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Full text of "Relation of phenolic compounds to germination of peach seeds"

RELATION OF PHENOLIC COMPOUNDS 
TO GERMINATION OF PEACH SEEDS 



By 

JAMES BRUCE AITKEN 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 

THE UNIVERSITY OF FLORIDA 
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 

August, 1967 



AGRI. 

CULTURAL 
LI9RARY 



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V 



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^iiiliii 



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. 0. 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. 



11 



TABLE OF CONTENTS 

Page 

ACKNOWLEDGEMENT 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 

MATERIALS AND METHODS 21 

EXPERIMENTAL RESULTS .^ 31 

DISCUSSION 67 

SUMMARY AND CONCLUSIONS 73 

APPENDIX: GAS CHROMATOGRAMS OF STANDARDS 75 

LITERATURE CITED 81 

BIOGRAPHICAL SKETCH 90 



iii 



LIST OF TABLES 
Table Page 

1. Influence of thiourea concentrations on germination and 

per cent of production of abnormal seedlings from 

'Okinawa' peach seeds 32 

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 33 

3. Per cent germination of 'Okinawa' peach seeds 7 and 20 

days after start of imbibition as influenced by cyanide ... 34 

4. Per cent germination of 'Okinawa' peach seeds 7 and 12 

days after start of imbibition as influenced by 

mandelonitrile 36 

5. Per cent germination of 'Okinawa' peach seeds 7 and 12 

days after start of imbibition as influenced by 

benzaldehyde 37 

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 40 

/ 

7. Paper chromatographic separation of the inhibitory 

complex from dormant peach seeds 42 

8. Influence of acids, bases and heat on the inhibitory 

complex from peach seeds after paper chromatography 43 

9. Solubility of the inhibitor-complex in various organic 

solvents as determined by the alfalfa bioassay 44 

10. Mean per cent germination of dormant 'Okinawa' peach 

seeds 30 days after start of imbibition as influenced 

by benzaldehyde and mandelonitrile concentrations 57 

11. Mean per cent germination of dormant 'Okinawa' peach 

seeds 30 days after start of imbibition as influenced 

by benzoic and p-hydroxybenzoic acid concentrations 58 



IV 



12. Comparison of retention times of p-hydroxybenzoic acid 

and L-mandelic acid as influenced by various acetylation 
procedures ^^ 

13. Comparison of peak areas of p-hydroxybenzoic acid and 

L-mandelic acid as influenced by various acetylation 

procedures ^^ 



LIST OF FIGURES 
Figure Page 

1. Influence of benzaldehyde and mandelonitrile on peach 

seed germination 38 

2. Gas chromatogram of the propyl esters of the acidic 

fraction from an ethanol extract of peach seeds. 

Time is in minutes 39 

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 47 

4. Changes in benzaldehyde-mandelonitrile content of 

peach seeds as measured at various intervals after 

start of imbibition under the designated treatments 48 

5. Change in the content of mandelonitrile in peach 

seeds at various intervals after start of imbibition 

under the designated treatments 49 

6. Change in the content of benzaldehyde in peach seeds 

at various intervals after start of imbibition under 

the designated treatments 50 

7. Germination of peach seed as influenced by embryo 

excision, and thiourea treatments as determined 

periodically after the start of imbibition 52 

8. Relative inhibitory activity, as measured by the 

alfalfa bioassay, of the inhibitory complex in an 
ethanolic extract of peach seeds chromatographing 
between R^'s 0.6 to 0,8 54 

9. Germination of peach seeds as influenced by 5° C 

of varying durations 55 

10. Gas chromatogram of diazomethane-solvent control. 

Retention time is in minutes 60 

11. Gas chromatograms of L-mandelic acid(a) and 

p-hydroxybenzoic acid(b) treated for 30 minutes 

with diazomethane. Retention time is in minutes 61 



vx 



12. Gas chromatograra 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- 

benzoic acid(a) and p-hydroxybenzoic acid(b) . Reten- 
tion time in minutes 77 

15. Gas chromatograms of the propyl ester of 2, 6-dihydroxy- 

benzoic acid. Retention time in minutes 78 

16. Gas chromatograms of the propyl 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 



VI 1 



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 dormancy, 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 temi "dormancy" 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 18-30° 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 



environmental components precondition tho socvis, so th;\l gormination 
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 gem;inate readily without preconditioning. However, it 
is interesting to note that the latter will also lose their readi.ioss 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 COg 
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 tliey 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. 
Most 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 pr 
coumaric acids (53, 64, 92). 



