone

labeled 'dormin' by Lipe and Crane (58).

Bennet-Clark and Kefford (8) first described a complex cj, inhibitory

substances that appeared on paper chromatograms of plant extracts running

ahead of IAA when developed in a solvent of isopropanol/ammonia/water.

This inhibitory area, possessing Rf values of 0.6 to 0.8, has been clas-

sified as the beta-inhibitor complex (48, 49). This inhibitory complex

has been shown to be widespread in plants and has been related to both

dormancy and correlative growth. For instance, Varga (100) has reported

that the juice of lemons, strawberries and apricots contains inhibitors

which appear to correspond to the beta-inhibitor complex. Lipe (57)

found that the inhibitors in 'Lovell' peach seeds are similar to the








beta-inhibitor complex. Elution and rechromatography of the beta-in-

hibitor-complex has yielded both acidic and neutral substances (56).

Recently, the beta-inhibitor-complex concentrated as acidic compounds

from extracts of dormant maple buds was shown to be a complex of phenolic

substances (86). It includes coumarin and salicylic, ferulic, p- and

o-coumaric, m-oxybenzoic acid (93, 108) and 'dormin' (15).

Many of the previously mentioned phenolic compounds have been found

to occur in various plant tissues, especially in fruits (64, 108). For

example, Varga (100) and Koves and Varga (53) have shown that many

phenolic compounds such as salicylic, ferulic, caffeic, chlorogenic,

p-coumaric, protocatechuic and p-oxybenzoic acids are present in fruits.

Along these same lines, it is interesting to note that peach juice is

injurious to peach seed germination (85). It has been suggested that

the inhibition of seed germination in fruit was generally not due to a

single compound but was due to the synergistic action of several com-

pounds that might be present within the fruit or the seed itself (lOS).

The activity of endogenous inhibitors may not be solely directed

at the prevention of germination per se, but may also influence some of

they other facto;.s controlling dormancy. Black and Warei-ingr (10) reported

that the removal of the embryo from intact seed reduced the light re-

quirement for germination of seed of the Betula spp. They also suggested

that the inhibitor in the seed coat increased the oxygen requirement of

the embryo. WVareingr and Foda (109, 110) found that leaching the embryo

of Xanthium seed removed the inhibitor and that maintaining the seed in

a pure oxygen atmosp:here causs a reduction in the inhibitor within 30

hours. Elliott and Leopold (23) showed that the inhibitors from Avena

seeds inhibited alpha-amylase activity.








Villiers and WVareing (106, 107, 108) reported that chilling Frax-inus

excelsior seeds had no effect on the activity of the inhibitor but that

dormancy was overcome during chilling by production of a growth stimu-

lator in the embryo tissues. Flemion and De Silva (31) also deme -ated

with peach seeds that with the bionssay they were using they coul, lind

little correlation between growth inhibitors and the termination of

donnancy.

The promotive effects of oxygen on germination of seeds and the

parallel effects of light led Paech (73) to suggest, ,,zat dorniancy was

regulated by phenolic substances in the seed coat. The oxidative activi-

ties of phenolic compounds could trap oxygen, preventing its entry into

the seed. The action of the phenolics could be blocked by oxygen or

light through the photoox-idation of the phenolics themselves.

The effects of gibberellin in breaking the dormancy of many seeds

indicated that it could possibly be the stimulator of growth if it were

formed during the period in which dormancy was broken (35). MIurakami

(66) has shown gibberellin to be present in a wide diversity of seeds.

As seeds of Avena fatua emerged from dormiancy a growth-promoting sub-

stance suggestive of gibberellin was for-med (67). These seeds were also

brought out of dormancy if soaked in gibberellin solutices. Kahn (47)

reported gibberellin overcame doi-rmancy of lettuce seed regardless of

whether it was imposed by hi,;. temperature, by far-red light, or by

osmotic solutions.

Recently, a mode of action was suggested for gibberellic acid (99).

It ~as been reported that gibberellic acid stimulated alpha-amylase

production in the aleurone layer ol ... coat of cereals which in turn

increased the rate of starch hydrolysis. Thr stimulation alpha-








amylase was believed to be due to the direct influence of gibberellic

acid on messenger RNA polymerase, an enzyme that is involved in producing

the alpha-amylase enzyme (101).


Physiology of Seed Germination

The actual germination of a seed reflects the cumulative effect of

interactions between many factors both external and internal. These

factors range from hereditary traits to environmental influences during

development and storage. For simplicity of this review, the influencing

factors will be grouped into external and internal factors. Excellent

reviews have been published on the physiology of seed germination (16,

17, 24, 64, 96, 102).

EXTERN~AL FACTORS:

Among the external factors required for seed germination are an

adequate supply of moisture, a suitable temperature range and composi-

tion of gases in the atmosphere, light, and sometimes certain chemicals.

The requirement for these conditions varies according to the species

and variety and is determined by hereditary factors and by the condi-

tions which prevailed during seed formation. Frequently it appears

there is a correlation between the environmental requirement for germi-

nation and the ecological conditions occurring in the habitat of the

plant and the seeds (64).

WYater: One of the first processes which must occur for germination

of dry seeds is the uptake of water. The extent of this uptake is deter-

mined by (a) the composition of the seed coat, (b) the permeability of

the seed coat to water, (c) the availability of water (liquid or gaseous)

in the environment, and (d) soluble solids (64).







Gases: Germination, a process of living cells, requires an expendi-

ture of energy. Energy requiring processes in living cells are usually

supported by processes of oxidation, in the presence or absence of

oxygen. These processes, respiration and fermentation, involve an ex-

change of gases, an output of carbon dioxide in both cases and the uptake

of oxygen for respiration. Consequently, seed germination is markedly

affected by the composition of the ambient atmosphere (64).

The partial pressure of oxygen in the atmosphere can be reduced

considerably without greatly interfering with the rate of respiration.

In fact, the seeds of some water plants germinate better under lower

oxygen tensions than in air. Seeds of many terrestrial plants can

germinate under water where the concentration of oxygen often corresponds

to a partial pressure of oxygen very much less than that of the atmos-

phere (65).

In the early stages of germination of seeds of species such as

Pisum sativum, respiration is largely or almost totally anaerobic be-

cause of the relative impermeability of even hydrated seeds of such

species to oxygen. As soon as the seed coats are ruptured, aerobic

respiration replaces the anaerobic oxidative processes (65).

The influence of carbon dioxide concentration is usually the re-

verse of that of oxygen. Many seeds fail to germinate when the carbon

dioxide tension is high. There seems to be a minimal requirement for

carbon dioxide in order for germination to occur in Atriplex halimus

and Salsola as well as lettuce whereas some other species of Atriplex

are resistant to high levels of carbon dioxide as long as the oxygen

concentration is kept constant (7).








Temperature: Different kinds of seeds have specific ranges of

temperature within which they germinate. Very low and very high temper-

atures tend to prevent the germination of all seeds. A rise in temper-

ature does not necessarily cause an increase in either the rate or the

percentage of germination. Therefore, germination is not characterized

by a simple temperature coefficient (107).

Light: Among cultivated and non-cultivated plants there is con-

siderable evidence for light as a factor influencing germination. For

example, lettuce, tobacco and many crucifers require light to germinate

(33, 75, 77, 96). Seeds may be divided into those which germinate only

in the dark, those which germinate only in continuous light, those which

germinate after being given a brief illumination and those which are in-

different to the presence or absence of light during germination (96).

Studies have shown that different spectral zones affected germina-

tion differently. Light of wavelength less than 2900 Ao has inhibited

germination of all seeds tested (33). Between 2900 Ao and 4000 Ao the

germination of some seeds is inhibited (33). In the visible range,

4000 Ao 7000 AO, it was shown that light in the range of 5600 Ao

7000 Ao and especially red light, usually promoted germination (64, 75).

If seeds exposed to red light were followed promptly by an exposure to

far-red light (7350 Ao), germination was partially or totally inhibited

(65). An excellent review of the phytochrome system and its relation to

germination has been made by Siegelman and Butler (87).

INTERNAL FACTORS:

The changes which take place during the germination process are to

a certain extent determined by the type of seed and its chemical compo-

sition. The composition is in turn influenced by environmental condi-







tions present durir seed formation as well as the hereditary factors of

the species involved.

Once the germination process is initiated, there is mobilization

and translocation of compounds from storage organs to the actively grow-

ing meristematic tissues (64). Studies with tree peony embryo and endo-

sperm tissues reveal that biochemical changes which take place with

germination are different for tissue after-ripened at 50 C from those

that are kept in the greenhouse at 210 300 C. The latter can be con-

sidered dormant tissue (5, 6, 27). Major biochemical changes in organic

acids, amino acids and sugars were noted. These typify what has been

found with many seeds. A good discussion of this aspect of seed germi-

nation can; be found in the book by Mayer and Poljakoff-Mlayber (64).

Since phosphates play an extremely important role in a variety of

reactions of seeds, some discussion of the metabolism of phosphorus-

containing compounds would be in order. The phosphates are required for

the formation of nucleic acids which in turn are intimately concerned

with protein synthesis and the hereditary constitution of plant cells.

