Title: Studies of seed dormancy of Prunus persica
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Title: Studies of seed dormancy of Prunus persica
Physical Description: 103 leaves : ; 28 cm.
Language: English
Creator: Sauls, Julian Winnfield, 1943-
Copyright Date: 1972
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Subject: Peach   ( lcsh )
Prunus   ( lcsh )
Fruit Crops thesis Ph. D
Dissertations, Academic -- Fruit Crops -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Julian Winnfield Sauls.
Thesis: Thesis (Ph. D.)--University of Florida, 1972.
Bibliography: Includes bibliographical references (leaves 94-103).
General Note: Typescript.
General Note: Vita.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000415146
oclc - 37907948
notis - ACG2382

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STUDIES OF SEED DORMANCY OF PRUNUS PERSICA
















By

Julian Winnfield Sauls


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
1972


















ACKNOWLEDGMENTS


The author extends his sincerest appreciation to Dr. R. H. Biggs,

Professor, Department of Fruit Crops, who served as chairman of the

supervisory committee and suggested these studies and provided the

needed guidance and assistance for the completion of the research and

preparation of this manuscript.

For their advice, criticism, and assistance during the course of

graduate study and in the preparation of this manuscript, appreciation

is also extended to Dr. A. H. Krezdorn, Professor and Chairman of the

Department of Fruit Crops; to Dr. R. C. Smith, Associate Professor of

the Department of Botany; to Dr. W. J. Wiltbank, Assistant Professor,

Department of Fruit Crops; and to Dr. D. W. Buchanan, Assistant

Professor, Department of Fruit Crops.

Also, the author extends special thanks to his wife, Rana, for her

support, encouragement, and understanding during this period of graduate

study.

















TABLE OF CONTENTS


Page
ACKNOWLEDGMENTS . . . . . . . . . . . . ii

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

LIST OF FIGURES . . . .. . . . . .. .. v

KEY TO SYMBOLS OF ABBREVIATIONS . ... . . .. . . . .viii

ABSTRACT .. .. . . .. . ............ .. . ix

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

REVIEW OF LITERATURE . . . .. . .. . . .. . .. 3
Seed Dormancy in Peaches .. . .. .. .. . . 3
Hormonal Concept of Dormancy Regulation . . . . ... 7
Nucleic Acid Metabolism and Dormancy . .. .. .. . 10

MATERIALS AND METHODS . . . ...... ..... .. .. 16
Plant Materials . . . . . . . . . .. . 16
Inhibitor Application to Developing Seeds . . .. ... .16
Embryo Culture During Seed Development . . ... .. 18
Studies of Seed Dormancy... . .. . . ..... 20
Seed irradiation .. .. . .. . . . . 20
Inhibitor application to stratifying seeds . . .21
Nucleic acid changes during stratification and
germination . . . . . . . . . 22
Extraction and Isolation of Nucleic Acids . . . . 23
Procedure for Gel Electrophoresis . . .. . . .. 23
Nucleic Acid Determination ... . . . . .. 25
Radioactivity Measurements .... . .. . . 25

EXPERIMENTAL RESULTS . . . . . . .. .. . 27
Experiments with Developing Seeds . . . . . . 27
Seed Irradiation Studies . . . . . . . . 45
Inhibitor Application to Stratifying Seeds . . .. . 59
RNA Changes During Stratification and Germination .. . . 70

DISCUSSION . . . . . . . . . . . ........... 80

SUMMARY AND CONCLUSIONS ...... .. . .. .. .. . 88

APPENEIX: BUD-BREAK STUDY... ...... . .... . . . 90

LITERATURE CITED . . . . . . . .. . . . . . 94

BIOGRAPHICAL SKETCH . . . . . . . . . . . 104


iii

















LIST OF TABLES


Page
1. Chemicals used to inhibit nucleic acid and
protein synthesis . . ... . . . . 17

2. Dates of injection, dates of sampling, and time
intervals between injection and sampling .. .. . 19

3. Evaluations of the growth of embryos from 'Early
Amber' peach seeds, cultured in vitro from
fruit injected on April 6, 1971 .. .. . .. 30

4. Average seed weights at each sampling interval during
the development of 'Early Amber' peaches .. . . 47

5. Germination of 'Okinawa' peach seeds exposed to 60Co
gamma-radiation . . . . ... .. . . 49

6. Germination and subsequent growth of 'Okinawa' peach
seeds exposed to 60Co gamma-radiation . . . .. 49

7. Comparisons of the growth responses of 2 separate
controls with those of the 0 kR treatment for
each time of irradiation . . . . . . . 60

8. Incorporation of radioactive 32PO4 into RNA of
'Okinawa' peach seeds during stratification and
germination . .. .. . . .. ... . . . 77


APPENDIX

9. Effects of several chemicals on bud dormancy of
'Okinawa' seedlings . . . .. .... .. . 91

















LIST OF FIGURES


Page
1. Procedure for nucleic acid extraction. ... . . . 24

2. Incorporation of 32PO4 into 'Early Amber' peach seeds
during development .. . .. . . . . . 28

3. Typical electrophoretogram of polymeric RNA fractions
of 'Early Amber' peach seeds . .. .. . ..... 31

4. Total RNA of 'Early Amber' peach seeds treated with
inhibitors during development . . .. .. .. . 33

5. Magnitude of change of total RNA of 'Early Amber' peach
seeds treated with inhibitors during development . . 34

6. Concentration of the 25S subunit of rRNA of 'Early
Amber' peach seeds treated with inhibitors during
development. .... .. .. ... .. .. .. 35

7. Concentration of the 18S subunit of rRNA of 'Early
Amber' peach seeds treated with inhibitors during
development . . . . .. . . . . 36

8. Total rRNA of 'Early Amber' peach seeds treated with
inhibitors during development . . .. . . 37

9. sRNA of 'Early Amber' peach seeds treated with
inhibitors during development . .. .. .. . 38

10. Ratios of rRNA/sRNA of 'Early Amber' peach seeds
treated with inhibitors during development . ... .39

11. Ratios of 25S/18S rRNA of 'Early Amber' peach seeds
treated with inhibitors during development . .. . 40

12. Average length of the 'Early Amber' peach embryo
during its development . .. .. .. .. .. . 46

13. Germination of 'Okinawa' peach seeds exposed to
gamma-radiation during stratification . . . . .51

14. Emergence of 'Okinawa' peach seedlings exposed to
gamma-radiation during stratification . . . .51

15. Rate of emergence of 'Okinawa' peach seedlings exposed
to gamma-radiation during stratification .. . . .52











16. Average weights of 'Okinawa' peach seedlings exposed
to gamma-radiation during stratification .. . .. . 53

17. Average shoot weights of 'Okinawa' peach seedlings
exposed to gamma-radiation during stratification .. . 54

18. Average root weights of 'Okinawa' peach seedlings
exposed to gamma-radiation during stratification . . 55

19. Average shoot lengths of 'Okinawa' peach seedlings
exposed to gamma-radiation during stratification . .56

20. Germination of 'Okinawa' peach seeds in response to
inhibitor application during stratification .... . .61

21. Emergence of 'Okinawa' peach seedlings in response to
inhibitor application during stratification . ... 61

22. Rate of emergence of 'Okinawa' peach seedlings in
response to inhibitor application during
stratification . . . . ... . . . . . 62

23. Average weights of 'Okinawa' peach seedlings in
response to inhibitor application during
stratification . . .... . . . . . . 63

24. Average shoot weights of 'Okinawa' peach seedlings
in response to inhibitor application during
stratification . . . . . . . . . . 64

25. Average root weights of 'Okinawa' peach seedlings
in response to inhibitor application during
stratification . . .. . . . . . . . 65

26. Average shoot lengths of 'Okinawa' peach seedlings
in response to inhibitor application during
stratification . . . . . .. .. . . . 66

27. Comparisons between a non-injected control and the
0-day and 20-day injected controls for each
growth parameter . . . . . . . . 67

28. Changes in total RNA during stratification and
germination of 'Okinawa' peach seeds . . . . . 71

29. Changes in rRNA and sRNA during stratification and
germination of 'Okinawa' peach seeds .... . ... .73

30. Changes in the 25S and 18S subunits of rRNA during
stratification and germination of 'Okinawa'
peach seeds . . . . . . . . . . 74











31. Ratios of 25S/18S rRNA and rRNA/sRNA during
stratification and germination of 'Okinawa'
peach seeds . . .... .. .. . . . 75

32. Typical electrophoretogram-histogram of polymeric
RNA fractions of 'Okinawa' peach seeds . . .. . 78

















KEY TO SYMBOLS OF ABBREVIATIONS


ABA Abscisic acid

ACTD Actinomycin D

CHI Cycloheximide

DNA Deoxyribonucleic acid

IE Electrophoresis buffer, 20%

GA Gibberellic acid

FUDR 5-Fluorodeoxyuridine

HC1 Hydrochloric acid

IAA Indole-3-acetic acid

RNA Ribonucleic acid

mRNA Messenger RNA

rRNA Ribosomal RNA

sRNA Soluble RNA

tRNA Transfer RNA









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

STUDIES OF SEED DORMANCY OF PRUNUS PERSICA

By

Julian Winnfield Sauls

June, 1972

Chairman: Dr. Robert H. Biggs
Major Department: Fruit Crops

Studies were conducted to explore the concept of nucleic acid

involvement in the mechanism of seed dormancy of peaches (Prunus persica

Batsch). Ribonucleic acid (RNA) levels in 'Early Amber' peach seeds

were assayed during the embryo development period from cytokinesis to

fruit maturity to determine when major changes in RNA levels occur.

Further, inhibitors of the nucleic acid-protein synthesis mechanism

were applied to ascertain the effects of such chemicals on the embryos

and to determine the effects on subsequent seed germination. RNA fluc-

tuations during seed development could not be related to changes in

embryo length or seed weight. Applications of 5-fluorodeoxyuridine,

actinomycin D, and cycloheximide early in the developmental period

generally caused decreases in RNA, but later applications had little

effect or. in several cases, resulted in substantial increases. The

inhibitors also reduced the growth of excised embryos in vitro.