Another possible function of the phenolic acids in seed genr.:: na- 
tion may be their role in the synthesis and degradation of indoleacetic 
acid (lAA) (74, S3). Pilet (78, 79) reported that the mono-hydroxyben- 
zoic acids increased the iii vitro destruction of lAA. 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 lAA. By growth experiments with IAA-l--'-'^C 
and determination of the -^^CO^ evolved, it was shown that monophenols 
stimulate the decarboxylation of lAA under conditions where growth was 
suppressed (95). When 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 form lAA . 

Probably the most active and most widely used gemiination 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 an 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 aj^'plication 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 'dormin' and 'abscisin II' are the same (15, 71). 
Eagles and Wareing showed that an inhibitor v dormin') concentrated from 
an extract 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 inV.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 Kef ford (8) first described a complex o^ inhibitory 
substances that appeared on paper chromatograms of plant extracts running 
ahead of lAA when developed in a solvent of i sopropanol/ammoni a/water . 
This inhibitory area, possessing R^ 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-in'iibitor complex. Lipe (57) 
found that the inhibitors in 'Lovell' peach seeds are similar to the 



beta-inhibitor complex. Elution and rechroraatography 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). ?or 
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 
the other factor's controlling dormancy. Black and Warding (10) reported 
that the removal of the embryo from intacx 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. Wareing and Foda (109, 110) found that leaching the embryo 
of Xanthium seed removed the inhibitor and that maintaining the seed in 
a pure oxygen atmosphere cause.' a reduction in the inhibitor within 30 
hours. Elliott and Leopold (23) showed that the inhibitors from Avena 
seeds inhibited alpha-amylase activity. 



11 

Villiers and Wareing (106, 107, lOS) reported that chilling Fraxinus 
excelsior seeds had no effect on the activity of the inhibitor but that 
dormancy was overcome during chilling by production of a growth stirau- 
lator in the embryo tissues. Flemion and De Silva (31) also demo. rated 
with peach seeds that with the bioassay they were using they coul^ I'ind 
little correlation between growth inhibitors and the termination of 
dormancy. 

The promotive effects of oxygen on germination of seeds and the 
parallel effects of light led Paech (73) to suggest ^r.a'c dorr.iancy 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 photooxidation 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 donriancy was broken (35) . Murakami 
(66) has shown gibberellin to be present in a wide diversity of seeds. 
As seeds of Avena f atua emerged from dormancy a growth-promoting sub- 
stance suggestive of gibberellin was formed (67). These seeds were also 
brought out of dormancy if soaked in gibberellin solutic.-s. Kahn (47) 
reported gibberellin overcame dormancy 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 has been reported that gibberellic acid stimulated alpha-araylase 
production in the aleurone layer of _ coat of cereals which in turn 
increased the rate of starch hydrolysis. Th ' stimulation of alpha- 



12 

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). 
EXTERNAL 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). 

Water ; 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). 



13 

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 
Pi sum 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). 



14 

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 A° has inhibited 
germination of all seeds tested (33). Between 2900 A° and 4000 A° the 
germination of some seeds is inhibited (33). In the visible range, 
4000 AO - 7000 A°, it was shown that light in the range of 5600 A° - 
7000 A 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 A ), 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- 



15 

tions present durin^ 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 af xer-ripened at 5° C from those 
that are kept in the greenhouse at 21° - 30° C. The latter can be con- 
si-dered 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 Poljakof f-Mayber (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 .;ey compounds including phospholipids 
which function in controlling surface p:.-operties 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). 

Phosphoi-us pi'imar^ly appears in seeds as organic phosphorus, v.'ith 
very little being present as inorganic orthophosphate. Phytin is fre- 
quently present and may constitute up to S0% of the total phosphorus 
content of ..e seed (64). Since most of the phosphate :.3 present in the 
bovind foi^, o.'jaophosphate may be the limiting factor in certain of the 



16 

reactions of the germination process. With this in mind the large amount 
of phytin present may be considered as a reserve of inorganic phosphate 
which can be liberated t. germinatio. proceeds by phosphatase activity 
or more specii' cally phytase activity. Phytir, 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 pr^^sents a possible limiting facxor for 
the race of germination and subsequent seedling development. 