They are components of many other .say compounds including phospholipids

which function in controlling surface properties and permeability of

cell membranes. Also, the various phosphorylated sugars and nucleotides

are very closely linked with the energy-producing processes in the cell

during germination (64).

Phosphorus primary ly appears in seeds as organic phosphorus, with

very little being present as inorganic orthophosphate. Phytin is fre-

quently present and may constitute up to 80% of the total phosphorus

content of a~ seed (64). Since mnost of the phosphate ,a present in the

bound form~, o Exophosphate may be the limiting factor in certain of the









reactions of the germination process. With this in mind the large amount

of phytin present may be considered as a * u7rve of inorganic phosphate

which can be liberated c,. germninatio- proceeds by phosphatase activity

or more special cally phytase activity. Phyti, is also present in the

embryo, disappearing rapidly during germination. The phosphorus is

replenished by transport from the endosperm~ to the embryo during germi-

nation (2). The rate of phytin hydrolysis and subsequent transport of

phosphorus to the growing sites presents a possible limiting factor for

the rate of germination and subsequent seedling development.

Recently, reports of myo-inositol acting as a growth factor in

plant tissue have ,2en made (3). This is of particular interest in re-

gard to phytin since it is the salt of phytic acid or inositol eseaphos-

phate. The Ilneutral fraction"l of coconut milk contains myo-inositol

along with scyllo-inositol and sorbitol, but myo-inositol was regarded

as the most important, as far as activity in growth-stimulation was

concerned. M~yo-inositol may stimulate the growth of seedlings and the

germination of certain seeds. In addition, myo-i;;ositol has stimulated

growth of callus in cultures of elm (U'lmus campestris), N'orway spruce

(Picea abies), tobacco (Nicotiana tabacium), Vinca rose, and carrot

(normal and tumorous) tissues (3).

Studies on the nucleotide content of seeds during germination dis-

closed that the ATP content rose initially during imbibition and then

decreased (34). The content of nicotinamide adenine dinucleotide (NAD)

and nicotinamide aienine dinucleotide phosphate (NAZDP) in seeds and

ss ings rises in all cases during germination. During the earlSy

stages of genuination there was a not increase in the RNlA of peanut

cotyledons (102). Some of this RNZA synthesis was thought to be associ-








ated with increased numbers of mitochondria, or in mitochondrial function,

and the ability of the cells to form chloroplasts. However, a part of

the increase was postulated to be associated with the appearance of

enzymes required for metabolism of the storage materials in the peanut.

A peak was reached in about 8 days followed by a more or less parallel

decline in RNA content and enzymic activities. These declines were con-

comitant with an increase in R~Uase activity.

As germination proceeded there was a sharp rise in carbon dioxide

evolution and a gradual rise in oxygen uptake of pea seeds. However,

after 24 hours there was a sharp decrease in the respiratory quotient

(88). This same pattern was observed for wheat for both carbon dioxide

evolution and oxygen uptake (67).

The energy pathways in seeds have been studied in some detail.

Both glycolysis and the organic acid metabolism have been observed in

germinating seeds (68, 91). Evidence for the presence of the pentose

phosphate-shunt pattern of metabolism has been found in mung beans (13).

In seeds containing large quantities of fats and oils, the tricarboxylic

acid pathway of metabolism may be partially replaced by the glyoxylate

pathway of metabolism which is a modified form of the tricarboxylic acid

cycle (52, 62, 68, 114). The glyoxylate pathway functions in the con-

version of fats to sugars.


Physiology of Peach Seed Germination

Peach seeds are characterized by a requirement for a period of low

temperature for natural termination of dormancy. Chemicals have been

found that wvill induce germination of dormant seed. These factors and

others are discussed below.








ENVIRONAE~TTL FACTORS:

The optimum temperature of 50 C with a range of 5-100 C for 60-90

days has been found best suited for the termination of dormancy of peach

seeds (12, 16, 19, 29).1 The duration needed varies with varieties.

Some varieties require fewer hours of chilling to break dormancy than do

others (12). If a warm temperature treatment immediately follows ex-

posure of seeds to low temperatures, the growth capacity of the seeds

will be greatly reduced (14, 80). The reduction in growth capacity can

subsequently be restored by subjecting the seeds to additional exposures

to low temperatures.

Observations indicate that peach seeds are indifferent or day-

neutral toward the influence of light on germination (R. H. Biggs, Un-

published data).

It has been observed that the amount of free water present during

germination will influence the process. If seeds were allowed to be in

contact with free water, as in a petri dish, they generally became

bloated as a result of too rapid an uptake of water. However, if the

seeds were placed in moist vermiculite, they were not bloated (36).

This has been shown to occur with other types of seeds, particularly the

legumes (64).

CHEMlICAL FACTORS:

External: Tukey and Carlson (97) showed that applications of

thiourea to dormant 'Lovell' peach seeds induced germination. Evidence

obtained by Garrard (36) indicated that both the sulfhydryl and the

imido group are requisite to the activity of thiourea. Mercaptoethanol,

mercaptoethylamine, and urea were not effective either alone or in com-

bination in promoting germination of 'Okinawa' peach seed (76). The








induction of germination of dor-mant peach seeds has resulted in the

formation of abnormal seedlings wlhen induction was by means of thiourea

or seed coat excision (2S, 36, 37, 97). However, it has been recently

shown that the temperature during germina,.on plays a major role in the

development of abnorma~lities in the seedlings (SO) and that warm temper-

atures during treatments with chemicals or by embryo excision was re-

sponsible for increasing the severity of abnormalities (9) and not the

treatments. Thus, it is possible that two mechanisms are functioning

within the embryo; one that breaks dormancy and initiates germination,

and another which controls the development of the epicotyl.

Gibberellic acid has been found to induce the germination of dormant

peach seeds but by a different mode of action than that of thiourea (76).

Gibberellic acid can decrease, to some extent, the occurrence of leaf

anomalies on peach seedlings and stimulate stem elongation (30). It is

possible that gibberellic acid has a modifying influence on both germi-

nation and epicotyl development.

Internal: Pollock and 01ney (72, 81) have studied extensively ,he

rest period of seeds of sour cherry, Prunus cerasus, with respect to

metabolic changes and growth. Their results showed that during low

temperature treatment to terminate dolrmiancy, nitrogen and phosphorus

are translocated from the cotyledons to the embryonic axis of the embryo.

The rate of translocation of nitrogen was equal to the rate of cell

division; therefore, the nitrogen content per cell seemed to remain

constant. The rate of translocation of phosphorus was in excess of

cell division and the phosphorus concentration in the cells increased.

The experiments indicated that the translocated phorphorus was incorpo-

rated into all phosphate compounds in the cells. In fully ,;urgid seeds








kept at warm; temperatures, phosphorus tended to accumulate as inorganic

phosphate rather than in organic metabolites. These authors suggested

that the rest period may be associated w' :-, a block in the phosphate

metabolism of the cells. This h. not .en substantiated at t:- present

time.

Pollock (80), using 'Elberta' peach seed, suggests that the causal

agent of the dwarfing effect in seedlings is independent of the growth

inhit tor content of the seed. The physiological and anatomical a,...cts

of dwarfing suggested a control by a self-duplicating system localized

in a limited region of the apical meristem and transmitted on~ly by cell

division. ";?is system was temperature sensitive during the time between

the first visible root 5-owth and shoot elongation.

Investigations have been made into the effect of the degradation

products of the glucoside amygdalin within the seed. Upon imbibition,

mandelonitrile could be detected in the seeds (1); it was assumed to

have ari.,an from the hydrolysis of an~ygdalin to mandelonitrile and

glucose by prunsin. 'I"!e mando-^onitrile was further hydroly::ed to

cyanide and benzaldehyde (104), presumably by emulsin.

The presence of benzaldehyde during i::bibition, as a result of the

degradation of amnygdalin, suggested that possibly ..enzoic acid and some

of its derivatives r:.y be foir~ed (26, 104). An alternative to this

pathw,,y is that in which rion, slic acid, foinned from ... .:Colon~itrile,

undergoes enzyr,:atic conversion to benzoic acids (40, 41, 89).

This study will be concerned with the changes in phenolic compounds

during the breaking of j ;;mancy s.;8 subsequent germination of the seed.















MATERIALSS AND `rIT ODS3~


All seeds were obtained from the 1965 and 1966 crops of Prunus

persica cy. 'Oktinawa'. This m~. 31ial was chosen for several reasons.

Principally, the seeds are relatively hom~ozygous in respect to the chill-

ing requirement to terminate seed do~rmancy (9), the seeds require a rel-

atively short period of low temperature stratification to overcome tnm

dormant state (9), and when the embryos are excised they germinate readi-

ly without any apparent abnormalities if the temperature range during

germination is 18-250 C (76).

The seeds were removed from the endocarp just prior to each experi-

ment and allowed to imbibe water from moistened vermiculite. Depending

upon the nature of the experiment, the time in moistened vermiiculite

varied. The seeds were planted in seed flats containing a 2:1 mixture

of perlite: vermiculite. Techniques for each experiment will be dis-

cussed separately.