Seed germination in response to cobalt-60 gamma-radiation was

studied. On the basis of growth responses, cell division and deoxy-

ribonucleic acid synthesis were assumed to be inhibited at the irradi-

ation dosages tested. Dosages up to 200 kR permitted 'Okinawa' seed

germination, but 50 kR or more prevented epicotyl growth and root pro-

liferation. Shoot growth occurred at dosages below 50 kR, but was a

ix










reflection of cell elongation. Proliferation of the root system

occurred at dosages below 50 kR, so cell division of the root system

was not inhibited.

Seeds were irradiated at various times during stratification to

ascertain whether a variable sensitivity existed as dormancy was termi-

nated. Dosages up to 20 kR did not inhibit seed germination, but germi-

nation decreased linearly from irradiation at the outset of stratifi-

cation to irradiation after stratification. Imbibed seeds were more

sensitive to gamma-radiation than dry seeds.

Injections of 5-fluorodeoxyuridine, actinomycin D, and cycloheximide

into seeds at various times during stratification were conducted to

determine if their effects on nucleic acid and protein synthesis would

be reflected in the termination of dormancy and subsequent seedling

growth. The concentrations tested did not appreciably alter germination

or emergence, but some differences in seedling weight and shoot height

occurred at certain times of application. Injection alone was respon-

sible for some differences in response.

RNA concentrations were followed during stratification and the

early stages of germination of peach seeds. RNA levels generally

declined until the sixteenth day of stratification, but increased by

the end of stratification. Levels of all polymeric RNA fractions were

high during germination. Abscisic acid and thiourea did not appreciably

alter the levels of RNA. Limited synthesis of RNA occurred during

stratification, and both abscisic acid and thiourea enhanced synthesis

slightly. No messenger RNA synthesis was detected.
















INTRODUCTION


The various phenomena of dormancy in plants have so interested

plant scientists that an extensive literature on the subject has

accumulated. The causative factors and the conditions which break

dormancy are numerous and diverse, as are the different manifestations

of dormancy in nature (4, 117, 126). Dormant organs possess a high

resistance to such adverse conditions as cold, drought, or heat, so the

inception and termination of dormancy in temporal relation to an

unfavorable season or condition represent practical means of survival.

Due to economic importance and the resultant efforts of plant

breeding and selection, many plants which exhibit dormancy are grown

throughout the world, frequently in climates which are unlike those of

the native habitats. Such is the case with many temperate fruit crops.

Consequently, dormancy of cultivated plants becomes a problem to man

in growing plants under certain conditions.

Of the various general theories as to the mechanism of dormancy of

seeds and buds, the most recent and prominent one assumes that the

regulation of dormancy is principally hormonal (4, 117, 121, 127). By

definition, the true resting condition must result from some internal

block to growth, as growth fails to occur even though external conditions

are considered favorable. Two lines of experimental evidence to support

the concept of hormonal regulation of dormancy include the effects of

exogenous growth regulators in breaking or imposing dormancy, and

correlations between the state of dormancy and levels of endogenous

growth regulators.












There is increasing evidence to support the concept that the mode

of action of hormones is either through the alteration of nucleic acid

metabolism and subsequent protein synthesis or through a direct involve-

ment in protein synthesis or metabolism (2, 12, 45, 67, 84, 119, 127,

128). In addition, many substances which are known to affect dormancy

have also been shown to alter nucleic acid and protein metabolism.

The preceding ideas and evidence, in combination with the recog-

nition that plant growth and development is principally under genetic

control in response to environmental stimuli, would indicate that a

logical area to study dormancy would be the mechanism of protein

metabolism, particularly the steps leading to protein synthesis. Thus,

these studies were initiated to determine the effects of various treat-

ments and chemicals on dormancy and on nucleic acid levels of peach

seeds, and to try to elucidate the role of nucleic acids in the

regulation of peach seed dormancy. The chemicals and treatments were

selected on the basis of their documented effects on either dormancy or

some phase of nucleic acid metabolism or protein synthesis.


















REVIEW OF LITERATURE


Although it is difficult to delineate a concise definition of

dormancy, in common usage it simply means a temporary suspension of

visible growth and development, without regard to causal factors. This

meaning is sufficient to describe annual rhythms of growth activity,

but a more specific terminology is warranted to define specific physio-

logical conditions that exist in potentially meristematic tissues. In

this review, dormancy will be used to denote rest, the true dormancy

caused and maintained by agents or conditions within the organ itself.

Rest is, therefore, the situation in which growth cannot he induced

under any set of environmental conditions normal for growth.

Chandler (14) described rest in a broad sense, but Samish (100)

defined the term in this more limited connotation. Rest is synonymous

with winter-dormancy (28, 95, 124), innate-dormancy (126), and deep-

dormancy (82). For an excellent discussion of dormancy terminology,

the review by Romberger (98) is suggested,



Seed Dormancy in Peaches

Dormancy in seeds generally denotes the failure of viable seeds to

germinate within a reasonable length of time after having been placed in

adequate moisture, temperature, and atmospheric gas composition to

facilitate growth. Such a designation is rather broad in that the

failure to germinate could be due to seed coat impermeability to water

and/or gases, embryo immaturity, mechanical restrictions of embryo












growth, endogenous inhibitors, or to special requirements for temperature

or light (4, 79, 82, 104, 111, 117, 125, 126, 127). It is obvious,

therefore, that a failure to germinate is not necessarily indicative of

the occurrence of an endogenous condition establishing the dormant state.

That the seed coat could present a mechanical barrier to embryo

growth is theoretically feasible and would be expected in peach seeds.

Vegis (117) asserts that the removal of the mechanical pressure that

the seed coat exerts on the swollen embryo is evidently of some signi-

ficance. Unfortunately, the elucidation of the function of seed coats

as mechanical obstacles to germination is confounded by the inherent

difficulty of separating mechanical influences from the possible

existence of other factors, i. e., permeability limitations and growth

inhibitors, so the question of mechanical resistance to embryo growth

has remained unanswered in a quantitative sense.

Because intact peach seeds imbibe water readily, there appears to

be no limitation to water permeability by the seed coat. However,

permeability to oxygen is another question. Nikolaeva (82) has presented

convincing evidence that the seed-covering structures of numerous species

constitute a tremendous barrier to oxygen diffusion. This is true

whether the seeds are considered to be deeply-dormant (rest) or non-

deeply-dormant (seeds requiring light, scarification, or other non-

chilling treatment to germinate). For example, the presence of the seed

coat and endospermal membrane greatly inhibited the respiration of the

apricot embryo. As compared to the respiration of excised embryos

cotyledonss intact), the respiration of seeds in which the seed coats

were broken at the radicle end was reduced by only 12%, whereas the

respiration of intact seeds was reduced by 57%.












As Nikolaeva (82) has concluded, gas exchange is particularly

hampered by living membranes; thus, freshly harvested seeds exhibit a

particularly deep dormancy (rest), as is generally the case in rosaceous

seeds. She postulated that the depth of seed dormancy that is respon-

sive to cold is determined by the degree of gas permeability of the

covering structures surrounding the embryo, i. e., fairly severe

impairment of gas exchange reduces the capacity of the embryo to grow

vigorously and normally. This explanation could be applied to the

occurrence of dwarfing in peach seedlings grown without chilling (6, 25,

36, 37, 38, 105, 130). Regardless of the attractiveness of this theory,

published growth curves of stone fruits indicate that the development of

the integuments precedes the period of rapid growth of the embryo (29,

105, 106, 107). This does not preclude the possibility that hardening

of the endocarp, which commences during the latter stages of the embryo

growth period, may prohibit oxygen diffusion and thereby be responsible

for the cessation of growth of the embryo after the endocarp has

completely hardened.

Nikolaeva's conclusions are in close accord with the general theory

of dormancy proposed by Vegis (115, 116, 117). Vegis contends that high

temperatures, in association with restricted oxygen uptake due to

covering structures, are the primary causes of dormancy. An essential

assumption of this theory, as pointed out by Wareing (126), is that

primary (rest) and secondary (imposed) dormancy are similar in nature,

such that factors which can induce secondary dormancy experimentally are

responsible for dormancy in general. Direct evidence to support either

the theory or the assumption is generally limited. However, that secon-

dary dormancy imposed by high temperature is very similar to dormancy is












indicated by the fact that to break secondary dormancy of peach seeds

requires a repeat chilling stratification of somewhat less duration than

required to break dormancy (1, 16, 126).

In addition, although physiological dwarfing of seedlings is

generally regarded as a consequence of insufficient chilling or of

treatments which circumvent the requirement for chilling, Pollock (91,

92) has observed that dwarfing in peach seedlings is not obligatory.

Non-chilled peach seeds, from which the basal 25% of the seed coat had

been removed, produced almost entirely normal seedlings when germinated

at or below 230C during the first 8-9 days. Temperatures above that

resulted in dwarfed seedlings. Biggs and Langan (10) have shown similar

effects in that high temperatures limited the growth capacity of

unchilled 'Okinawa' peach seedlings. Also, Flemion and Prober (41)

obtained apparently normal peach seedlings from excised embryonic axes

without the necessity of a chilling treatment. Thus, in peach seeds at

least, Vegis' theory and assumption may be entirely valid, in which case

the existence of embryo dormancy in peach seeds is questionable. At

best, the embryo may be weakly dormant.

Nikolaeva (82) studied respiratory changes in seeds during strati-

fication and concluded that at chilling temperatures, respiration is

reduced to such low levels that the restriction of oxygen penetration to

the embryo is no longer limiting to growth. Thus, dormancy of seeds

would be connected with a high respiration rate under conditions of

impeded gas exchange. The main physiological basis of stratification

would then be a temporary reduction of the respiration rate and a

simultaneous improvement of aeration.