Recently, reports of myo -inositol acting as a growth factor in 
plant tissue have .^een made (3). This is of particular interest in re- 
gard to phytin since it is the salt of phytic acid or inositol .exaphos- 
phate. The "neutral fraction" 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 grov/th-stimulation was 
concerned. Myo- inositol may stiir.ulate the growth of seedlings and the 
germination of certain seeds. In addition, myo -i..ositol has stimulated 
growth of callus in cultures of elm ( Ulmus campestris ), Norway spi'uce 
( Picea abies ) , tobacco ( Xicotiana tabacium ) , Vinca rosea , 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 (54). The content of nicotinamide adenine dinucleotide (NAD) 
and nicotinamide auonine dinucleotide phosphate (NADP) in seeds and 
sec "lings rises in all cases during germination. During the early 
stages of germination there was a net increase in the RNA of peanut 
cotyledons (102). Some of this RNA synthesis was thought to be associ- 



17 

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 RNase 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 
(SS) . 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 will induce germination of dormant seed. These factors and 
others are discussed below. 



18 

ENVIRONMENTAL FACTORS: 

The optimum temperature of 5° C with a range of 5-10 C for 60-90 
days has been found best suited for the termination of dormancy of peach 
seeds (12, 16, 19, 29). 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) . 
CHEIIICAL 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 



19 

induction of germination of dormant peach seeds has resulted in the 
formation of abnormal seedlings when induction was by means of thiourea 
or seed coat excision (2S, 36, 37, 97). However, it has been recently 
sho\vn that the temperature during germina"c;.on plays a major role in the 
development of abnormalities 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 dorraant 
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 Olney (72, 81) have studied extensively -.le 
i-est 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 donaancy, 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 call seemed to remain ' '' v 
constant. The rate .^f 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 'curgid seeds 



20 



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 with a block in the phosphate 
metabolism of the cells. This hr -• not "aen substantiated at th : present 
time. 

Pollock (SO), using 'Elberta' peach seed, suggests that the causal 
agent of the dwarfing effect in seedlings is independent of the growth 
inhibitor content of the seed. The physiological and anatomical a._ .octs 
of dwarfing suggested a control by a self-duplicating system loci:.lized 
in a limited region of the apical meristem and transmitted only by cell 
division. This system was temperature sensitive during the time between 
the first visible root growth 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 arisen from the hydrolysis of amygdalin to mandelonitrile and 
glucose by prunsin. The mande_onitrile was further hydrolyzed to 
cyanide and benzaldehyde (104), presumably by emulsin. 

The presence of benzaldehyde during imbibition, as a result of the 
degradation of ainygdalin, suggested that possibly .^enzoic acid and some 
cf its derivatives m.\y be formed (25, 104). An alternative to this 
pathway is that in which man^lelic acid, formed from r..- ndelonitrile, 
undergoes enzymatic conversion to benzoic acids (40, 41, S9). 

This study will be concerned with the changes in phenolic compounds 
during the breaking of iorraancy and subsequent germination of the seed. 



MATERIALS AND METIiODS 



All seeds were obtained from the I960 and 1966 crops of Prunuf 



persica cv, 'Okinawa'. This m:.'^orial was chosen for several reasons. 
Principally, the seeds are relatively homozygous in respect to the chill- 
ing requirement to temiinate seed dormancy (9), the seeds require a rel- 
atively shoi-t period of low temperature stratification to overcome the 
dormant state (9), and when the embryos are excised they germinate readi- 
ly without any apparent abnormalities if the temperature range during 
germination is 18-25° C (76). 