Tests for interaction I thiourea and seed coat excision on germi-

nation: In order to determine the most effective thiourea concentration

to promote the greatest amount of germination with the least amount of

anomolous growth, a range of concentrations was tested. Th~;is was as

follows: 0.0, 1.0, 3 x 10-1, 10-2 and 10-3 AI thiourea. The seeds were

kept in a moist medium ;or 42 hours and then followoo~ uy 6 hours' soaking

in the respective thiourea concentrations. Before imbibition, the seeds

were surface sterilized for 3 minutes with a 1,000 ppm merthiolate in









25% ethanol: water solution. After the soaking period, the seeds were

blotted and planted in flats and kept in the dark at 200 C for 16 days

before beings placed in a greenhouse. Each treatment was replicated 3

times with 40 seeds per replication.

A second experiment was designed to determine if any interaction

existed between thiourea and the seed coat on the degree of anomalous

development of the subsequent seedlings. Thiourea concentrations of

0.0, 10-1 and 3 x 10- M were applied to intact seeds and to excised

embryos after 42 hours imbibition. After a 6 hour treatment period,

the seeds were removed from the solutions, blotted, and planted in seed

flats. At 3 time intervals of 24, 48 and 72 hours, seed coats were re-

moved from samples of intact seeds treated with thiourea and the excised

embryos replanted. All treatments were kept in the dark at 200 C for 10

days, except for brief period of examination. After 10 days the flats

were moved to the greenhouse. Each treatment was replicated 3 times with

9 seeds per replication.

Testing chemicals for modification of germination of peach seeds:

Benzaldehyde, benzoic acid, cyanide, p-hydroxybenzoic acid and mandelo-

nitrile in a series of concentrations were tested on seed germination.

Because of volatility and water solubility of the chemicals, methods of

treatment varied. Each treatment was replicated 3 times with a random-

ized block design and observation on germination were taken at 7 and 12

days after the start of seed imbibition in all cases. Data was analyzed

statistically using F test and Duncan's multiple range (90).

In the cyanide treatments, the concentrations used were 0.0, 1.0,

10-1 3 x 12,and 10-2 Mi made with potassium cyanide. Seeds were

allowed to imbibe for 42 hours, seed coat removed and the embryos placed








in an aqueous solution of the chemical for 6 hours. They were then

planted in seed flats and placed in a growth chamber with a controlled

temperature of 200 1 2o C and a 12-hour- day of approx-imately 900 ft-C

light inten;sity.

Benzoic acid and p-hydrox ~;nzoic acid were tested at concentrations

of 0.0, 10-1, 3 x 10-2, 10-2, and 10-3 5.~ To test the respective concen-

trations of each compound, a, roximately 5 g of dry perlite were placed

in 100 ml beakers and the perlite saturated with the solution of c:;emical

to be tested. After equilibration of the mixture, fully turgid seeii ,

attaining this condition in moist vermiculite in 4S hours at 200 C, were

placed in the perlite plus chemical media, and maintained under aerobic

conditions. After 5 days in the inedia, the seeds were transferred to

flats c..nitainingr a 2:1 mixturee of perlite: vermiculite. All ~ of

the experiments were conducted in growth chambers at 200 1 20 C with a

12-hour day of 900 ft-C. light intensity.

Since benzaldehyde and mandelonitrile are only slightly soluble in

water, the method of treatment was modified. For these tests, a logra-

rithmic range of quantities of the material per unit of perlite was used.

A measured amount of the chemical was sbsorbeC onto fine perlite and

water added to the medium. The concentrations noted are based on the

amount that was available to the water phase. Five grams of the mixture

were used per container per treatment and care was taken to maintain

aerobic conditions. For preparation of the seed belore treatment, they

were allowed to imbibe for 4S hours, embryos excised and placed in the

perlite-chemical xsture. The embryos wcl- left in the c.
days at 200 C. Thou~, They were removed from the chemical envilolronen~ts,

planted in flats and placed in a grocculouse for the remainider of the

observational period.








Extraction and preparation of fractions from seeds for gas chroma-

tography: The isolation of the fractions was made from seeds that were

fully turgid after 4S hours in moist vermiculite at 200 C. The seeds

(10 g) were ground in a Servall Omni-mixer at 16,000 rpm for 3 minutes

in 30 ml of S0O% ethanol. The homogenate was filtered, the subsequent

filtrate dried under vacuum and the residue dissolved in 0.1 Mi tartaric

acid. This aqueous solution was partitioned against ethyl ether and the

ether phase separated and partitioned against an aqueous solution of

0.1 M sodium bicarbonate. The aqueous bicarbonate phase was acidified

with tartaric acid to pH 2.0 and then partitioned again with 100 ml

ethyl ether. The resulting ether solution was concentrated under nitro-

gen gas. The ether-soluble acidic fraction was subjected to gas chroma-

tography before and after treatment with acetylating agents.

Diazopropane was prepared with slight modification by the method

of WVilcox (112). Briefly, N-propyl-N-nitrosourea ( obtained from Dr.

Mlerrill Wilcox, Agronomy Department, University of Florida) was reacted

with 40% KOH in water and trapped in peroxide-free ethyl ether. The

etheral solution was stored over sodium sulfate in a polyethylene bottle

in a freezer. To acetylate a sample, sufficient amounts of the solution

were added so that a straw-yellow color persisted at the end of the re-

action period.

Alternative esterification methods with diazomethane and diazobutane

were used to aid in the identification of aromatic acids. The diazo-

methane reagent was prepared as outlined by Williams (113). Briefly,

N-methyl-N'-nitro-N'-nitrosoguanidine (Aldrich Chemical Co., Milwaukee)

was added to 20% KOH and trapped in ethyl ether. The diazobutane was

prepared in a manner similar to the diazopropane except substituting N,







N-butyl-N-ni trosourea (obtained fromt Dr. Mocrrill W~ilcox, Agronomny

Department, University of Florida) for the Nl-propyl-N-nitrosourea With

both diazomethane and diazobutane, the initial esterification period,

30 minutes, was the same as with diazopropane.

In tests where esterification was slow for the carboxyl group or

where acetylation of hydroxyl groups on the ring was slow or non-existent,

O.7%o methanolic boron trifluoride was added to these diazo-compounds and

the reaction was allowed to proceed at room temperature for 3 hours.

Standards of chemicals and fractions of extracts were dissolved in

ethyl acetate for gas chromatography. Weights and volume on seed and

solvent fractions were kept so that quantities could be expressed as

seed equivalents. Standards had a final concentration of 1 mg per ml.

Conditions for gas chromatography: Separation of compounds of the

acidic fraction of the ethanol extract was on a model 400 F and MI gas

chromatograph equipped with a flame-ionization detector. The column

consisted of 1/4 inch stainless steel tubing 6 feet long packed with So

S.E. 30 on 60-80 mesh Chromosorb WV. Helium was used as a carrier gas with

flow rate of 70 ml per minute. Temperatures for the system were as

follows: oven, 1800 C; injection port, 2600 C; and detector, 2500 C,

except as noted in the results.

Identification of extracted compounds was made by comparison of

their retention times with those of the known compounds. Matched reten-

tion times of several derivatives of knowns to those of identically treat-

ed unknowvns lent greater support to tentative identification.

Alfalfa bioassay: Peruvian alfalfa seed were separated into red and

yellow seeds. The red seeds were discarded because of their low germina-

tion capability (111) and the yellow seeds were used for the bioaissay.








The bioassay was conducted in petri dishes with either filter paper

disks or chromatography paper strips as a moisture holding absorbent,

depending upon the test. Generally 40-50 seeds per dish were used for

each assayed fraction. Before placing the seeds on the moistened paper,

they were soaked in distilled water for a few seconds to improve the

rate of imbibition of the seeds. Once the seeds had been placed in the

dishes, the dishes were placed in the dark at 200 f 20 C. After 24

hours, observations were made on the number of germinated and non-germi-

nated seeds per dish. A seed was considered to have germinated upon

protrusion of the radicle.

Inhibitor characterization: Bioassays were conducted on 80%"

ethanolic extracts and fractions paper chromatographed in isopropanol:

ammonia: water (80:1:19, v/v/v) solvent on WVhatman 3 MM~ chromat'ogrr apl.i

paper. Chromatograms were divided into sections of 10 Rf units and

assayed, using the alfalfa seed bionssay (18).

Ex-tracts of peach seeds were also subjected to acid hydrolysis

(pH 2) with acetic acid, alkraline hydrolysis (pH 10) with ammonium

hydroxide, dialysis against distilled water for 24 hours; and heating

for 10 minutes at 50, 75, and 1000 C. Changes in inhibitory activity

were monitored, using the alfalfa bionssay.