Roberts (96, 97) discovered that pretreatment of dormant rice seeds











with cytochrome oxidase inhibitors (carbon monoxide, cyanide) stimulated

the breaking of dormancy. Respiratory inhibitors which did not affect

the terminal oxidase system had little effect on dormancy. Thus, it was

concluded that some unknown oxidation reaction must occur to a certain

stage before germination can proceed.

Because cytochrome oxidase is a strong competitor for oxygen, and

because the oxygen tension within a seed is undoubtedly low, the inhi-

bition of cytochrome oxidase would alleviate the competition for oxygen,

such that the proposed oxidation reaction could proceed more rapidly.

Paech (86) suggested that the oxidation of phenolics in the seed coat

would effectively prohibit the entry of oxygen to the embryo. Numerous

phenolics exist in peach seed coats (3), so partial removal of the coat

would facilitate oxygen entry to the peach embryo. Even so, the fact

that physiological dwarfing usually occurs as a result of this treatment

would appear to eliminate the phenolic oxidation proposal. It is

tempting to speculate that the unknown oxidation reaction may be

involved in the incapacitation or destruction of some endogenous

germination inhibitors.


Hormonal Concept of Dormancy Regulation

The prominent hypothesis of dormancy in seeds and buds assumes that

the regulation of dormancy is principally hormonal. By definition, the

true resting condition must result from some internal block to growth,

so it is not difficult to envision that growth may be prevented by an

unfavorable balance of growth promoters and inhibitors. Amen (4) has

compiled extensive evidence to show that the induction, maintenance,

and termination of seed dormancy are under hormonal control. Although











feasible for certain types of dormancy, Amen could not adequately

explain the effects of a chilling period as a trigger agent to break

dormancy. The contention that the regulation of seed dormancy may be

hormonal was reiterated by Villiers and Wareing (120) on the basis of

work with Fraxinus, which requires chilling to break dormancy.

Since Hemberg (50, 51) correlated the presence of growth inhibiting

substances and dormancy in the potato tuber and Fraxinus buds, much

research has been directed toward the detection of inhibitory substances

in dormant organs. Consequently, many growth inhibitors and promoters

have been discovered and identified in many species. Initially,

attempts were made to correlate the levels of endogenous inhibitors with

the emergence from rest, but the results were frequently variable and

there existed considerable disagreement. In addition, growth promoting

substances were found to be present in many dormant organs, and their

levels generally increased sharply upon emergence from dormancy. This

increase could well be the result rather than the cause of emergence.

Blommaert (11) reported that an inhibitor in dormant peach buds

decreased more rapidly in chilled buds. Nevins and Hemphill (81)

extracted an unidentified inhibitor and auxins from peach flower buds.

Hendershott and Bailey (52) showed an inhibitor, later identified as

naringenin (53), in dormant peach flower buds. The levels of naringenin

apparently correlated well with the state of dormancy (54). However,

other workers took exception to the correlation, claiming that the

reduction of naringenin levels near the end of dormancy was due to

dilution caused by flower enlargement (23, 26). Naringenin levels were

subsequently re-examined and were found to be highest during the peak

intensity of dormancy in peach leaf buds (32, 49).











Jones and others (62, 63, 64) extracted organic cyanides from peach

flower buds, some of which were inhibitory in bioassays. Aitken (3) has

shown the existence of inhibitory phenolics in peach seeds. Prunin has

been identified and observed in dormant peach buds (24, 33). Flemion

(39) found an inhibitor in the cotyledons of unchilled peach seeds and

postulated that it may be responsible for physiological dwarfing in

peach embryos.

Biggs (8) reported that non-after-ripened peach embryos contained

substantially larger quantities of growth inhibitors than after-ripened

embryos. Furthermore, the concentrations of growth promoters were not

changed by chilling. Flemion and deSilva (40) reported no correlation

between dormancy and the growth promoting or inhibiting substances in

peach seeds. Liao, as reported by Walker (121), observed that an in-

hibitor, thought to be naringenin, extracted from peach seed coats and

cotyledons was highest in concentration before chilling and decreased

during chilling. In addition, the levels of 3 wheat coleoptile growth

promoters were essentially unchanged during chilling. Gibberellin acti-

vity was reported in dormant after-ripened seeds of Prunus avium (93).

An inhibitor thought to be ABA was identified in the integuments of

peach seeds (75). It disappeared by the sixth week of stratification.

Both ABA and the inhibitory extract caused similar effects on seedlings

and excised embryos, suggesting that physiological dwarfing in peach

seedlings is caused by a certain concentration of ABA which is not

sufficiently high to prevent seed germination. Ryugo (99) observed an

inhibitory substance, identified as ABA, in peach, cherry, and apricot

seeds. The inhibitor could be removed by sustained leaching. The ABA

content of peach flower buds was observed to fluctuate during dormancy











but did not disappear with the termination of rest (121).

Exogenous GA has been shown to break the dormancy of peach seeds

(15, 16, 27, 47) and peach leaf buds (27, 49, 122). Kinetin breaks the

dormancy of peach leaf and flower buds (121). Thiourea will also cause

germination of unchilled, intact peach seeds (44, 88, 108).

Naringenin was reported to inhibit the dormancy-breaking action of

GA. Additionally, the degree of inhibition was dependent upon the

relative concentrations of the 2 materials (89). Exogenously applied

aqueous solutions of naringenin failed to inhibit peach bud-break (26).

Lipe and Crane (75) reported that ABA was antagonistic to GA3 in peach

seed germination. An antagonism between ABA and gibbercllins has been

noted in other species as well (4, 48, 117, 127).


Nucleic Acid Metabolism and Dormancy

In view of the considerable evidence as to the involvement of

growth substances in dormancy regulation, it is natural to attempt to

ascertain the mechanism of action of such substances. Although consid-

erable stress has been placed on respiratory metabolism, more recent

attention has been directed at differences in nucleic acid metabolism

in dormant and non-dormant tissues. There is substantial evidence that

growth regulatory substances influence nucleic acid metabolism (67) and

it has been proposed that the mode of action of hormones may be mediated

through nucleic acid metabolism; so this approach to dormancy is

consistent with the concept of hormonal control of dormancy.

Two major components of growth and development, cell division and

cell enlargement or elongation, have been shown to be dependent upon the

synthesis of structural and catalytic proteins (60, 65, 74, 85).











Protein synthesis is directed by nucleic acids, so it represents the

best-defined correlation between nucleic acids and plant growth.

According to the current concept (22, 46), protein synthesis

requires various enzymes, cofactors, amino acids, phosphorylated nucleo-

tides, and 3 species of polymeric RNA. The different RNA species have

been isolated and identified by gel electrophoresis and spectroscopy

(56, 58, 76, 77, 113). They are: mRNA, rRNA, and sRNA. In higher

plant tissue, there are 3 types of rRNA--cytoplastic, chloroplastic, and

mitochondrial. Cytoplastic rRNA is comprised of 25S and 18S subunits;

whereas the other 2 types contain 23S and 16S subunits. The rRNA

combines with protein to form ribosomes. Only sRNA and the subunits of

rRNA can be distinguished on gel electrophoretograms by spectroscopic

means. As mRNA is usually present in low quantities and is relatively

unstable, it cannot be distinguished.

Recent detailed reviews on the relations of hormones and dormancy

(127), nucleic acids (67), and protein synthesis (45) are recommended

for additional information and pertinent discussion of the evidence

which purports to link hormonal action with nucleic acid metabolism and

dormancy.

Silberger and Skoog (102) were the first to demonstrate that auxin

affects nucleic acid metabolism. Promotion of cell elongation in

excised soybean hypocotyl by IAA was accompanied by enhanced incorpor-

14
ation of 1C-labelled nucleotides into RNA (69). Venis (118) showed

that IAA induced the production of benzoylaspartate and suggested that

IAA caused the synthesis of new mRNA. The action of IAA was inhibited

by ACTD and puromycin, inhibitors of RNA and protein synthesis,

respectively. Nooden and Thimann (85) concluded that the locus of action











of IAA in cell enlargement could be on the nucleic acid system which

controls protein synthesis. Holm et al. (55) reported that auxin caused

marked accumulation of DNA and RNA, and apparently stimulated an increase

in chromatin, accompanied by an increased capacity for RNA synthesis.

Fan and Maclachlan (34) concluded that IAA selectively brings into

operation the coding systems) for cellulase biosynthesis due to the

fact that exogenous IAA on etiolated pea epicotyls resulted in a marked

increase in the amount and specific activity of cellulase, which could

be inhibited by inhibitors of protein synthesis. They later showed (35)

that IAA quantitatively increased the DNA, RNA, and proteins (partic-

ularly cellulase) in peas. It was concluded that IAA-induced RNA

synthesis is required for cellulase synthesis and lateral cell expansion,

irrespective of cell division. Whether cellulase is actually the cell

wall-modifying factor or some other factor is needed prior to cellulase

activity, IAA promotes constituents controlling protein metabolism.

In barley, exogenously applied GA results in the de novo synthesis

of alpha-amylase, ribonuclease, and protease (18, 19, 20, 87, 112). The

production of these enzymes is inhibited by ACTD during the early stages,

which suggests control of transcription by GA. Afterwards, if GA is

removed, alpha-amylase synthesis stops, which suggests that GA is also

acting during translation.

Lang and Nitsan (74) have shown that the increase in DNA in lentil

epicotyl in the absence of cell division was due to synthesis induced by

exogenous GA. Also, rRNA synthesis was increased by GA. An inhibitor

of DNA synthesis, FUDR, suppressed the increase in RNA, which would

indicate that the increase in RNA synthesis was dependent on DNA syn-

thesis. Furthermore, cell elongation was enhanced by GA and suppressed










by FUDR. They suggested that DNA synthesis is essential for GA-regulated

cell elongation, and that IAA-regulated growth depends on RNA and protein

synthesis, but not on DNA synthesis.