The seeds were removed from the endocarp just prior to each experi- 
ment and allowed to imbibe water from moistened vermiculixe. Depending 
upon the nature of the expei'iment, the time in moistened vermiculite 
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 of thiourea and seed coat excision on germi- 
nation : In order to determine the most effective thiourea concentration 
to promote the greatest amount of gerrainatioii with the least amount of 
anomolous growth, a range of concentrations was tested. This was as 
follows: 0.0, 1.0, 3 x lO"-*-, 10" and 10""^ M thiourea. The seeds were 
kept in a moist medium for 42 hours and then followed by 6 hours' soaking 
in the respective thiourea concentrations. Before imbibition, the seeds 
were surface sterilized for 3 minutes with a 1,000 ppm raerthiolate in 



21 



22 

25% ethanol: water solution. After the soaking period, the seeds were 
blotted and planted in flats and kept in the dark at 20° C for 16 days 
before being 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" , 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 20° 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, 

-1 -9 -2 

10 , 3 X 10 , and 10 M made with potassium cyanide. Seeds were 

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



23 



in an aqueous solution of the chemical for 6 hours. They were then 
planted in seed flats and placed in a growth charaber with a controlled 
temperature of 20° 1 2° C and a 12-hour day of approximately 900 ft-C 
lig;ht intensity. 

Benzoic acid and p-hydrox; ;;enzoic acid were tested at concentrations 
of 0.0, 10"^, 3 X 10~2, 10"^, and 10"^ M. To test the respective concen- 
trations of each compound, approximately 5 g- of dry perlite were placed 
in 100 ml beakers and the perlite saturated with the solution of che.uical 
to be tested. After equilibration of the mixture, fully turgid seeds, 
attaining this condition in moist vermiculite in 48 hours at 20° C, were 
placed in the perlite plus chemical media, and maintained under aerobic 
conditions. After 5 days in the media, the seeds were transferred to 
flats i:untaining a 2:1 mixture of perlite: vermiculite. All ;.. .?es of 
the experiments were conducted in growth chambers at 20° ^ 2° 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 loga- 
rithmic range of quantities of the material per unit of perlite was used. 
A measured amount of the chemical was absorbed 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 ti'eatment and care was taken to maititain 
aerobic conditions. For preparation of the seed before treatment, they 
were allowed to imbibe for 4S hours, embryos excised and placed in the 
perlite-chemical ...rixture. The embryos were left in the media for 5 
days at 20 C. Thei. they were removed fi'om the chemical environments, 
planted in flats and placed in a greenhouse for the remainder of the 
observational period. 



24 

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 20 C. The seeds 
(10 g) were ground in a Servall Omni-mixer at 15,000 rpm for 3 minutes 
in 30 ml of 80% ethanol . The homogenate was filtered, the subsequent 
filtrate dried under vacuum and the residue dissolved in 0.1 M tartaric 
acid. This aqueous solution was partitioned against ethyl ether and the 
ether phase separated and partitioned against an aqueous solution of 
0.1 AI 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 Wilcox (112). Briefly, N-propyl-N-nitrosourea ( obtained from Dr. 
Merrill 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 esterif ication 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, 



25 

X-butyl-N-nitrosourea (obtained from Dr. Merrill Wilcox, Agronomy 
Department, University of Florida) for the N-propyl-N-nitrosourea. With 
both diazomethane and diazobutane, the initial esterif ication period, 
30 minutes, was the same as with diazopropane. 

In tests where esterif ication was slow for the carboxyl group or 
where acetylation of hydroxyl groups on the ring was slow or non-existent, 
0,7% 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 M gas 
chromatograph equipped with a f lame-ionization detector. The column 
consisted of 1/4 inch stainless steel tubing 6 feet long packed with 8% 
S.E. 30 on 60-80 mesh Chromosorb W. Helium was used as a carrier gas with 
flow rate of 70 ml per minute. Temperatures for the system were as 
follows: oven, 180° C; injection port, 260 C; and detector, 250 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 unknowns 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 bioassay. 



26 

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 dishete were placed in the dark at 20° t 2° 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 TOiatman 3 MM chromatography 
paper. Chromatograms were divided into sections of 10 R^ units and 
assayed, using the alfalfa seed bioassay (18) . 

Extracts of peach seeds were also subjected to acid hydrolysis 
(pH 2) with acetic acid, alkaline hydrolysis (pH 10) with ammonium 
hydroxide, dialysis against distilled water for 24 hours; and heating 
for 10 minutes at 50, 75, and 100*-" C. Changes in inhibitory activity 
were moiiitored, using the alfalfa bioassay. 