Solubility of components of the inhibitor~ complex in various organic

solvents was investigated. Sections of the paper chromotograms contain-

ing the inhibitory zone were cut into strips representing the equivalent

of a 0.5 g seed sample. These strips were steeped in various solvents

for 2 hours. The solvents were decanted into small petri dishes con-

tainingr a Whnatman No. 4 filter paper diskr and the residue deposited on

the paper by evaporation. Distilled water (1.5 ml) was added to the








petri dishes, and to appropriate controls, and then bioassayed. Redis-

tilled solvents of water, hexane, acetonitrile, ethyl ether, chloroform,

methanol, ethyl acetate and carbon disulfide were used for the solubility

studies.

MIeasurement of benzaldehyde and mandelonitrile: The quantity of

benzaldehyde and mandelonitrile present in seeds under various treat-

ments was determined. All seeds were fully turgid since they were placed

in moist vermiculite for 42 hours at 200 C prior to treatment. Treat-

ment I was seeds steeped in 3 x 10-2 M thiourea for 6 hours, blotted

and kept in a moist medium until sampled. Treatment 2 was embryos re-

moved from the seed coat and associated tissue after 48 hours from the

start of the experiment. Treatment 3 was the control of intact seeds.

Seeds in each treatment were kept at 200 C and a 4.8 g sample wet

weight, equivalent to approximately 3 g dry weight, were taken at the

following times from the start of seed imbibition: 48, 60, 72, 80, 88,

96, 104, 112, 120, 132, 144, 156, and 168 hours. The samples were

frozen immediately to -700 C and then placed in a freezer at -300 C

until ground, approximately 8 hours. The frozen seeds were ground in a

WViley mill with a 20-mesh sieve. The mill had been thoroughly cooled by

passing large quantities of dry ice through it before the samples were

ground. Also, sufficient amounts of powdered dry ice were passed

through the mill along with the frozen seeds to keep the grinding head

at approximately the temperature of the dry ice. The ground seeds plus

powdered dry ice were collected together and added to ethyl ether at

-700 C. After the dry ice had sublimed from the ethyl ether (generally

30-40 minutes at room temperature) the solutions were allowed to warm

to approximately -50 C before they were placed in a -300 C environment









for 3 hours. This warming and steeping in a freezer was needed to obtain

benzaldehyde and mandelonitrile in the ether phase. This etheral solu-

tion was subjected to gas chromatography under conditions noted with the

results. Weight and volume were taken quantitatively so the data could

be expressed in the amount of chemical per seed equivalent.

Under the conditions of gas chromatography, benzaldehyde and

mandelonitrile chromatographed as benzaldehyde since heat caused mandelo-

nitrile to decompose to HCN and benzaldehyde. Therefore, the following

series of reactions were used to separate the 2 components. Firstly,

the quantity of both compounds was obtained from gas chromatographic

analysis of an aliquot of an extract. Secondly, the quantity of

mandelonitrile remaining in an etheral solution was determined after

quantitatively removing benzaldehyde by reacting with sodium bisulfite.

This was accomplished by solvent partitioning between the etheral solu-

tion and aqueous 40%o sodium bisulfite. Thirdly, quantitative analysis

was again done on the ether phase after 40% potassium cyanide was added

to the aqueous sample layered under ethyl ether and the mixture shaken

vigorously. This converted the sodium bisulfite addition product of

benzaldehyde to mandelonitrile which allowed it to pass back into the

ether phase. After allowing the mixture to stand for 5 minutes in the

cold, the ether phase was subjected to gas chromatographic analysis

the third time. Quantitative determinations were made using the area

under the peak as a measure of both compounds and the peak area of the

sample after addition of sodium bisulfite. The latter represents that

due to mandelonitrile. The difference between the two peak areas was

assumed to be that due to benzaldehyde.








The conversion of benzaldehyde to a sulfite derivative soluble in

water and then conversion to mandelonitrile is a well-known reactionr

(26). The sodium bisulfite reacts with the carbonyl group of La e-

hyde to form the sulfite addition product. Addition of potassium cyanide

acts as a base and neutralized the sodium bisulfite in equilibrium with

the bisulfite compound to form potassium bisulfite; the simultaneously

liberated benzaldehyde and hydrogen cyanide then combine to give mandelo-

nitrile (26).

Chilling study: Determinations were made of the inhibitor complex

benzaldehyde and mandelonitrile after periods of chilling. Fully turgid

seeds, attaining this condition after 48 hours in moistened vermiculite

at 200 C, were placed at 4o C for 0, 168, 336, 504, and 672 hours. At

the time of sampling, one sample was removed and extracted immediately

and another samnple was placed for an additional 40 hours at 200 C. A

control lot of seeds was maintained at 200 C for sampling at equivalent

times. At each time of sampling, seeds equivalent to 5 g dry weight

were taken in duplicate. The quantity of benzaldehyde and mandeloni-

trile in the seeds was determined as outlined previously and the level

of non-volatile inhibitors, presumably the beta-inhibitory complex (8),

was assayed as follows. A sample of treated seeds was subjected to

extraction with 80% ethanol after grinding, as previously noted. The

solution was taken to near dryness by vacuum distillation, keeping the

distilling chamber at less than 50 C. The residue was redissolved in

80% ethanol, applied to chromatogrraphic paper (WVhatman 3 ADII) and devel-

oped in isopropanol: ammonia: water (80:1:19 v/v/v) solvent, using de-

cending techniques. The inhibitory zone, as determined by Rf, w\ere

sectioned from the chromatogramus, and solutes eluted from thie paper with





glass distilled water. The eluates were then diluted in such a way that

equivalent seed weights in the solutions were 1.0 g, 500 mg, 300 mg,

100 mg and 0 mg. The solutions were placed in small petri dishes on

Wihatman No. 4 filter paper disk, frozen and water removed by sublimation

under vacuum. After again moistening the filter pads with 1.5 ml of


H20, they were bioassayed with the alfalfa bioassay using 40 seeds per

disk. Inhibitory levels were determined by calculations from a dilution

curve based on relative seed weight,





EXPERIMENTAL RESULTS


Thiourea and seed coat excision: The influence of thliourea on

germination of dormant 'Okinawa' peach seed and on anomalous seedling

development is shown in Table 1. It was quite evident from this data

that thiourea greatly increased the per cent geinuination, but enhanced

anomalous development: in the seedlings. In both cases, the higher the

concentration, the greater the effect. The data indicates further that

increases in germination and abnormal growth were statistically signifi-

cant with concentrations of thiourea stronger than 10-2 M.

In determining the possible interaction between thiourea and seed

coat on germination and subsequent seedling growth, the most strikiing

finding was the absence of abnormal seedlings in any of the treatments;

yet very good germination was obtained, as shown in Table 2. The length

of time after imbibition and thiourea treatment for embryo excision

seemed to have little effect on germination.

Influence of Benzaldehyde, cyanide, and mandolonitr-ile on seed

germination: The data in Table 3 indicates that cyanide does not dras-

tically reduce germination, except in very high conccontrations (1.0 Ml).

Data taken 20 days after start of imbibition showed that the 1.0 M con-

centration was still significantly different from the lower concentra-

tions used. No abnormalities were noted in the seedlings froml any of

the treatments, and, interestingly, the 1.0 M cyanide did not kiill the

seeds.























Miean
%o abnormal, z

0.0 a

0.0 a

21.9 b

40.1 b

61.4 c

79.0 c


Table 1.--Influence of thiourea concentrations on germination and
per cent of production of abnormal seedlings from
'Okinawa' peach seeds.


xEach treatment was replicated 3 times with 40 seed per replication.

Yleans not having a following letter in common are significantly differ-
ent at the 1% level.

zPercentage based on the total number of seed germinated.


Thiourea
concentration, Mx

0 (Control)

10-3

1-2
10-1

3 x101



1.0


M~ean
% germination?

5.8 a

5.8 a

26.8 ab

45.8 bc

62.5 c

58.3 c



































































xEach treatment consisted of 3 replications of 9 seed each.

Y~leans not having a following letter in common are significantly differ-
ent at the 1% level.

zHours after start of imbibition.


% atypical seedling

0.0

0.0

0.0

0.0

3.7


Treatments Mlean
Chemical Sood coat %0 germinationF


Table 2.--Effect of thiourea concentration and embryo excision on
germination of 'Okinawa' peach seeds 12 days after start
of imbibition and on abnormal seedling production 32
days after start of imbibition.


0.0 a

100.0 e

92.6 cd

100.0 e

100.0 e


Control












10-1RI
Thiourea


Intact

Excised;










Intact

Excised;


hrsz

hrs

hrs

hrs


7.4

0.0

0.0

0.0

3.7



3.7

0.0

0.0

0.0

3.7


81.5

96.3

92.6

100.0

100.0



88.9

96.3

100.0

100.0

100.0


b

de

cde

e

e


hrs

hrs

hrs

hrs


3 x10-14I
Thiourea


Intact

Excised;


c

de

e


hrs

hrs

hrs

hrs


WSeeds were imbibed 42
before planting.


hours, then treated with chemicals for 6 hours















Table 3.--Per cent germination of 'Okinawa' peach seeds 7 and 20 days
after start of imbibition as influenced by cyanidex



Cyanide Mlean %/ germinationz
concentration, MI 7 days 20 days

1.0 0.0 a 0.0 a

101 88.9 b 94.4 b

3 x 10- 100.0 c 100.0 b

10-2 100.0 c 94.4 b

Control 100.0 c 100.0 b


xSeeds were imbibed for 42 hours, then treated with the designated
cyanide concentrations for 6 hours.