Haber et al. (48) observed that GA3 stimulated the germination of

lettuce seeds in which DNA synthesis and cell division were eliminated

by 60Co gamma-radiation. Germination was inhibited by ABA whether in

the presence or absence of GA3. An additional role of GA, that of

retarding senescence, has been attributed to the ability of GA to main-

tain a high content of RNA (7, 42, 43).

ABA has been demonstrated to inhibit RNA and DNA synthesis in seeds

(119, 123) and potato tuber buds (101). Van Overbeek et al. (110)

reported that nucleic acid synthesis was suppressed by ABA and that

benzyladenine (a synthetic cytokinin) would counteract ABA, but that

auxin or GA would not. Also, ABA inhibited 32P04 incorporation into DNA,

rRNA, and sRNA in embryonic axes of bean (123).

Total RNA synthesis is much lower in dormant than in non-dormant

seeds (59, 90, 129). In buds of apple and cherry, it was reported that

RNA synthesis began to decrease in early fall and that DNA and RNA

content remained low during rest (5). In addition, the synthesis of

DNA and particularly RNA began after the termination of rest in

association with renewed growth. Moreover, Khan et al. (71, 73)

observed that the capacity of dormant pear embryos to synthesize nucleic

acids progressively increased during chilling, and that incorporation of

32P04 into sRNA, DNA-RNA, and light rRNA was enhanced as chilling

progressed. However, ABA prevented the incorporation of label, and

this effect was reversed by either kinetin or GA.

Tuan and Bonner (109) reported that the breaking of dormancy in the











potato tuber was accompanied by the ability to synthesize DNA-dependent

RNA. Moreover, DNA and RNA content increased prior to the increase in

fresh weight of potato buds induced to grow by ethylene chlorohydrin

treatment. Furthermore, they noted that chromatin isolated from dormant

potato buds showed a diminished ability to support RNA synthesis in

vitro. This was claimed to indicate decreased template availability or

more extensive gene repression in dormant organs.

In dormant hazel seeds, GA3 increased the incorporation of 32PO4

into RNA in the embryonic axis within 12 hr (90). In isolated chromatin

of hazel seeds, increased incorporation of 32PO4 was accompanied by

increased RNA polymerase activity after GA3 treatment (59). There was

also 3 times more available template of chromatin as a result of GA3

treatment (DNA template availability was assayed after saturation with

E. coli RNA polymerase). After GA3 treatment, embryonic axes of hazel

first showed greater template availability, then increased activity of

RNA polymerase, followed by increased RNA synthesis.

Villiers (119) showed that ABA inhibited the incorporation of 3H-

labelled nucleotides into nucleic acids of embryos of Fraxinus excelsior,

but that 14C-labelled precursors were not prevented from being incor-

porated into proteins. GA3 reversed the effect of ABA on RNA. Conse-

quently, he concluded that ABA maintains dormancy by inhibiting the

production of specific types of mRNA, which thus prevents the formation

of specific proteins which are involved in terminating dormancy.

Khan and Anojulu (72) reported that ABA altered the base composition

of rapidly labelled RNA species, and suggested that ABA changes the

readout pattern of the genome. Dure and Waters (31, 128) reported that

although protein synthesis was essential, RNA synthesis was apparently








15


unnecessary for the initial stages of cotton seed germination. They

suggested the presence of a stable mRNA in dry seeds. Chen et al. (17)

showed the existence of a stable mRNA in dry wheat embryos which became

functional during imbibition. Synthesis of new mRNA did not occur until

germination had begun.
















MATERIALS AND METHODS


Plant Materials

These studies were conducted using plant materials of 2 varieties

of Prunus persica Batsch. Seeds for embryo developmental studies were

taken from developing fruit of 4-year-old 'Early Amber' peach trees

located at the University of Florida Horticultural Unit near Gainesville.

'Okinawa' seeds for the seed germination experiments were collected from

tree-ripened fruit in early June, 1970, in an orchard near Hawthorne,

Florida. After the removal of the mesocarp, the stones were washed in

a mechanical potato peeler to remove any remnant flesh. After air-

drying at 200C for 1 wk, the seeds were stored at 60C until used.

Immediately prior to use, seeds were removed from the stony endocarp

and selected for treatment.


Inhibitor Application to Developing Seeds

Developing 'Early Amber' fruit were treated with 3 chemicals which

are known to inhibit different processes in the sequence of protein

synthesis (Table 1). The concentrations were as follows: 10-5 M FUDR,

10 pg/ml ACTD, and 104 M CHI. The chemicals were injected into the

basal end of the fruit from a microsyringe inserted to an appropriate

depth through the suture at a slight angle from the vertical. Before

release of the chemical, the needle was withdrawn slightly to alleviate

back-pressure. Thus, 3 pl of material were deposited in or near the main

vascular bundle which connects to the seed.






































Table 1. Chemicals used to inhibit nucleic acid and protein synthesis.


Chemical Abbreviation Process inhibited

5-Fluorodeoxyuridine FUDR DNA synthesis

Actinomycin D ACTD RNA synthesis

Cycloheximide CHI Protein synthesis











Treatment was begun about 2 days after cytokinesis and continued at

weekly intervals until commercial maturity (cytokinesis is that stage of

peach seed development in which the endosperm changes from the free-

nuclear to the cellular state). Samples for RNA determinations were

collected at 5-day intervals beginning 10 days after the initial

application. In the orchard, 10 seeds were removed from the endocarp,

immediately frozen with ethanol-dry ice, and then stored frozen in

plastic bags. Both brown (non-viable) seeds and those which had been

punctured by the syringe needle were discarded. Seeds from non-injected

fruit were used as controls. Additional seeds were collected for

measurements of the embryo length and seed weight. The dates of

injection, the dates of sampling, and the time intervals between

injection and sampling are summarized in Table 2.

Radioactive 32PO4 was injected at intervals to ascertain that

chemicals applied in the manner described were translocated to the

seeds, and to indicate when applications should be discontinued due to

insufficient translocation. Each of several fruit received 0.03 pc.

After 24 hr, the seeds were removed and assayed for radioactivity.


Embryo Culture During Seed Development

Non-treated fruit and fruit treated on the first date of application

of inhibitors were collected weekly for embryo culture. The freshly

harvested fruit were surface sterilized with aqueous tincture of

merthiolate (1:2000), after which the ovules were removed aseptically.

The innocula were transferred to 150 x 20 mm test tubes containing 15 ml

of a nutrient medium described by Brooks and Hough (13) for peach

embryo culture.






























Table 2. Dates of injection, dates of sampling, and time intervals
between injection and sampling.


Days between injection and sampling

Date of injection
Sampling Sampling
interval date 4/6 4/13 4/20 4/27 5/4

1 April 16 10 -- -- -- --

2 April 21 15 8 -- -

3 April 26 20 13 6 --

4 May 1 25 18 11 4

5 May 6 30 23 16 9 --

6 May 11 35 28 21 14 7

7 May 16 40 33 26 19 12











On the first sampling date, the entire micropylar end of the ovule

was cultured. On subsequent dates, however, the embryos were large

enough that only the embryo and a minimum of accompanying tissues were

taken. Cultures were grown at 23C under a 15-hr photoperiod until

June 9, 1971, when they were visually rated for discernible growth and

development.


Studies of Seed Dormancy

As a general procedure, seeds were allowed to imbibe in moistened

vermiculite for 48 hr at 23-240C, after which they were treated with

Captan and planted in metal flats of perlite or vermiculite. The flats

were then placed in appropriate growth chambers for either stratification

or germination. The period of stratification was 20 days at 5-60C in

the dark. Germination was accomplished in a 12-hr photoperiod at 21-

22C. Deviations from this general handling procedure will be noted.


Seed irradiation

To study the effects of gamma-radiation on seed germination, 3 seed

irradiation experiments were conducted. Seeds were irradiated at the

Co Irradiator of the University of Florida Agricultural Experiment

Station. Initially, 9 lots of 80 dry seeds were subjected to dosages

of 0, 25, 50, 100, 200, 400, 600, 800, and 1000 kR at a rate of 0.861

kR/min. After imbibition and stratification, the seeds were allowed to

germinate for 30 days. At that time, the number of seeds having

germinated and having rotted were recorded. Germinated seeds and firm

seeds which had not germinated were replanted for further observation.

Later, a lower range of dosages of gamma-radiation was tested.

Six lots of 40 dry seeds were irradiated at dosages of 0, 5, 10, 15, 20,











and 30 kR at a rate of 0.843 kR/min. Data for germination, shoot height,

and the number of leaves per shoot were taken 60 days after the end of

stratification.

In the third irradiation experiment, imbibed seeds were irradiated

at different stages of stratification. Four replications of 15 imbibed

seeds were irradiated after 0, 7, 14, and 21 days of stratification at

dosages of 0, 1, 3, 10, 13, and 20 kR, applied at a rate of 0.780 kR/min.

After irradiation, the seeds were allowed to complete 21 days of

stratification before transferral for germination.

Care was taken to preclude a possible high temperature reversal of

accumulated stratification. This was accomplished by using an insulated

ice water bath as a holding chamber and by placing plastic bags of dry

ice above and below the irradiation cannister in which the seeds were

located during irradiation. The air temperature was 70C in the holding

chamber and 9-100C within the irradiation cannister.

Beginning 12 days after the completion of stratification, emergence

data were recorded at 4-day intervals through 40 days. On the last day,

the seeds and seedlings were harvested and the following data were

recorded for each replication: average seedling weight, average shoot

weight, average root weight, average shoot length, and total germination

(emerged seedlings plus non-emerged but germinated seeds). These data

were subjected to statistical analysis as a factorial experiment, with

irradiation dosage as the primary factor and time of irradiation as the

secondary factor (103). The means were then compared for significance

by Duncan's test (30).


Inhibitor application to stratifying seeds

Distilled water, 10-5 M FUDR, 10 pg/ml ACTD, and 10-4 M CHI were











injected into 'Okinawa' peach seeds at various times during stratifi-

cation. Each of 60 selectively firm seeds received 2 pl of material

after 0, 2, 4, 8, 12, 16, and 20 days of stratification. The materials

used in this experiment were kept in the stratification chamber and the

injections were made in the chamber in order to circumvent any effects

of temperature changes.