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- 
taining a Wliatman No, 4 filter paper disk and the residue deposited on 
the paper by evaporation. Distilled water (1.5 ml) was added to the 



27 

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 . 

M easurement of benzaldehyde and mandclonitrile : The quantity of 
benzaldehyde and raandelonitrile 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 20° C prior to treatment. Treat- 
ment 1 was seeds steeped in 3 x 10 " 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 20° 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 -70° C and then placed in a freezer at -30° C 
until ground, approximately 8 hours. The frozen seeds were ground in a 
Wiley 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 
-70 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 -5° C before they were placed in a -30° C environment 



28 

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% sodium bisulfite, Thirdly, quantitative analysis 
was again done on the ether phase after 407o 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. 



29 

The conversion of benzaldehyde to a sulfite derivative soluble in 
water and then conversion to mandelonitrile is a well-knov/n reaction 
(26). The sodium bisulfite reacts with the carbonyl group of benz- Ide- 
hyde to forni 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 : Detemiinations 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 20° C, were placed at 4° C for 0, 16S, 336, 504, and 672 hours. At 
the time of sampling, one sample was removed and extracted immediately 
and another sample was placed for an additional 40 hours at 20° C. A 
control lot of seeds was maintained at 20 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 iiihibitors, presumably the beta-inhibitory complex (8), 
was assayed as follows. A sample of treated seeds was subjected to 
extraction with 807o ethanol after grinding, as previously noted. The 
solution was taken to near dryness by vacuum distillation, keeping the 
distilling chambei' at less than 5 C. The residue was redissolved in 
807o ethanol, applied to chromatographic paper (Whatman 3 MM) 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, were 
sectioned from the chroraatograms, and solutes eluted from the 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 mg. The solutions were placed in small petri dishes on 
Wliatman No. 4 filter paper disk, frozen and water removed by sublimation 
under vacuum. After again moistening the filter pads with 1.5 ml of 
H„0, 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 thiourea on 
gei'mination 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 germination, 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 M. 

In determining the possible interaction between thiourea and seed 
coat on germination and subsequent seedling growth, the most striking 
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 mandelonitrile on seed 
germination ; The data in Table 3 indicates that cyanide does not dras- 
tically reduce germination, except in very high concentrations (1.0 M) . 
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 from any of 
the tx-eatments, and, interestingly, the 1.0 M cyanide did not kill the 
seeds. 



31 



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



32 



Thiourea 
concentration, M' 



X 



Ale an 
% Kemiination^ 



Mean 

V 2 

% abnormal'' ' 



(Control) 
3 



10 



10-2 



10 

3 X 10 

1.0 



-1 



5.8 a 

5.8 a 
26.8 ab 
45.8 be 
62.5 c 
58.3 c 



0.0 a 

0.0 a 
21.9 b 
40.1 b 
61.4 c 
79.0 c 



^Each treatment was replicated 3 times with 40 seed per replication. 

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



"Percentage based on the total number of seed germinated. 



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. 



Treatments^ Mean 

Chemical Seed coat % germination^ % atypical seedling 

Control Intact 

Excised; 42 hrs 

72 hrs 

96 hrs 

120 hrs 

10"% 

Thiourea Intact 

Excised; 42 hrs 

72 hrs 

96 hrs 

120 hrs 100.0 



0.0 


a 


100.0 


e 


92.6 


cd 


100.0 


e 


100.0 


e 


81.5 


b 


96.3 


de 


92.6 


cde 


100.0 


e 



3 X 10"% 

Thiourea Intact 

Excised; 42 hrs 

72 hrs 

96 hrs 

120 hrs 



88.9 


c 


96.3 


de 


100.0 


e 


100.0 


e 


100.0 


e 



0. 





0. 





0. 





0. 





3. 


,7 


7. 


,4 


0, 


.0 


0, 


.0 


0, 


.0 


3, 


.7 


3 


.7 





.0 





.0 





.0 


3 


.7 



^Seeds were imbibed 42 hours, then treated with chemicals for 6 hours 
before planting. 

^Each treatment consisted of 3 replications of 9 seed each. 

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

^Hours after start of imbibition. 