Each concentration consisted of 3 replications with 6 seed per
replication.

Means not having a following letter in common are significantly
different at the 5% level.






The treating of samples of excised embryos with various concentra-

tions of mandelonitrile and benzaldehyde resulted in the inhibition of

germination with some of the stronger concentrations (Tables 4 and 5).

Mandelonitrile at 1.4 to 140.0 mg/g completely inhibited germination,

while all other concentrations except 0.42 mg/g inhibited only slightly.

Data taken 5 days after removing the seeds from the chemical showed

that 1.4 mg/g exhibited very little inhibitory influence. Concentrations

of 4.2 to 140.0 mg/g were still strongly inhibitory (Table 4). Concen-

trations of benzaldehyde of 11.0 and 110.0 mg/g completely inhibited

germination, while a concentration of 3.3 mg/g resulted in only 16.7%0

germination. Concentrations lower than 3.3 mg/g had no measurable in-

fluence (Table 5). Five days after removal of seeds from the benzaldehyde

media, germination occurred to an appreciable extent in the 3.3 mg/g

treatment but 11.0 and 110.0 mg/g were still inh~ibitory. As found with

cyanide, the anomalous growth patterns were not present on seedlings

produced from seeds treated with either mandelonitrile or benzaldehyde.

The influence of the benzaldehyde and mandelonitrile on seed germination

is portrayed graphically in Figure 1.

Aromatic acids investigation: Tentative identification of the com-

ponents isolated from the propyl esters of the acidic fraction of an

ethanol extract of peach seeds was made by comparing the retention times

on gas chromatograms with those of known compounds. A gas chromatogram

of the fractions is shown in Figure 2.

The phenolic compounds tentatively identified were benzoic, mandelic,

o-hydrox-ycinnamic, 2, 6-dihydroxybenzoic, o-hydroxybenzosic, p-hydroxyben-

zoic and 2, 4-dimethoxybenzoic acids (Table 6). The gas chromatograms of

the standards for the known compounds listed above can be found in the

Appendix.















Table 4.--Per cent germination of 'Okinawa' peach seeds 7 and 12 dlays
after start of imbibition as influenced by mandelonitrilex'y




Mlandelonitrile aainnl 5; g:ennlinationz
mg/g of perlite 7 days 12 days

140.0 0.0 a 0.0 a

14.0 0.0 a 0.0 a

4.2 0.0 a 0.0 a

1.4 0.0 a 83.3 b

0.42 100.0 c 100.0 c

0.14 94.4 bc 100.0 c

0.042 94.4 bc 100.0 c

0.014 94.4 bc 100.0 c

Control 88.9 b 88.9 bc


xSeeds were allowed to imbibe for 2 days, then placed
containing mandelonitrile for 5 days. The indicated
mandelonitrile was applied to the perlite.


in perlite
quantity of


YEach treatment was replicated 3 times with 6 seed per replication.

zIMeans not having a following letter in common are significantly
different at the 1% level.














Table 5.--Per cent germination of 'Okinawa' peach seeds 7 and 12 days
after start of imbibition as influenced by benzaldehydexty




Benzaldehyde M~ean %' germination
mg/g of perlite 7 days 12 days

110.0 0.0 a 0.0 a

11.0 0.0 a 0.0 a

3.3 16.7 b 94.4 b

1.1 100.0 c 100.0 c

0.33 100.0 c 100.0 c

0.11 100.0 c 100.0 c

0.033 100.0 c 100.0 c

0.011 100.0 c 100.0 c

Control 100.0 c 100.0 c


XSeeds were allowed to imbibe for 2 days, then placed in perlite contain-
ing benzaldehyde for 5 days. The indicated quantity of benzaldehyde
was applied to the perlite.

yEach treatment was replicated 3 times with 6 seed per replication.

IMeans not having a following letter in common are significantly
different at the 1% level.

















































a6z~ua~~ad 'uo!aeu!Lucla~ u~a~


--- 7


O

O
*,
>0








o


>ae


j O


SO
O d
O o


a.
+-


O
-0 r



oI


od


dehyde ---
Benza\
--- ~ y\\e
-~ de\on\
1 pan

I
I
I


O
O


~O


~t I I-~---.--1_-CJ---= -- ---
O O O O O O
O0 03 COd


i I
I


"c~














SI




Oi

Ul I/






O 8 1 2 3


II 1T1







Fig. 2. Gas chromatogram of the propyl esters of the acidic
fraction from an ethanol extract of peach seeds.
Time is in minutes. (See Table 6 for gas chromato-
graph parameters.)
















Table 6.--Relative retention time and possible identity of components
separated by gas chromatog~raphy of the propyl esters of the
acidic fraction from an eth~anolic ex-tract of peach seedsx




Peak No, Relative retention time7 Possible identity of acidsz

1 1.00 Benzoic
2 1.37 Succinic
3 1.91 Malic or mandelic?
4 2.13 o-hydroxycinnamic
5 2.50 Fumaric ?
6 2.S9 2,6-dihydroxybenzoic
7 3.59 o-hydroxybenzoic
S 4.99 p-hydroxybenzoic
9 5.89 2,4-dimethoxybenzoic
10 7.11 ?
11 10.21 Citric
12 19.29 ?


x -


Instrument: F & M model 400, flame detector.
Column: 870 S.E. 30 on 60-80 mesh chromasorb WY, acid washed,
silane treated; 1/4"1 O.D. stainless steel 6' in length.
Carrier gas: He. Outlet flow rate: 70 ml/min.
Oven temp: 1800 C. Injection port temp: 2600 C.
Detector temp: 250 C.
Range and attenuation: 10 x 8. Chart speed: 1/411 per man.


Relative retention time is bsdo ezi cd

z Possible identity based on matched retention times.








Inhibitor characterization: Using the Peruvian alfalfa bioassay,

it was found that extraction of 8 g wet weight of dormant peach seeds

with SO% ethanol yielded a strongly inhibitory complex (0%/ germination).

Specific gravity measurements indicated that the inhibitory influence

was due to factors other than osmotic ones. Paper chromatography of the

ethanol extract, using an isopropanol: ammonia: water (80:1:19 v/v/v)

solvent system, yielded a strong inhibitory complex between Rf's 0.6 and

0.8 when bionssayed with the alfalfa seed test (Table 7).

From tests on the influence of acids, bases and heat on the stabil-

ity of the inhibitory complex, the data on per cent germination from the

alfalfa bioassays (Table 8) would seem to indicate that the inhibitory

complex was reasonably stable since none of the treatments destroyed the

inhibitory capacity of the extract.

Comparison of various organic solvents (Table 9) for the solubil-

ization of the inhibitory complex showed that the more polar solvents

(water and alcohol) serve as suitable solvents for the inhibitor. The

data indicated also that the inhibitor may be only partially soluble

in acetonitrile.

Isolation and characterization of benzaldehyde and mandelonitrile

from peach seeds: Benzaldehyde and mandelonitrile were isolated and

characterized from peach seeds by several techniques. Crushed peach

seeds evolve an aroma similar to that of benzaldehyde and mandelonitrile.

Co-chromatography of the pure chemicals and the extract components from

peach seeds by chromatography yielded identical R 's and retention times,

respectively. Ultra-violet fluorescence (3200 Ao and 2537 Ao) of benz-

aldehyde and a fraction from an ethanol extract from peach seeds on

paper chromatograms were identical. Also, benzaldehyde and the extracted

















Table 7.--Paper chromatographic separation of the
inhibitory complex from dormant peach
seeds.


xSolvent system: Isopropanol: ammonia: water
(80:1:19 v/v/v).

YI~ioassayed with the alfalfa seed test.
Control = 90%.


Rf Valuex


% GerminationY


0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


-0.1

- 0.2

- 0.3

- 0.4

- 0.5

- 0.6

- 0.7

- 0.8

- 0.9

-1.0


0j2.5

85.0

87.5

85.0

55.0

0.0

0.0

0.0

85.0

80.0















Table 8.--Influence of acids, bases and heat on the
inhibitory complex from peach seeds after
paper chromatography.


%j Germinationx


Test


Acid hydrolysis (pH 2)

Alkaline hydrolysis (pH 10)

Dialysis, inside tubing

outside tubing

Heating for 10 minutes:


0.0

0.0

0.0

0.0


500 C

750 C

1000 C


0.0


0.0

0.0


Extract control

WVater control


90.0


XBioassayed with the alfalfa seed test.
equivalent of the extract was 0.5 g dry


Seed
weight.


Dialysis was conducted with seamless cellulose
tubing against distilled water for 24 hours.

















Table 9.--Solubility of the inhibitor-complex in
various organic solvents as determined
by the alfalfa bioassay.