Injection was accomplished by inserting the syringe needle com-

pletely through one cotyledon and into the other, then withdrawing it

slightly before the material was injected. In this way, the material

had direct access to the area between the cotyledons, to each cotyledon,

and to the embryonic axis. Injection was into the distal end of the

seed.

Emergence, germination, and growth measurements were recorded as

described in the previous experiment. Statistical analysis was

conducted with chemical treatment as the primary factor to determine

differences due to chemical treatment or time of treatment.



Nucleic acid changes during stratification and germination

To determine the changes in nucleic acids during stratification and

germination, 10 firm seeds were collected after 0, 2, 4, 8, 12, 16, and

20 days of stratification and after 2, 4, and 8 days of germination.

The samples were immediately frozen with dry ice and stored frozen until

nucleic acid determinations could be made. To determine changes in the

synthesis of nucleic acids and to detect mRNA, 1 pc of 32P04 was injected

into the distal end of each of the 10 selected seeds 24 hr prior to

certain sampling intervals. The samples receiving radioactivity were

those taken after 4, 12, 20, 22, 24, and 28 days.











In addition, 3 x 10-3 M ABA and 10 M thiourea were applied to

separate lots of seeds as a 6-hr soak prior to stratification. The

sampling intervals and radioactivity treatments were the same as

described in the preceding paragraph.


Extraction and Isolation of Nucleic Acids

A Sorvall Omni-Mixer was used to grind the seeds at 0-50C for 3 min

at 8,000 rpm in 25 ml of a phenol-buffer grinding medium as described by

Loening (76). The subsequent extraction procedure is essentially that

of Loening (76) and is described in Figure 1.




Procedure for Gel Electrophoresis

Polyacrylamide gels of 2.4% acrylamide with 5% cross-linking with

bis-acrylamide were prepared according to the procedure described by

Loening (76). Materials were obtained from Bio-Rad Laboratories.

Initially, 65 x 6 mm gels were cast in 75 mm glass tubes, but some

difficulty was encountered in the removal of the gels for scanning.

Consequently, 65 x 6.5 mm gels were cast in plastic tubes. The lower

ends of the plastic tubes were closed with a dialysis membrane (cellu-

lose acetate) to prevent the gels from sliding out during storage and

electrophoresis. The gels were stored in IE tris-phosphate buffer

prepared according to Loeining (76).

The gels were pre-electrophoresed in IE buffer at room temperature

for 1 hr at 40 v to remove the polymerization catalyst and other impur-

ities. Subsequently, 50 pl of the nucleic acid solution (25 pl from

'Okinawa' extracts) were layered on the gel. Electrophoresis was con-

tinued for 70-75 min. Only 8 gels were electrophoresed at one time.












Frozen seeds

Grind in 25 ml phenol-buffer 3 min, 8,000 rpm, 0-50C
Centrifuge 12 min, 18,000 g, 0-50C





I
Aqueous Phenol

Wash 2X with: Discard
0.2 ml 10% sodium lauryl sulfate
0.6 ml 3 M sodium acetate
12.5 ml phenol-cresol
Centrifuge 12 min, 18,000 g, 0-50C


Aqueous Phenol

Precipitate with 25 ml cold 95% ethanol, Discard
2 hr, -50C
Centrifuge 12 min, 18,000 g, -50C


Pellet Ethanol

I
Dissolve in 3 ml 0.5% sodium lauryl sulfate- Discard
0.15 M sodium acetate solution
Precipitate with 8 ml cold 95% ethanol
Centrifuge 15 min, 18,000 g, -50C


Pellet Ethanol

Wash with 10 ml cold 80% ethanol Discard
Centrifuge 15 min, 18,000 g, -50C


Pellet Ethanol

Dissolve in 2.5 ml IE buffer with 0.2% Discard
sodium lauryl sulfate and 6% sucrose

Store in refrigerator for electrophoresis




Fig. 1. Procedure for nucleic acid extraction. Volumes are based on
4 g of tissue.











In addition, a marker of pyronin B dye in 6% sucrose was layered on one

gel of each run 5 min before the nucleic acid solutions were layered.


Nucleic Acid Determination

A Beckman DU Spectrophotometer in conjunction with a Gilford Gel

Scanner (Model 2000) was used to scan the gels and produce a line graph

of ultraviolet absorbancy. The gels were scanned at 260 nm at a rate of

2 cm/min. Areas under the RNA peaks were measured with a Keuffel &

Esser planimeter. Quantitation of RNA fractions was described in terms

of area under the peak.


Radioactivity Measurements

Measurement of the radioactivity present in the seeds of 'Early

Amber' fruit injected with 32pO4 was accomplished with a Nuclear Chicago

Gas Flow Counter (Model D-47). The seeds were ground using a mortar and

pestle and hydrolyzed for 8 hr in 25 ml IN HC1. Aliquots were placed in

metal planchets and dried under infrared light. Counting efficiency was

approximately 3%. Measurement of the radioactivity in the nucleic acid

fractions of the gels was accomplished by liquid scintillation. After

the gels had been scanned, they were immediately frozen with dry ice

and stored frozen in closed vials.

The frozen gels were imbedded in Tissue-Tek in 72 x 8 mm plastic

tubes. The tubes were then placed on dry ice to freeze the Tissue-Tek

and to prevent the gels from thawing. A micrometer equipped with a

guillotine was used to cut the frozen, imbedded gels into 2-mm sections.

The sections were digested for 24 hr at 600C in 0.5 ml of 30% hydrogen

peroxide in scintillation vials. Subsequently, 15 ml of Aquasol (New

England Nuclear Corp.) were added to each vial, and they were stored








26


in the dark at 60C for 24 hr prior to counting. The samples were counted

with 72% efficiency in a Packard Tri-Carb Liquid Scintillation Spectro-

meter (Model 3380). Loading into the counter was accomplished in the

dark.


















EXPERIMENTAL RESULTS


Experiments with Developing Seeds

In consideration that the changes in major polymeric RNA concen-

trations during seed development might provide some insight into the

inception of dormancy in peach seeds, net RNA concentrations were

followed from cytokinesis until commercial fruit maturity for seeds of

'Early Amber' peaches. Attempts to alter the normal pattern involved

the application, at weekly intervals during development, of 3 specific

inhibitors of the nucleic acid-protein synthesizing system (Table 1).

Seeds which were not sampled for RNA determinations were used for

germination tests to ascertain whether the inhibitors altered the

germination of seeds.

To verify that the applied inhibitors were actually reaching the

seeds, radioactive 32P04 was similarly applied to several fruit during

the period studied. The uptake of 32P04 by the seeds is shown in

Figure 2. Appreciable radioactivity was detected in the seeds during

the early stages of development, but incorporation was reduced near

fruit maturity. This would indicate that the vascular bundle which goes

through the endocarp to the seed is still functional after the endocarp

has hardened and that the method of application of the inhibitors is

sufficient for introducing chemicals into the seeds. However, the

quantity of chemical actually being translocated to the seeds varied, as

shown by the decrease in 32PO4 uptake.

In order to determine whether the inhibitors were affecting growth

27












1000


800

U\
UJ 600
U)

2 400
C-)



0 I I
200


0
4/11 4/23 5/5

DATE


Fig. 2. Incorporation of
during development.


32PO4 into 'Early Amber' peach seeds











potential and the inception of dormancy in the developing seeds, samples

were taken weekly from fruit which had been injected on April 6. The

embryos were then cultured in vitro until June 9, at which time they

were evaluated by visual comparison for growth and development. The

primary criterion for development was the elongation and proliferation

of roots. Of secondary consideration were the enlargement and greening

of the cotyledons and extension of the epicotyl. If no root or shoot

development was evident, the tissues were evaluated solely on the basis

of cotyledonary development. Tie results of these evaluations are

presented in Table 3.

Samples taken on April 14 were too immature to grow sufficiently

in vitro, as evidenced by the failure of root or shoot elongation.

However, some embryo enlargement and development was noted. On subse-

quent sampling dates, the majority of control embryos did develop roots.

The number of elongated shoots was initially low, but steadily increased

until May 11. On that and the following sampling date, several of the

tubes containing control embryos became contaminated, as small insects

were able to enter beneath the plastic caps of the test tubes.

FUDR inhibited root formation and shoot elongation until near the

end of the experiment. The same was generally true of the other treat-

ments. Root formation in all treatments was usually better than shoot

growth. For the May 18 samples, however, all of the embryos which

showed signs of good growth developed extensive root and shoot systems.

Even at that time, however, the embryos were not yet mature.

Total RNA concentrations were assessed during seed development,

using techniques of gel electrophoresis. Figure 3 is a typical electro-

phoretogram which shows the various fractions of RNA that were separated



















Table 3. Evaluations of the growth of embryos from 'Early Amber' peach
seeds, cultured in vitro from fruit injected on April 6, 1971.



Sampling No. of Coty-
date Chemical embryos Rootx Shooty ledonz


4/14


4/21


CONT
FUDR
ACTD
CHI

CONT
FUDR
ACTD
CHI

CONT
FUDR
ACTD
CHI

CONT
FUDR
ACTED
CHI

CONT
FUDR
ACTD
CHI

CONT
FUDR
ACTD
CHI


5/18


XRoot elongation or proliferation.
YShoot elongation.
ZCotyledon enlargement, without regard


to root or shoot development.