34 



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



Cyanide 


M^ 




Mean " 


% germination^ 


concentration, 


7 davs 




20 davs 



1.0 

10-1 

3 X 10 



-2 



-2 



10 



Control 



0.0 a 

88. 9 b 

100.0 c 

100.0 c 

100.0 c 



0.0 a 
94.4 b 

100.0 b 
94.4 b 

100.0 b 



^Seeds 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 57o level. 



u^ 



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 shov;ed 
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 16.77o 
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 inhibitory. 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-hydroxycinnamic, 2, 6-dihydroxybenzoic, o-hydroxybenzoic, 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. 



36 



Table 4. — Per ceiit germination of 'Okinawa' peach seeds 7 and 12 days 

after start of imbibition as influenced by mandelonitrile^'^. 



Mandelonitrile 




Mean % germination 


mg/g of perlite 


7 days 


i:.'. days 



140.0 0.0 a 0.0 a 

14.0 0.0a 0.0a 

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 be 100.0 c 

0.042 ■' 94.4 be 100.0 c 

0.014 94.4 be 100.0 c 

Control 88.9 b 88.9 be 



X 



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

^Each treatment was replicated 3 times with 6 seed per replication. 

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



37 



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



Benzaldehyde Mean % 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 

Seeds 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. 

^Each treatment was replicated 3 times with 6 seed per replication, 

z 

Means not having a following letter in common are significantly 

different at the 1% level. 



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Gas chroraatogram 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.) 



40 



Table 6. — Relative retention time and possible identity of components 
separated by gas chromatography of the propyl esters of the 
acidic fraction from an ethanolic extract of peach seeds^. 



Peak No. 



Relative retention timc'^ 



Possible identity of acids'' 



1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 



1.00 
1.37 



1. 
2, 
2, 
2, 
3, 
4, 
5, 
7, 



91 
13 
50 
89 
59 
99 
89 
11 



10.21 
19.29 



Benzoic 
Succinic 

Malic or mandelic? 

o-hydroxycinnamic 

Fumaric ? 

2, 6-dihydroxybenzoic 

o-hydroxy benzoic 

p-hydroxybenzoic 

2, 4-diraethoxybenzoic 

? 
Citric 

? 



- Instrument ; F &. M model 400, flame detector. 

Column ; 87o S.E. 30 on 60-80 mesh chromasorb W, acid washed, 

silane treated; 1/4" 0,D. stainless steel 6' in length. 

Carrier gas ; He. Outlet flow rate ; 70 ml/min. 

Oven temp; 180° C. Injection port temp; 260° C. 
Detector temp ; 250° C. 

Range and attenuation ; 10 x 8. Chart speed ; 1/4" per rain. 

- Relative retention time is based on benzoic acid. 



Possible identity based on matched retention times, 



41 

Inhibitor chai-acterization ; Using the Peruvian alfalfa bioassay, 
it was found that extraction of 8 g wet weight of dormant peach seeds 
with 80% 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 R^ ' s 0.6 and 
0.8 when bioassayed 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 A° and 2537 A°) of benz- 
aldehyde and a fraction from an ethanol extract from peach seeds on 
paper chromatograms were identical. Also, benzaldehyde and the extracted 



42 



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



Rf Value-^ % Germination^ 



0,0 - 0,1 92,5 

0,1-0,2 85,0 

0,2-0.3 87,5 

0,3-0,4 85.0 

0.4 - 0.5 55,0 

0,5 - 0,6 0,0 

0.6 - 0,7 0,0 

0.7 - 0,8 0.0 

0.8-0.9 85.0 

0,9 - 1.0 80.0 



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

^Bioassayed with the alfalfa seed test. 
Control = 90%. 



43 



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



Test % Germination^ 

Acid hydrolysis (pH 2) 0.0 

Alkaline hydrolysis (pH 10) 0.0 

y 

Dialysis, inside tubing 0.0 

outside tubing 0.0 

Heating for 10 minutes: 

50° C 0.0 

75° C 0.0 

100° C 0.0 

Extract control 0,0 

Water control 90.0 



Bioassayed with the alfalfa seed test. Seed 



equivalent of the extract was 0.5 g dry 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. 