%~l Germination
Chromatogram
Solvent Eluatex Sectiony
(Rf 0.6-0.8)

WVater 0.0 80.0

Hexane 92.5 0.0

Acetonitrile 30.0 0.0

Ethyl ether 85.0 0.0

Chloroform 90.0 0.0

Methanol 0.0 82.5

Ethyl acetate 87.5 0.0

Carbon disulfide 92.5 0.0


xElution fraction from
0.8.


chromatogram section of Rf 0.6-


Chromatogram section containing the inhibitory complex
after eluting with the respective solvent.








component reacted similarly to aldehyde indicators. Component of an

ethanol extract and benzaldehyde formed a sodium bisulfite addition

product which then generated mandelonitrile on treatment with KCN.

Ultra-violet fluorescence (3200 Ao and 2537 Ao) of mandelonitrile

and a fraction from an ethanol extract from peach seeds on paper chroma-

tograms were identical. The extract components and mandelonitrile form

benzaldehyde and cyanide when subjected to high temperatures (200-2500 C).

Mlandelonitrile and components of the extract reacted alike when tested

with hydrocyanin indicators.

Quantitative determinations of benzaldehyde and mandelonitrile:

Benzaldehyde and mandelonitrile were determined using procedures estab-

lished in identifying the 2 compounds. Briefly, this was gas chromatog-

raphic analysis of the ethereal extract before making a sodium bisulfite-

addition product, after the reaction to assay the level of decrease and

again after converting the benzaldehyde to mandelonitrile by KCN. A

chromatogram of the composite of both compounds is shown in Figure 3a.

After treating with sodium bisulfite, the peak is reduced (Figure 3b)

and increased after the subsequent addition of potassium cyanide

(Figure 3c).

Comparison of the influence of thiourea treatment, and embryo exci-

sion, as compared to a non-germinating control of intact seeds, on the

rate of release of benzaldehyde and mandelonitrile both collectively and

individually is shown in Figures 4, 5 and 6. The graphs indicated that

intact seeds and thiourea-treated seeds had peak times of production of

benzaldehyde and mandelonitrile at about the same time, 72 hours, while

the excised seeds had a delay in the maximum period of production by 16

hours. The thiourea-treated seeds had a second peak of production at






























Fig. 3. Gas chromatogramns of a known composite sample of an etheral
solution of benzaldehyde-mandelonitrile: (a) initial solution;
(b) after addition of a solution of sodium bisulfite; and (c)
after addition of potassium cyanide.

Gas chromatograph parameters.


F & M model 400, flamne detector.
8%o S.E. 30 on 60-80 mesh Chromosorb WY, acid-
washed, silane treated; 1/4"' O.D. stainless
steel 6' in length.
Helium. Outlet flow rate: 70 ml/min.
Oven, 1000 C; Injection port, 1500 C; Detector,
1600 C.

10 X 8
1/4n per minute.


Instrument:
Column:



Carrier gas:
Temperatures:

Range and
Attenuation:
Chart Speed:

















U .


-


asuods-


0





00





I~F~~~CI~=~C~--U ~-----~C-S--- ----L~R-._~~ .i~-~~~_~5~-~7~i-~


1__1_ __I~...~. _i~~


11I
1-
!!
ii


U')
L
O
.c
o'
E


.- I


1. a
Il

Oi


paas ~~ p 6/~LU




49






I





I



U



z ~O a


L a
IE O

CO -a
1 0 H S
a =
I ?y
a~ rso
Ek 'jC
00





1s u

11B o

no I

cm~ o
0 0 0































































co
d


-cO



















O
- ev














. to


C~P-~--CC"-rm~-T~-~----~3~-3~- I--- -~C----l --~_----


i __


O,




t o
c,


oa
r
*Hd
09
f
o
S3
Hd
On


s:
cm


o t0
rJs


0 0


<0
3
O




E


C\






IICO


1 .1EL;L~
v o
Cj C







120 hours after the start of imbibition. Relatively larger amounts of

mandelonlitr-ile than benzaldehyde were in the extracts from the seeds.

The effect of the 3 treatments on peach seed germination is shown

in Figure 7. The excised seeds attained 100% germination at approxi-

mately 132 hours or about 44 hours after the peak in production of benz-

aldehyde and mandelonitrile. However, those seeds treated with thiourea

required a much longer period of time after the peak production time in

order to attain nearly 100%0 germination. This was true even when the

time from the second peak at 120 hours was considered.

Quantitative determination of benzaldehyde, mandelonitrile and the

inhibitory complex of seeds subjected to various degrees of chilling:

Gas chromatographic determination of the quantities of benzaldehyde and

mandelonitrile in peach seeds at weekly intervals during the chilling

period indicated only trace amounts were present. Calculations indicated

that the tissue level of both chemicals avas below 1.0 ug/gm dry weight

of tissue. Only trace amounts of benzaldehyde and mandelonitrile were

detected by gas chromatography on seeds placed at 200 C for 40 hours

after removal from various intervals of chilling.

The level of the 80% ethanol soluble inhibitory complex of peach

seeds, as determined by the alfalfa bionssay, does not decrease during

the chilling period (Figure 8). Slight week-to-week fluctuations were

present but the overall analysis showed little change in the level.

However, the per cent germination of seed periodically removed from the

chilling temperatures and placed at 200 C indicated that 504 hours of

chilling was sufficient to terminate dormancy in over 80% (Figure 9) of

the population of the seeds.






F -~-- ~---- ---
I `-


EXCISED


100 -
















I


CI
O



on


o
O,


~ NTACTCONTROL)


100 160 220 280


Tirne hours


Fig. 7. Germination of peach seed as influencocl by embryo excision,
and thiourea treatments as determined periodically after the
start of imbibition. (Growth of excised embryos wa~s taken
to be equivalent to germination when the radicl<. ha~d elongated
to 2 mm.)


HIO UR EA





























Fig. 8. Relative inhibitory activity, as measured by the alfalfa
bioassay, of the inhibitory complex in an ethanolic extract
of peach seeds chromatographing between Rf's 0.6 to 0.8.
(Hours of chilling were just prior to extraction; and seed
equivalents were A= 1.0 g. B= 0.5 g, C= 0.3 g, D= 0.1 g and
E= control.)











0O
uL




C3
U
m<




oU


U


n


L


O O O O
O 00 CO 4


-C
U
O


O
I


10
I0


O


O O
cu









100 -



















20~-




O -
I I1 ~ I_ _I I_~
O 168 336 504 672


Hours of chilling


Fig. 9. Germination of peach seeds as influenced by 50 C of varying
durations.








Influence of chemicals on possible stimulation of peach seed grermi-

nation: The influence of benzaldehyde, mandelonitrile, benzoic acid and

p-hydroxybenzoic acid on the breaking of seed dormancy was determined

and the test differed from that for inhibition of germination in that

they were applied to intact seeds. Using benzaldehyde and mandelonitrile

at various concentrations, there was no evidence for a stimulatory effect

on seed germination (Table 10). However, the 2 chemicals were active

in inhibiting the weak capacity for germination, which supported the

data of an inhibitory influence as shown earlier.

Benzoic and p-hydroxybenzoic acids were used on intact seed at

concentrations ranging from 103 to 10- M. The data in Table 11 in-

dicates that p-hydroxybenzoic acid at 10-1 and 3 x 10-2 M may have

slightly stimulated seed germination as compared to the control; yet

this is of doubtful significance since the control had a weak capacity

to germinate (compare Tables 10 and 11). Benzoic acid had little in-

fluence on germination under the conditions of these tests.

L-mandelic and p-hydroxybenzoic acid determinations: Gas chroma-

tograms of the control, peach seed extract, L-mandelic acid and p-hy-

droxybenzoic acid after treating with diazomethane for 30 minutes are

shown in Figures 10, 11, and 12. These should be primarily the estorss

of the aromatic acids. These were separated on the gas chromatograph

at an oven temperature of 1800 C. The methoxy and butoxy asters woro

separated by gas chromatography after acetylation by diazomethane and

diazobutane, respectively. Since these derivatives were more volatile,

an oven temperature of 1500 C was used.

Based upon the comparison of retention times of the components in

the extract with those of the standards (Table 12), it was concluded















Table 10.--Mlean per cent germination of dormant 'Okinawa'
peach seeds 30 days after start of imbibition
as influenced by benzaldehyde and mandelo-
nitrile concentrations,



% Germination
Concentration, Ml Benzaldehydez Mandelonitrilez

1.0 0.0 a 0.0 a

10-1 0.0 a 0.0 a

3 x 10-2 16.7 b 0.0 a

102 22.2 b 38.9 b

0 33.4 b 33.4 b


xSeeds were imbibed for 2 days, then
concentrations for 5 days.


exposed to chemical


Each treatment was
replication.


replicated 3 times with 6 seed per


zMeans not having a following letter in common were
significantly different at the 5%0 level.
















Table 11.--Mlean per cent germination of dormant 'Okinawa'
peach seeds 30 days after start of imbibition
as influenced by benzoic and p-hydroxybenzoic
acid concentrations.