1.5
rRNA

25S


01.0 AsRNA
N 18S
Lu



16S
0.5
23S




0- I
0 0.5 1.0

Rf

Fig. 3. Typical electrophoretogram of polymeric RNA fractions of 'Early
Amber' peach seeds, separated on 2.4% polyacrylamide gels and scanned
at 260 nm.











on 2.4% acrylamide gels for developing 'Early Amber' peach seeds. The

first small peak may be comprised of a number of components, possible:

including DNA, but no attempt was made to quantitate it for further

comparison. The second peak represents absorption of ultraviolet light

by the 25S subunit of rRNA. The next major peak corresponds to the 18S

subunit of rRNA. Each of these peaks may have either a shoulder or a

smaller peak to the right. Such peaks correspond to the 23S and 16S

subunits of chloroplastic rRNA. Generally, these peaks were too small

or insufficiently separated from the cytoplastic rRNA peaks to be

measured separately, so they were considered as a part of the 25S and

18S subunits, respectively. In most cases, the rRNA of the chloroplasts

was completely masked by the rRNA of the cytoplasm. The final peak on

the electrophoretogram represents sRNA.

The areas of the peaks which represent the major RNA fractions,

i.e., sRNA and the 25S and 18S subunits of rRNA, were measured on the

electrophoretograms, recalculated as areas per gram of tissue, and

summarized graphically (Figs. 4, 5, 6, 7, 8, 9, 10, and 11). Each

illustration was designed to present the data for each treatment at

each sampling interval for each date of injection. Data for the controls

are repeated for each treatment in each illustration to facilitate

comparisons.

Total RNA (Fig. 4) represents simply the sum of rRNA and sRNA. In

seeds of the controls, total RNA did not fluctuate appreciably during

the first 5 sampling dates, but there was an increase at the sixth

sampling interval (May 11) and a slight decrease at the last sampling

(May 16). Although each of the 3 treatments for the April 6 injection

initially contained about the same quantities of RNA as the controls,











FUDR
















ACTED
















CHI


234567 1234567 1 234567 34567
CONTROL 4/6 4/13 4/20


4567
4/27


167
5/4


SAMPLING INTERVAL a INJECTION DATE

Fig. 4. Total RNA of 'Early Amber' peach seeds treated with inhibitors
during development. The sampling intervals were as follows: 1--
April 16; 2--April 21; 3--April 26; 4--May 1; 5--May 6; 6--May 11;
and 7--May 16.


1Jv
I


c50











100


50





S -50

z -100
0


o 50








0 100
cr

a. 50





-50

1 357 357 357 57 7
I-









-100






246 246 46 46 6

4/6 4/13 4/20 4/27 5/4

SAMPLING INTERVAL & INJECTION DATE

Fig. 5. Magnitude of change of total RNA of 'Early Amber'
peach seeds treated with inhibitors during development.
Top: FUDR; middle: ACTD; bottom: CHI. The sampling
intervals were as follows: 1--April 16; 2--April 21;
3--April 26; 4--May 1; 5--Mav 6; 6--May 11; and 7--May 16.









140
FUDR
120

100

80

60

40

20


140
120 ACT D
-y120
E
100

< 80
Z
' 60

Wn 40

N 20


140
CHI
120

100

80

60

40


0
1234567 1234567 234567 i 34567 I 4567 67
CONTROL 4/6 4/13 4/20 4/27 5/4

SAMPLING INTERVAL & INJECTION DATE

Fig. 6. Concentration of the 25S subunit of rRNA of 'Early Amber' peach
seeds treated with inhibitors during development. The sampling
intervals were as follows: 1--April 16; 2--April 21; 3--April 26;
4--May 1; 5--May 6; 6--May 11; and 7--May 16.









FUDR









Ft 1. 1i 1 -

ACTD











CHI









. i 1 1 1 -


2346 2-3 U
4/6 4/13


,3567of 4 567 i3o [
4/20 4/27


SAMPLING INTERVAL 8 INJECTION DATE
Fig. 7. Concentration of the 18S subunit of rRNA of 'Early Amber'
peach seeds treated with inhibitors during development. The
sampling intervals were as follows: 1--April 16; 2--April 21;
3--April 26; 4--May 1; 5--May 6; 6--May 11; and 7--May 16.


eI 03450 I
CONTROL


5/4








240

200

160

120

80

40

0

S200
E
160

z
Z
S120

_j 80

0 40
I-
0

200

160

120

80

40

0


FUDR













ACTED













CHI






-j IT-U -I-TI


1 234567 1 34567 i 4567 1 67


4A3


4/20 4/27 5/4


SAMPLING INTERVAL & INJECTION DATE
Fig. 8. Total rRNA of 'Early Amber' peach seeds treated with inhibitors
during development. The sampling intervals were as follows: 1--
April 16; 2--April 21; 3--April 26; 4--May 1; 5--May 6; 6--May 11;
and 7--MaN 16.


1234567 1 234567
CONTROL 4/6









600


500 FUOR


400

300


200 -

100

0


500 ACTOR


400

300

200 r-

100

0


500 CHI


400


300

200 -

100

r. _ - - - - L -


1234567 I 1234567 1 234567 I 34567 I
CONTROL 4/6 4/13 4/20

SAMPLING INTERVAL S INJECTION


4567 67
4/27 5/4

DATE


Fig. 9. sRNA of 'Early Amber' peach seeds treated with inhibitors
during development. The sampling intervals \ere as follows:
1--April Id; 2--April 21; 3--April 26; 4--May 1; 5--May 6; 6--May 11;
and 7--May 16.









FUDR












ACTD












CHI









I .1I I


1234567 1 1234567
CONTROL 4/6


i 234567 34567 4567 67
4/13 4/20 4/27 5/4


SAMPLING INTERVAL & INJECTION DATE
Fig. 10. Ratios of rRNA/sRNA of 'Early Amber' peach seeds treated with
inhibitors during development. The sampling intervals were as
follows: 1--April 16; 2--April 21; 3--April 26; 4-May 1; 5--ay 6;
6--May 11; and 7--May 16.





























3.0

2.5

2.0

1.5

1.0

.5


1234567 I 1234567 1 234567 34567 1 4567 67
CONTROL 4/6 4/13 4/20 4/27 5/4

SAMPLING INTERVAL & INJECTION DATE
Fig. 11. Ratios of 25S/18S rRNA of 'Early Amber' peach seeds treated
with inhibitors during development. The sampling intervals were as
follows: 1--April 16; 2--April 21; 3--April 26; 4--May 1; 5--May 6;
6--May 11; and 7--May 16.











there were considerable fluctuations subsequently. FUDR caused a

decrease through 3 samplings, followed by a steady increase through the

sixth sampling (May 11), and then showed a decline at the last sampling

on May 16. At all but the fifth sampling, however, there was less RNA

than in the corresponding control. ACTD-treated seeds parallelled the

control for the first 3 samplings, but deviated substantially afterwards.

There was less RNA at the second, fourth, and seventh samplings, but

there was substantially more at the fifth and sixth samplings than in the

corresponding controls. CHI, however, appeared to cause the greatest

reduction in total RNA, as all but the first and fifth samples contained

less RNA than the controls.

A drastic reduction of total RNA in seeds treated with ACTD and CHI

occurred at the fourth sampling on May 1. About 50% of the fruit treated

on April 6 abscised. Fruit abscission in these 2 treatments occurred

between April 30 and May 4, but fruit abscission in the FUDR treatment

occurred over several weeks. It is possible that the reduction in RNA

for ACTD and CHI on May 1 could have resulted because several fruit in

those samples were senescing and about to abscise. Fruit abscission for

the other dates of injection was only about 10-15%, and there was no

discernible drop period.

Injection on April 13 generally resulted in less RNA/g for each

treatment than was present in corresponding controls. The only excep-

tions were the fifth sampling of ACTD and CHI on May 6. For the April

20 injection, however, the reverse was true, in that only the sixth

sampling for FUDR and CHI contained less total RNA than the corresponding

control. It is noteworthy that the CHI sample injected on April 20 and

taken on May 1 (interval 4) contained the most RNA of all samples of the











CHI treatment. Similarly, the ACTD sample injected on April 20 and taken

on May 16 (interval 7) contained the most RNA of all samples in the

experiment. The 4 samples taken from the April 27 injection were of

interest inasmuch as the first 2 samples in each treatment contained

more RNA than the controls. The first of the 2 samples of the May 4

injection showed a drastic reduction of total RNA for each of the treat-

ments, but only FUDR-treated seeds failed to completely recover by the

next and last sampling.

Having examined the patterns of total RNA as a result of application

of the inhibitors, the magnitude of change from the control was consid-

ered for each treatment. For the April 6 and April 13 injections, the

application of FUDR generally caused decreases in the total RNA present

(Fig. 5). These decreases ranged from 15% to 45% less RNA than was

present in the controls. For the April 20 injection, the changes from

control varied from +70% on May 6 (interval 5) to -20% on May 11 (interval

6). The greatest deviation as a result of FUDR treatment was a -75%

deviation for the May 11 sample of the May 4 injection. Only the April

20 and April 27 injections of FUDR caused increases of RNA in relation

to the control.

ACTD injected on April 6 caused less than 20% change from the

controls for the first 3 and the sixth samplings. The change which

occurred at the fourth sampling on May 1 represented a 60% reduction of

RNA, whereas the fifth sampling (May 6) showed 75% more total RNA than

the control. Total RNA at the fifth sampling interval for the April 13

injection was 67% greater than the control, but the other samples of

that treatment contained equal or less RNA than the corresponding

control. The seeds collected at the fifth and seventh sampling intervals











for the April 20 injection of ACTD contained 77% and 83% more RNA,

respectively, than the controls. The other samples of that treatment

did not vary from the controls. Samples from the April 27 injection

varied by less than 35% from the controls, and only 1 sample, the sixth,

represented less RNA than was present in the corresponding control.

The May 4 injection of ACTD resulted in a 55% decrease of RNA on May 11

and a 25% increase on May 16.

The CHI treatments on April 6 and April 13 caused 9 of the 13

samples to contain at least 25% less RNA than the controls. Samples

taken at the fifth interval on May 6 showed RNA increases of 45% and

70%, respectively, for the 2 injection dates. Although the fifth

sampling for the April 20 injection contained 50% more RNA than the

control, the seeds of the fourth sampling date contained 127% more.