44 



% Germination 



Solvent 



Eluate^ 


Chromatogram 

Section^ 
(Rj 0.6-0.8) 


0.0 


80.0 


92.5 


0.0 


30,0 


0.0 


85.0 


0.0 


90.0 


0.0 


0.0 


82.5 


87.5 


0.0 


92.5 


0.0 



Water 

Hexane 

Acetonitrile 

Ethyl ether 

Chloroform 

Methanol 

Ethyl acetate 

Carbon disulfide 



^Elution fraction from chromatogram section of R^ 0.6- 
0.8. 

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



45 



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 A° and 2537 A°) 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 (200-250° C) , 
Mandelonitrile and components of the extract reacted alike when tested 
with hydrocyanin indicators. 

Quantitative detemiinations 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 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. 

Gas chromatograph parameters. 



Instrument : 
Column: 



Carrier gas : 
Temperatures : 

Range and 
Attenuation : 
Chart Speed: 



F &, M model 400, flame detector. 
8% S.E. 30 on 60-80 mesh Chromosorb W, acid- 
washed, silane treated; 1/4" 0,D. stainless 
steel 6' in length. 

Helium. Outlet flow rate : 70 ml/min. 
Oven, 100° C; Injection port, 150° C; Detector, 
160° C. 

10 X 8 

1/4" per minute. 



47 




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51 

120 hours after the start of imbibition. Relatively larger amounts of 
mandelonitrile 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 1007o 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 ,was below 1.0 ug/gra dry weight 
of tissue. Only trace amounts of benzaldehyde and mandelonitrile were 
detected by gas chromatography on seeds placed at 20° 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 bioassay, 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 20° C indicated that 504 hours of 
chilling was sufficient to terminate dormancy in over 807o (Figure 9) of 
the population of the seeds. • 'i 



52 



loo- 



se - 



60 ;r 



c 
q 

03 



C7) 40- 



20 



EXCISED 

o o — o 



Oir 




THIOUREA 



INTACT(CONTROL) 

o — o o 



^ 






5 



-I , ^- 






100 



160 



220 






280 



Time, hours 



Fig, 7, Germination of peach seed as influenced by embryo excision, 
and thiourea treatments as detennined periodically after the 
start of imbibition, (Growth of excised embryos was taken 
to be equivalent to germination when the radicle had elongated 
to 2 mm. ) 



Fig. 8. Relative inhibitory activity, as measured by the alfalfa 

bioassay, of the inhibitory complex in an ethanolic extract 
of peach seeds chromatographing between R^ ' 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 . ) 



54 



























CM 

o 

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40- 


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y 




Hours of chilling 



Fig. 9. Germination of peach seeds as influenced by 5 C of varying 
durations. 



56 

Influence of chemicals on possible stimulation of peach seed germi - 
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 

-T -1 
concentrations ranging from 10 to 10 M. The data in Table 11 in- 

-1 -2 

dicates that p-hydroxybenzoic acid at 10 and 3 x 10 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 esiors 
of the aromatic acids. These were separated on the gas chromatograph 
at an oven temperature of 180° C. The methoxy and butoxy esters were 
separated by gas chromatography after acetylation by diazomethane and 
diazobutane, respectively. Since those derivatives were more volatile, 
an oven temperature of 150° 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 



57 



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



% Germination 



Concentration, M^ Benzaldehyde^ Mandelonitrile^ 



1.0 0,0 a 0.0 a 

10 0.0 a 0.0 a 

3 X 10~ 16.7 b 0.0 a 

-2 

10 22.2 b 38.9 b 

33.4 b 33.4 b 



X 



Seeds were imbibed for 2 days, then exposed to chemical 
concentrations for 5 days. 

^Each treatment was replicated 3 times with 6 seed per 
replication. 

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



58 



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



acid concentrations^, 



% Germination 



2 

Concentration, My Benzoic^ p-Hydroxybenzoic 

lO"-"- 5,5 a 38.9 b 

3 X 10"^ 0.0 a 38.9 b 

lO"^ 16.7 a 11.2 a 

10""^ 16.7 a 11.2 a 

16.7 a 16.7 a 



'^Seeds were in moist medium for 2 days, then exposed to 
chemical concentrations for 5 days. 