%/ Germination

Concentration, MY Benzoicz p-Hydroxybenzoicz
1-1 55a38.9 b


3 x 10-2 0.0 a 38.9 b

1-2167a1.a
103 16.7 a 11.2 a


0 16.7 a 16.7 a


xSeeds were in moist medium for 2 days, then exposed to
chemical concentrations for 5 days.

yEach treatment was replicated 3 times with 6 seed per
replication.

zbleans not having a following letter in common are
significantly different at the 5% level.





























Fig. 10. Gas chromatogram of diazomethane-solvent control. Retention
time is in minutes. (See Table 6 for parameters of gas
chromatograph.)






































a s u o d sa ~




































O 4 8


O
CL


O 4 8 12


Time


Fig. 11. Gas chromatograms of L-mandelic acid(a) and
p-hydroxybenzoic acid(b) treated for 30 minutes
with diazomethane. Retention time is in minutes.
(See Table 6 for parameters of gas chromatograph.)






























Fig. 12. Gas chromatogram of an ethanol extract from peach seeds treated
for 30 minutes with diazomethane. Retention time is in minutes.
(See Table 6 for parameters of gas chromatograph.) (MI=
L-mandelic acid and pHBA= p-hydroxybenzoic acid.)

















O




C~l







to






00




o


asuodsa~

















Table 12.--Comparison of retention times of p-hydroxybenzoic
acid and L-mandelic acid as influenced by various
acetylation procedures.



Retention times (minutes)

Acetylation procedures Extract Standard

1-diazomethane, 30 minutes:
p-Hydroxybenzoic acid 4.10 4.10
L-Mlandelic acid 2.05 2.05

2-diazomethane, 3 hours:
p-Hydroxybenzoic acid 11.80 11.80
L-Mlandelic acid 4.57 4.57

3-diazobutane, 3 hours:
p-Hydroxybenzoic acid 12.60 12.90
L-M"/andelic acid
Methyl derivative 4.48 4.48
Butyl derivative 6.70 6.35


xRefer to Materials and Methods for the details of each
procedure.





65



that L-mandelic acid and p-hydroxybenzoic acid were present in peach

seeds after 48 hours' imbibition. However, only trace amounts of L-

mandelic acid could be detected in the extract (Table 13). On the other

hand, sufficient quantities of p-hydroxybenzoic acid were present for an

estimation of amounts in the tissue. Based on peak area comparison of

components of the extract with the standard, it was estimated that ap-

proximately 0.12 ug of p-hydroxybenzoic acid was present in 1 g wet

weight of seed tissue under the conditions of the tests.















Table 13.--Comparison of peak areas of p-hydroxybenzoic
acid and L-mandelic acid as influenced by~
various acetylation procedures.


xRefer to Materials and Methods for the details of each
procedure.

YEach standard represents 2 ug of the respective compound.


Peak area, mm2
Extract StandardY


Acetylation procedures


1-diazomethane, 30 minutes:
p-Hydroxybenzoic acid
L-Mandelic acid

2-diazomethane, 3 hours:
p-Hydroxybenzoic acid
L-Mandelic acid

3-diazobutane, 3 hours:
p-Hydroxybenzoic acid
L-Mandelic acid
Methyl derivative
Buty1 derivative


45.0
Approx. 5.0


49.0
Approx. 5.0


30.0

Approx. 5.0
Approx. 5.0


540.0
597.0


342.0
675.0


545.0

996.0
812.0















DISCUSSION


A quantitative method was devised to determine the amount of benz-

aldehyde and mandelonitrile in peach seeds. The method was designed to

quantitatively obtain the 2 volatile components in an ethereal solution

so it could be analyzed by gas chromatography. Grinding of the frozen

tissue and extraction at a low temperature prevented enzymatic release

and destruction of benzaldehyde and mandelonitrile. Keeping the ethereal

solution cool and immediate analysis prevented loss by volatilization.

Also, the analysis by gas chromatograph was done before and after reac-

tion with aqueous sodium bisulfite and again after reacting the benz-

aldehyde-sulfite addition product with potassium cyanide to form mandelo-

nitrile. The chemical reactions used are well known (26), and the deter-

mination of pure benzaldehyde and mandelonitrile by the technique de-

scribed was shown to be quantitative.

Using this procedure, the quantity and rate of release of mandelo-

nitrile and benzaldehyde were studied in relation to germination. The

data indicated that a lag time existed in excised seeds for the maximum

release of mandelonitrile and benzaldehyde. The intact seeds and those

treated with thiourea differed only in magnitude and the thiourea-treated

seeds exhibited a secondary peak at 120 hours.

The maximum period of germination of the excised seeds was about

48 hours after the maximum period of release of benzaldehyde and mandelo-

nitrile whereas the greatest period of germination of seeds treated with

thiourea occurred about 160 hours after the second peak of release of









benzaldehyde and mandelonitrile. Thus, there was no indication that

either benzaldehyde or mandelonitrile was correlated with an inhibition

of germination under conditions of these tests. Yet, when concentrations

of benzaldehyde and mandelonitrile are present in tissues at concentra-

tions of 11.0 and 4.2 mg/g respectively, they would be affecting the

system. Thus, the 2 compounds may have a temporary influence on germi-

nation, and it could be possible under certain circumstances a factor

contributing to dormancy.

Determinations were also made of the content of mandelonitrile and

benzaldehyde present in peach seeds at weekly intervals during chilling.

Only trace amounts were observed at the various times of sampling. Thus,

it seems that the majority of mandelonitrile and benzaldehyde was re-

leased between about 72 and 96 hours after the start of imbibition.

This was the first reported instance of the detection and measure-

ment of mandelonitrile in peach seeds. It may be significant that the

quantity of mandelonitrile present was much greater than that of benz-

aldehyde. This would indicate that the hydrolysis of amygdalin in in-

tact seeds was not the same as in seed homogenates. In the latter case

the products formed are benzaldehyde and cyanide with the mandelonitrile

considered an unstable intermediate (104).

Interest was also directed at the presence of phenolic acidls in

peach seeds. Using gas chromatographic techniques, benzoic, o-hydroxy-

cinnamic, 2,6-dihydroxybenzoic, o-hydroxybonzoic, p-hydroxybonzoic,

2,4-dimethoxybenzoic and mandelic acids were isolated and tentatively

identified. Of primary interest was p-hydroxybenzoic acid since it had

been reported in the literature as having growth regulator actions (105).

With this in mind, it was necessary to determine if p-hydroxybenzoic acid







existed in peach seeds. Using extraction procedure for phenolic acids

(113), gas chromatographic analysis of various derivatives were made of

the extracted components. Positive identification was made for p-hydrox-

ybenzoic and a strong indication was noted that L-mandelic acid was

present in the seeds. The latter has been reported to occur in peach

seeds (R. H. Biggs, Unpublished data), and found to inhibit germination
of alfalfa sed t1-6 M c ce rai n Jones and Enzie (46) identi-


fied a growth-inhibiting substance from peach flower buds as being

mandelonitrile.

Since degradation products of amygdalin were found to occur in

seeds, attempts were made to assess the influence of cyanide, benzalde-

hyde and mandelonitrile on germination. Cyanide treatments indicated

that only at the highest concentration tested, 1.0 M, was an inhibitory

influence shown. Recently, it has been reported that some plants, par-

ticularly Vicia sp., have the capability of metabolizing cyanide and

converting it into non-toxic compounds (34, 69). It was observed that

hydrogen cyanide (14C) was incorporated into asparagine in a number of

plant species. This was thought to be accomplished by cyanide coupling

with serine directly to form the 4-carbon chain of beta-cyanoalanine.

The beta-cyanoalanine could then form asparagine, or by addition of a

gamma-glutamyl group, form gamma-glutamyl-beta-cyanoalanine It was

concluded that cyanide had little influence on germination, except at

concentrations considered quite high. Interestingly, this indicates

that peach seed do contain a cyanide-resistant mechanism for respiration.

Furthermore, the subsequent seedlings were much greener and exhibited

other characteristics that accompany nitrogen fertilization. Thus, it

was concluded that the tissues were incorporating cyanide.









In contrast to the results of cyanide treatments, concentrations of

4.2 mg/g mandelonitrile and 11.0 mg/g benzaldehyde completely inhibited

germination of excised embryos. It was noted that mandelonitrile in-

hibited at a weaker concentration and that the intermediate concentra-

tion of both compounds had an action that was reversible. Thus, if high

enough concentrations of benzaldehyde or mandelonitrile did occur in

seeds they could be inhibitory and the action could be transitory if the

compounds were subsequently degraded (104).

The possibility that subsequent derivatives of benzaldehyde could

be involved in seed germination was investigated. Thus, the influence

of benzoic and p-hydroxybenzoic acids on dormant peach seeds was tested.

Benzoic acid had little influence, but concentrations of p-hydroxybenzoic

acid at 3 x 10-2 and 10-1 M significantly increased the degree of germi-

nation as compared to the control.