Generally, CHI caused greater reductions of RNA than the other 2 inhib-

itors during the April 6, April 13, and April 20 injections. The April

20 injections, however, represented the most drastic changes from the

control for each inhibitor, and those changes were actually increases

of total RNA.

The same general relationships that were established for total RNA

are applicable to its components (Figs. 6, 7, 8, 9). However, sRNA did

not fluctuate greatly during the season (Fig. 9). Consequently, as

shown in Figure 8, the highest total RNA in the controls was associated

with the highest rRNA. Also, it appeared that the highest total RNA in

the April 6 and April 13 injected seeds seemed to be more closely

associated with the higher quantities of rRNA than sRNA, with the excep-

tion of the CHI treatment of April 13. However, sRNA seemed to con-

tribute the most to the total RNA pattern for the April 20 injections.











Total RNA for the April 27 injection was due to a varied contribution of

both rRNA and sRNA, whereas each was important for the May 4 injection.

The rRNA/sRNA ratios (Fig. 10) generally support the previous observa-

tions as to the composition of total RNA.

The 25S and 18S subunits of rRNA appeared to be closely associated,

as the patterns of each closely parallelled the patterns of rRNA (Figs.

6, 7, and 8, respectively). Because the 25S/18S ratios have been con-

sidered to indicate metabolic stability and activity of RNA, the ratios

are presented in Figure 11. For the untreated controls, ratios varied

from 0.83 at the first sampling on April 16 to 1.71 at the sixth sampling

on May 11. Although the ratios never reached the range of 1.8-2.0, which

is considered to represent metabolic stability, there were 3 distinct

levels of activity indicated. Initially, the ratios were below 0.9 for

samples collected on April 16 and April 21. The samples collected on

April 26, May 1, and May 6 ranged between 1.1 and 1.4. Finally, the last

2 samples had ratios of about 1.7. Consequently, RNA metabolism was

progressing toward stability at the end of the season, but considerable

growth activity was indicated between cytokinesis and maturity.

In view of the different levels of activity of RNA metabolism,

these levels should be related to possible growth activity of the seeds.

Full bloom occurred on February 25 and cytokinesis occurred about 40

days later on April 4. The final samples were collected 40 days after

the first injection, which was on April 6. Consequently, the only

development of the seed during the 40 days of the experiment was that of

the embryo, as the nucellus and integuments had completed development

prior to cytokinesis.

The average lengths of the embryos of the controls steadily











increased during the 40 days of this experiment, but had not completely

filled the seed coats by the last sampling on May 16 (Fig. 12). Seed

weights of the treatments fluctuated somewhat, but the fluctuations

could perhaps be attributed to moisture changes during development (Table

4). Even so, the increases in embryo size and seed weight as the fruit

matured did not reflect the different levels of RNA and growth activity

shown by the 25S/18S ratios. Size per se was seemingly not related to

the patterns of total RNA.

The 25S/18S ratios for each of the treatments are also presented in

Figure 11. Although there were exceptions, the ratios for each treat-

ment generally increased toward the last sampling date within each time

of injection, and increased as time of injection approached the end of

the experiment.

The injected fruit which remained on the trees after the final

sampling were collected so that the seeds could be germinated to observe

the effects of the inhibitors on the termination of dormancy and on

subsequent growth. However, none of the seeds, including controls,

germinated, but rotted in the germination flats. The seeds dried to

about 2 mm in thickness during after-ripening. When imbibed, all were

soft and spongy--indicating the lack of viability. Too, the seeds were

still immature when the fruit had attained commercial maturity, as the

embryos had not completely filled the seed coats by May 21.


Seed Irradiation Studies

To test the hypothesis that seeds can germinate without concurrent

cell division, seeds were subjected to 60Co gamma-radiation. Initially,

it was necessary to determine a dosage which would inhibit cell division





























4/21 5/1 5/11


5/21


DATE


Fig. 12. Average length of the 'Early Amber' peach
embryo during its development.






























Table 4. Average seed weights at each sampling interval during the
development of 'Early Amber' peaches.


Weight (g)
Sampling
date CONT FUDR ACTD CHI

April 16 .384 .392 .327 .342

April 21 .372 .375 .374 .420

April 26 .355 .382 .390 .345

May 1 .418 .395 .412 .421

May 6 .467 .369 .408 .415

May 11 .513 .371 .406 .401

May 16 .420 .382 .359 .387











but would not result in non-viable seeds, so a range of 25 to 1000 kR

was tested on dry seeds. The germination data for this experiment are

presented in Table 5.

Dosages of 400 kR or more completely prevented seed germination as

determined by radicle emergence from the seed coat. Such dosages also

caused high percentages of non-viable seeds, as was evidenced by rot.

Of the seeds which received 200, 100, or 50 kR, less than 50% germinated,

although rotted seeds accounted for only about 20% of the total. Seeds

of the latter 3 dosages, however, did not develop into seedlings, as the

epicotyls appeared to be dead. Generally, the radicles were swollen and

necrotic or brown in color, whereas the cotyledons were elongated and

white, with some greening. No branching of the radicle was apparent.

About 50% of the seeds which received 25 kR germinated and produced

seedlings. The root systems were branched and extensive, though less so

than the controls. Shoot growth was inhibited and stunting was apparent.

All firm seeds and germinated seeds were replanted for further

observation, but after an additional 30 days, no further germination or

growth was noted. However, the seedlings that were already growing did

continue to grow slowly, but did not overtake the growth of the controls.

To obtain a better picture of interaction between irradiation and

subsequent seedling growth immediately following germination, a lower

range of 5-30 kR was tested next. Data for germination, shoot elon-

gation, and the average number of leaves per seedling were recorded

after 60 days at suitable growth conditions (Table 6). Although seed

germination was generally about 50%, subsequent growth was limited in

accordance with the dosage applied. For example, subsequent seedling

height decreased as irradiation dosage increased. Also, the number of


















Table 5. Germination of 'Okinawa' peach seeds
exposed to 60Co gamma-radiation.



Percentage
kR Germinated Firm Rotted

0 64 16 20
25 50 33 17
50 43 41 16
100 33 45 22
200 43 39 18
400 0 63 37
600 0 51 49
800 0 34 66
1000 0 20 80


Table 6. Germination and subsequent growth of
'Okinawa' peach seeds exposed to 60Co gamma-
radiation.



Germination Height Number
kR (%) (cm) leaves

0 49 8.6 15.0
5 56 5.4 10.6
10 49 5.9 11.1
15 44 4.4 8.1
20 39 4.1 6.2
30 50 2.8 5.5










leaves ppr seedling was inversely related to irradiation dosage.

A third Irradiation experiment was conducted to determine whether

there was a differential sensitivity to irradiation during stratifi-

cation. Consequently, imbibed seeds were subjected to irradiation at

varying stages of stratification. Data were recorded for germination,

emergence, ani subsequent seedling growth, the means of which are

presented graphically (Figs. 13, 14, 15, 16, 17, 18, and 19).

Statistical analysis of total germination data revealed that there

was no significant interaction between irradiation treatment and time of

irradiation, so the data could be examined on the basis of the means of

one factor as an average of all levels of the other factor. For emphasis

and clarity, the data are presented both ways (Fig. 13). Although there

were no significant differences among the irradiation dosages, the

obtained F value for time of irradiation was highly significant.

Irradiation after 14 days of stratification was not significantly

different from irradiation after either 7 or 21 days, but each of the

other times of irradiation was significantly different from each other.

Germination decreased as the time of gamma-radiation application was

delayed during stratification, which would suggest a sensitivity of seeds

to irradiation during the termination of dormancy.

The percentages of emergence for both factors are presented in

Figure 14. Because none of the germinated seeds of the 10, 13, or 20 kR

treatments emerged from the medium, those treatments were excluded from

statistical analysis. A significant number of embryonic shoots of those

treatments appeared to be viable and were not necrotic, but they failed

to elongate.

There was 63% emergence of the 0 kR seeds, and this was signifi-












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8 16 24 32


40


DAYS


Fig. 15. Rate of emergence of 'Okinawa' peach seedlings exposed to
gamma-radiation during stratification.







1.0
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IRRADIATION (kR)

Fig. 16. Average weights of 'Okinawa' peach seedlings exposed to
gamma-radiation during stratification. Letters in common within
each group indicate lack of significance at the .05 level.







.9
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IRRADIATION (kR)

Fig. 17. Average shoot weights of 'Okinawa' peach seedlings exposed
to gamma-radiation during stratification. Letters in common within
each group indicate lack of significance at the .05 level.







.7
00
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IRRADIATION (kR)

Fig. 18. Average root weights of 'Okinawa' peach seedlings exposed to
gamma-radiation during stratification. Letters in common within
each group indicate lack of significance at the .05 level.







18
0O
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IRRADIATION (kR)

Fig. 19. Average shoot lengths of 'Okinawa' peach seedlings exposed to
gamma-radiation during stratification. Letters in common within each
group indicate lack of significance at the .05 level.











cantly different from the 3 kR treatment. The 1 kR treatment was not

statistically different from either of the other 2 treatments. The

means of seedling emergence expressed by time of irradiation closely

parallelled the results obtained for total germination.

The effects of gamma-radiation were partially reflected in the rate

of seedling emergence, with irradiated seedlings being slower to emerge.

However, there was no apparent effect of irradiation of 1 kR on the rate

of emergence, but the seedlings which had received 3 kR were slower to

emerge than seedlings of either 1 kR or 0 kR (Fig. 15).

The data for average seedling weights are presented in Figure 16.

Obtained F values for time of irradiation and for interaction were

highly significant, but irradiation dosage was insignificant. Conse-

quently, it is possible to ignore the interaction and compare times of

irradiation as averages of all irradiation dosages. Irradiation after

stratification was completed (21 days) resulted in seedlings of signi-

ficantly higher weight than at any other time. Also, irradiation at the

outset of stratification (0 days) caused significantly lower average

seedling weight. For irradiation during stratification, there was no

discernible difference between irradiation after 7 or 14 days.