^Each treatment was replicated 3 times with 6 seed per 
replication. 

^Means 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 , ) 



60 



9SUOClSG^ 



00 



O 



00 



C\J 







CD 



00 



O 



61 



If) 
c 
O 
Q. 
t/) 
<D 

q: 




_M 



J 




4 



8 



Time 



4 8 12 



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. ) (M= 
L-mandelic acid and pHBA= p-hydroxybenzoic acid.) 



63 







^i-v.-Z^ 



o 



CM 

en 



c\j 



CD 



CD 



00 



^ 



O 



GSUOdSG^ 



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



64 



Acetylation procedure 



Retention times (minutes) 



Extract 



Standard 



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

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



4.10 


4.10 


2.05 


2.05 


11.80 


11.80 


4.57 


4.57 



3-diazobutane, 3 hours: 

p-Hydroxybenzoic acid 
L-Mandelic acid 

Methyl derivative 
Butyl derivative 



12.60 

4,48 
6.70 



12.90 

4.48 
6.35 



^Refer 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. 



66 



Acetylation procedure'^ 



9 

Peak area, mm 



Extract Standard^ 



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

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



45.0 


540.0 


Approx . 5.0 


597,0 


49.0 


342.0 


Approx. 5.0 


675.0 



3-diazobutane, 3 hours: 
p-Hydroxybenzoic acid 
L-Mandelic acid 

Methyl derivative 
Butyl derivative 



30.0 

Approx. 5.0 
Approx. 5.0 



545.0 

996.0 
812.0 



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

^Each standard represents 2 ug of the respective compound. 



DISCUSSION 

A quantitative method was devised to determine the amount of benz- 
aldehyde and raandelonitrile 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-sulf ite 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 gemiination 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 

67 



68 

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 acids in 
peach seeds. Using gas chromatographic techniques, benzoic, o-hydroxy- 
cinnamic, 2, 6-dihydroxybenzoic, o-hydroxybenzoic, p-hydroxybcnzoic, 
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 



69 

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 seeds at 10~° M concentrations. 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 (■'■'^C) 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. 



70 

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"^ and 10~ 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 lAA-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 



71 

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~ 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 

-2 -1 
acid would stimulate peach seed germination at 3 x 10 to 10 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 R^ 
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. 



72 

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 promotors 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 



74 

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 seeds, 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"^ to lO"-"" 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 R^ 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 



/ 



76 



0) 

en 

c 

O 
Q. 

to 

q: 




4 



4 8 



Time 



Fig. 13. Gas chromatograms of the propyl esters of benzoic 
acicl(a) and mandelic acid(b). Retention time in 
minutes. (See Table 6 for parameters of gas 
chromatograph . ) 



77 







u 



4 






!! 

i ! 



I « 



iFii 



i-i'l 






I 



--J \j 



U 



%^ 



10 

I &9 



14. 



Gas Ghroinatogi*ai.is ei the propyl ggtoi'g of o-liydroxybenseie 
acid(a) and p-hydroxyboHKoio acid(b). Retention time in 
minutes. (See Table 6 for parameters of gats ehromatogi-aph. ) 



78 



(D 

c 

o 

CO 

q: 








4 8 

Time 



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 chromatograph . ) 



79 



0) 
(J) 

C 

o 

Q. 
if) 
(D 

Q:: 




4 8 12 16 

Time 



Fig, 16. Gas chromatograms of the propyl ester of 

2,4-dimethoxybenzoic acid. Retention time 
in minutes. (See Table 6 for parameters of 
gas chromatograph, ) 



80 



0) 
fj) 

c 

o 

Q. 

CO 



q: 




4 8 12 

Time 



Fig. 17, Gas chromatograms of the propyl ester of 
o-hydroxycinnamic acid. Retention time 
in minutes. (See Table 6 for parameters 
of gas chromatograph. ) 



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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 Clerason, 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. 



90 



This dissertation was prepared under the direction of the chairman 
of the candidate's supervisory conunittee and has been approved by all 
niembers of that conuiiittee. It was submitted to the Dean of the College 
of Agriculture and to the Graduate Council, and was approved as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 



August, 1967 



Supervisory Committee; 



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Dean, College of Agriculture 



Dean. Graduate School 




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