The fact that p-hydroxybenzoic acid has been found to have growth

regulatory properties (105) and its presence in peach seeds suggested

that it may play a role in dormancy. The growth regulatory activity of

p-hydroxybenzoic acid has been established for woody cuttings of Ribes

rubrum (105). Pilet (78) has reported that p-hydroxybenzoic acid at

low concentrations causes a stimulation of the growth of stem sections,

while at high concentrations it inhibits growth. The inhibition was

apparently due to the stimulation of IAA-oxidase and subsequent decrease

in auxin level (116). Also, the activation observed for lower concentra-

tions of p-hydroxybenzoic acid indicated that it acted on several other

biochemical processes which were connected with growth (78).

In Ribes rubrum, p-hydroxybenzoic acid was present in the range of

0.2 1.0 ug/g of fresh tissue (105). The quantity found in peach seeds

was approximately 0.12 ug/g of fresh tissue. Thus it appears that the







tissue-levels of p-hydroxybenzoic acid in both dormant Ribes woody stems

and dormant peach seeds were similar. In the case of Lens stems, inter-

node sections were stimulated to elongate at 10-6 M concentration (78).

At higher concentrations, the growth of stem sections was inhibited.

The quantity isolated from peach seeds was in the range that was inhibi-

tory in the Lens bioassay. Thus, it could be inhibitory to the seeds.

However the data was such with peach seeds that this point can be con-

sidered a matter of conjectual. Yet, it was shown that p-hydroxybenzoic

acid would stimulate peach seed germination at 3 x 102 to 101 M con-

centration. This stimulation was from adding p-hydroxybenzoic acid to

the external media for 120 hours. Thus, the internal concentration could

have been much lower. The bioassay would not demonstrate an inhibitory

effect.

The ethanolic extracted inhibitory-complex obtained from peach

seeds was studied with the use of paper chromatography and other physical

treatments in order to obtain some clues as to its identity. The Rf

values obtained on paper chromatography correspond with the inhibitory

area obtained by Bennet-Clark and Kefford (8) using the same solvent

with an alcoholic extract from Ribes sp. This inhibitory area was termed

the beta-inhibitor complex. Recently, the beta-inhibitor concentrated

from an acidic fraction from extracts of dormant maple buds was thought

to be a complex of phenolic substances (86). However, the phenolic com-

pounds described were found not to be identical with any of the phenolic

compound previously proposed as being members of the beta-inhibitor

complex. Recently, many of the phenolic compounds associated with the

beta-inhibitor complex have been identified. Koves and Varga (53) re-

ported the identification of many phenolic compounds, among which were

several of the hydroxybenzoic acids.








The data on the level of the inhibitor-complex during the chilling

period showed that it did not change drastically. In fact, at the end

of chilling period, the level seemed to be greater than anytime during

chilling. This finding was in line with that found by Villiers and

Wareing (106, 107) for dormant organs of Fraxinus excelsior. Briefly,

chilling has no effect on the level of inhibitors in the tissue but ter-

mination of dormancy was accompanied by a buildup in growth promoters in

the seed. This may be the case for peach seeds since the capacity to

germinate increased with increases in the duration of the chilling period.

The products of amygdalin degradation, mandelonitrile, benzaldehyde

and cyanide do not appear to directly influence the breaking of peach

seed dormancy, However, it would seem that a hydroxylated derivative,

p-hydroxybenzoic acid, of the benzaldehyde oxidation product, benzoic

acid, exhibits some stimulatory influence upon dormant peach seeds.

Furthermore, L-mandelic acid, as well as other phenolic compounds, may

be involved in peach seed dormancy.

The induction of germination of peach seed by thiourea substantiated

previous reports that this chemical will terminate seed dormancy (76,

97). Furthermore, it supported earlier observations (30, 80) that the

growth of the subsequent seedlings was not abnormal if the proper envi-

ronmental condition were maintained during germination.














SUMMARY AND CONCLUSIONS


Investigations were initiated to determine the relation of certain

phenolic compounds to peach seed germination. The phenolic compounds

of primary interest were those which are degradation products of the

glucoside, amygdalin, namely, mandelonitrile and benzaldehyde, and their

immediate by-products. The following conclusions were made based on the

research conducted:

1. Mandelonitrile and benzaldehyde at 1.4 to 11.0 mg/g of perlite

inhibited germination of excised embryos, but did not stimulate dormant

seeds to germinate. Quantitative determinations of these 2 compounds

from peach seeds by gas chromatography indicated that the majority of the

mandelonitrile and benzaldehyde was released between 72 and 96 hours

after the start of imbibition and thereafter only trace amounts could be

observed. Only at this time was the tissue-level high enough to be con-

sidered inhibitory to germination, yet it showed no correlation with

germination. Furthermore, determinations made at weekly intervals during

chilling indicated that only trace amounts were present at any of the

sampling times during chilling. Therefore, it was concluded that mandelo-

nitrile and benzaldehyde have no direct, inhibitory or promotive influ-

ence on the germination of peach seeds.

Cyanide had little effect on reducing the per cent germination at

concentrations less than 1.0 M. From observations on the increased size

of seedlings in several cyanide treatments, it was postulated that the

tissues were incorporating cyanide, however no measurements of the in-

crease in glucoside content was made.

73







2. The fact that phenolic acids could be produced in dormant peach

seeds as a result of the metabolism of mandelonitrile and benzaldehyde,

led to an investigation of phenolic acids in dormant peach soods, and

several phenolic acids, including the hydroxy and methoxy derivatives of

benzoic acid, were found. Of prime interest was the finding that p-hydrox-

ybenzoic acid at concentrations of 3 x 10-2 to 10-1 M would slightly stim-

ulate the germination of dormant peach seeds.. However, quantitative de-

terminations showed that approximately 0.12 ug of p-hydroxybenzoic acid

was present per 1.0 g of tissue on a wet weight basis which was below

that found to be necessary in the external media for germination. Deter-

minations of the tissue-levels of p-hydroxybenzoic acid in germinated

seed was not conducted. L-mandelic acid, a product of mandelonitrile

hydrolysis, was also shown to be present in amounts in the range of 0.005

to 0.05 ug/g of fresh tissue. The influence of L-mandelic acid on peach

seed germination was not studied.

3. The inhibitor-complex level of peach seeds which appeared on

paper chromatograms at an Rf of 0.6 0.8 was found to be essentially the

same after chilling as prior to chilling. Thus this complex does not

appear to be involved in the maintenance of dormancy of peach seeds.

The inhibitory-complex had similar characteristics to the beta-inhibitor

complex reported to be found in other plant tissues.

4. Experiments with thiourea supported previous research and showed

that the time of embryo removal from the seed coat and associative tissue

after seed imbibition had little influence on the amount of abnormal

seedling production.






































APPENDIX: GAS CHROMATOGRAMS OF STANDARDS








































O 4 8


O 4


Time



Fig. 13. Gas chromatograms of the propyl esters of benzoic
acid(a) and mandelic acid(b). Retention time in
minutes. (See Table 6 for parameters of gas
chromatograph.)


















































i
i
"1

~i~~

"; rri
"r ~ z


0l 8


aCi.r nd I; nnr ;21; yd x 1 0 .
ranues (SLCo 000 r arutr..0amood


























O

CL













O 4 8 12 16





Fig. 15. Gas chromatograms of the propyl ester of
2, 6-dihydroxybenzoic acid. Retention time
in minutes. (See Table 6 for parameters
of gas chromaltograph.)
























C)
O














O 4 8 12 16
T imre



Fig. 16. Gas chromatogramns of the propyl ester of
2,4-dimethoxybenzoic acid. Retention time
in minutes. (See Table 6 for parameters of
gas chromatograph.)























v3

O
CL













O 4 8 12

Time



Fig. 17. Gas chromatograms of the propyl ester of
o-hydroxycinnam~ic acid. Retention timo
in minutes. (See Table 6 for parameters
of gas chromatograph.)
















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89



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BIOGRAPHICAL SKETCH


The author, James Bruce Aitken, was born August 1, 1938, in Orlando,

Florida. He received his secondary education at the Lakeview High School

in Winter Garden, Florida between the years of 1953 and 1956. He at-

tended Clemson University in Clemson, South Carolina and was granted the

degree of Bachelor of Science with major in Agriculture in January, 1962

and was also granted the degree of Master of Science with major in

Horticulture from Clemson University in January, 1964.

In 1964, he was granted an assistantship from the Department of

Fruit Crops, University of Florida to study toward the degree of Doctor

of Philosophy. He entered the University of Florida in April, 1964, and

completed his work towards the degree of Doctor of Philosophy in August

1967.

He is a member of Alpha Zeta, Phi Sigma and Gamma Sigma Delta hon-

orary fraternities. He is also a member of the American Association for

the Advancement of Science, and The American Society for Horticultural

Science.

He is married to the former Patricia Ann Dillard and they have one

daughter, Amy.







This dissertation was prepared under the direction of the chairman

of the candidate's supervisory committee and has been approved by all

members of that commiittee. It was submitted to the Dean of the College

of Agriculture and to the Graduate Council, and was approved as partial

fulfillment of th~e requirements for the degree of Doctor of Philosophy.






August, 1967










Dean, College of Agriculture









Dean, Graduate School




Supervisory Committee:




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