The data for average shoot and root weights are shown in Figure 17

and Figure 18, respectively. The statistical significance of the F

values was identical to that just presented for average seedling

weights. Moreover, the same differences were noted and the interpre-

tations were unchanged.

In Figure 19 are presented the data for average shoot lengths in

response to irradiation dosage and time. All F values were highly

significant. However, close inspection of the data revealed that the










presence of a significant interaction actually made no difference in the

statistical arrangement of the means, whether they were compared by

dosage or by time of irradiation. Thus, comparisons of the means of

either factor as averages of all levels of the other factor should be

valid. The 1 kR treatment was not statistically different from the 0 kR

treatment, but seedlings in both attained a greater height than those of

the 3 kR treatment. Also, irradiation at the end of stratification was

less damaging to shoot elongation than at other times, and irradiation

at the outset of stratification was most damaging. During stratifi-

cation, it made no difference in shoot length whether the seeds were

irradiated after 7 or 14 days.

One point of interest of the growth responses concerns the 0 kR

treatment. Because that treatment was a control, time of irradiation

should not have influenced its growth. However, there was a wide

divergence of response between the 0 kR seeds which were irradiated at

the outset and the end of stratification, and between each of those and

irradiation during the course of stratification (Figs. 16, 17, 18, and

19). Inasmuch as the seeds were all treated on the same date, the times

when seeds were placed in stratification were staggered so that the

exact number of days of stratification had been attained by the date of

irradiation. Thus, stratification was proceeding during a 6-wk period,

as the 21-day seeds had completed stratification before the 0-day seeds

had begun stratification.

Apparently, there was a malfunction of the refrigeration unit of

the growth chamber which resulted in temperature fluctuations. The

degree of fluctuation could not be ascertained, however. The mal-

function apparently occurred soon after the irradiation date, as the











seedlings of those treatments which still required stratification showed

effects similar to the physiological dwarfing which results from either

insufficient chilling or temperature reversal of chilling.

This was substantiated by comparison of these results with those

obtained for 2 separate controls (Table 7). One set of controls (I)

completed stratification 5 days after the 21-day seeds and 2 days before

the 14-day seeds. Another set of controls (II) was stratified 2 days

later than the 0-day seeds. The results for control I, which received

only 5 days of stratification after the date of irradiation, generally

ranged between the results for the 14-day and 21-day irradiation treat-

ments. The growth responses for control II, however, fell between

those for the 0-day and 7-day irradiation treatments.

Because the experiment was initiated in June, 1971, and the data

were not analyzed until September, these discrepancies were not dis-

covered until the supply of seeds became limited. Also, since germina-

tion per se was not influenced, a decision was made not to retest using

the same method.


Inhibitor Application to Stratifying Seeds

The objectives of this experiment were to determine if FUDR, ACTD,

and CHI would exert an influence on seed dormancy; to ascertain if there

was a time sensitivity of stratifying seeds to these inhibitors; and to

discern whether the inhibitors caused any adverse effects on the subse-

quent growth of the resultant seedlings. Germination, emergence, and

growth responses of the emerged seedlings as a result of treatment with

the inhibitors are summarized in Figures 20, 21, 22, 23, 24, 25, 26,

and 27.































Table 7. Comparisons of the growth responses of 2 separate controls with
those of the 0 kR treatment for each time of irradiation.


Time of irradiation, days Separate
after stratification began controls

Parameter 0 7 14 21 I II

Seedling weight (g) .41 .68 .72 1.46 .86 .66

Shoot weight (g) .30 .50 .48 .82 .62 .43

Root weight (g) .11 .28 .24 .64 .24 .23

Shoot length (cm) .74 10.45 11.40 16.41 14.23 8.91




XControl I completed stratification 5 days after the 21-day seeds and 2
days before the 14-day seeds. Control II completed stratification 2
days after the 0-day seeds.




































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Fig. 22. Rate of emergence of 'Okinawa' peach seedlings in response to
inhibitor application during stratification.


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For total germination, neither the F value for interaction between

The 2 factors nor for chemical treatments was statistically significant.

Consequently, times of injection were compared as averages of all

chemical treatments. The germination ranged from 50 to 67%, but the

only significant differences were between injection after 0 and 4 or

16 days of stratification (Fig. 20).

The F value for seedling emergence was significant for chemicals

and times of injection, but not for interaction between the 2 factors.

The results show the same relationships that were just presented for

germination (Fig. 21). The only clear differences among times of

injection were between injection after 0 days and after 4 or 8 days of

stratification. FUDR and CHI were statistically different from each

other, but neither was different from ACTD or the control.

The inhibitors caused no appreciable differences in the rate of

emergence, nor did the time of injection alter the rate. The overall

rate of emergence for this experiment is virtually identical to that

obtained for the irradiation experiment (Figs. 22 and 15, respectively).

The only difference is that the final emergence of seedlings in this

experiment was slightly lower quantitatively.

The average weights of the emerged seedlings requires a more

complex interpretation, due to the fact that the F values for treat-

ments, times of injection, and interaction were all statistically

significant. Consequently, the data were interpreted for chemical

treatments at each time of application separately. A significant

difference could not be detected with the Duncan test, so the less

precise LSD test of significance was employed.

Average seedling weights ranged from 0.505 g for ACTD injected










after 16 days to 1.066 g for FUDR injected after 12 days of stratifi-

cation (Fig. 23). There were no significant differences between treat-

ments at 8 or 20 days of the experiment. At the other times of injec-

tion, ACTD generally caused the lowest seedling weights. The only

exception was the ACTD treatment at 4 days, which resulted in seedlings

of the highest weight. Weights of seedlings of the control and ACTD

treatments injected at 0 days were clearly lower than either FUDR or

CHI. At 4 days, only the control seedlings were statistically lower

in weight than the FUDR and CHI treatments. At 12 days, ACTD seedlings

weighed less than those of all other treatments, but only seedlings of

the control weighed more at 16 days.

The same interpretations and relationships between treatments and

times of application just presented for seedling weights are appli-

cable to both average shoot weights and average root weights (Figs. 24

and 25, respectively).

Data for the average shoot lengths of the emerged seedlings had

the same statistical significance of the F values as the previously

presented growth parameters. However, there were no differences among

treatments injected at 2, 8, 12, or 20 days of stratification (Fig. 26).

At 0 days, CHI-treated seedlings were longer than seedlings of ACTD

only, whereas the ACTD treatment resulted in seedlings which were

shorter than all but the control at 2 days. Control seedlings were

significantly taller than both FUDR and ACTD at 16 days. With only

one exception, at 4 days, ACTD-treated seedlings were always shorter

than seedlings of the other treatments at all times of injection.

The effects of injection on the performance of the seedlings in

this experiment were large enough to be considered, so a non-injected











control was compared to the 0-day and 20-day injected controls for each

of the parameters (Fig. 27). The responses by seeds and seedlings of

the non-injected control were greater than the injected controls. The

20-day injected control was greater than or equal to the 0-day injected

control in each case. Statistically, there was no significance in the

differences for emergence, seedling weights, shoot weights, or root

weights. For total germination, the non-injected control was statisti-

cally different from the injected controls, which were not different

from each other. For shoot length, however, all 3 were statistically

different from each other.


RNA Changes During Stratification and Germination

Changes in RNA and RNA synthesis were followed through stratifi-

cation and into germination of seeds of 'Okinawa' peaches. ABA (3 x

10-3 M) and thiourea (10-1 M) were applied to seeds prior to the

beginning of stratification to determine what effects these chemicals

would cause in the RNA patterns during stratification. RNA synthesis

was determined by incorporation of radioactive 32PO4.

Total RNA fluctuated appreciably within and between treatments

until 12 or 16 days of stratification (Fig. 28). Substantial increases

occurred at the end of stratification and were continued through the

germination period. The lowest quantities of RNA in seeds treated with

ABA and thiourea occurred at 12 days and were 153 and 144 cm2/g, respec-

tively; whereas the lowest concentration in untreated seeds, 147 cm2/g,

was at the initial sampling immediately prior to the start of stratifi-

cation. At 16 and 20 days, seeds treated with ABA contained more RNA

than either the control or the thiourea treatment. After stratification,



















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however, the ranking in magnitude was thiourea, control, and ABA, in

descending order. It is noteworthy that the most consistent trend of

total RNA was that of the thiourea treatment, which showed an almost

linear decline to 12 days and a curvilinear increase through 24 days.

The summarized data for rRNA and sRNA, which were the 2 major RNA

fractions measured in these studies, exhibit changes that are quite

similar to those of total RNA (Fig. 29). Because there was much more

rRNA than sRNA, it would be expected that changes in rRNA would be

reflected to a greater degree in total RNA, as was the case for the

decrease in total RNA between 20 and 22 days for the ABA-treated seeds.

Although sRNA did increase in all treatments after 12 days of stratifi-

cation, slight decreases occurred between 16 and 20 days for ABA and

control seeds.

The fluctuations of total RNA and rRNA during stratification are

further emphasized in comparisons of the 25S and 18S subunits of rRNA

(Fig. 30). Increases in the relative amounts of each subunit began

about 16 days after stratification was begun, and continued until the

end of the experiment. Although the increases in the 18S subunit were

not so great as that for the 25S subunit, it is possible that, in order

to maintain a metabolically active system of stable RNA levels, this

shift occurs.

Because the ratios of the major RNA fractions have been used as

indicators of the status of RNA metabolism and growth activity, the

ratios were considered (Fig. 31). For control seeds, the 25S/18S ratios

deviated from 1.5 at 22 days to 2.2 at 24 days. Inasmuch as ratios

deviating from 1.8-2.0 indicate a lack of stability of RNA levels, there

was instability in the system at 4, 8, and 12 days during stratification,































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and at 22 days--2 days after the end of stratification. The ratios for

ABA-treated seeds gradually increased from 1.4 at 0 days to 2.3 at 20

days, and fluctuated during the early stages of germination. For the

thiourea treatment, ratios ranged from an initial low of 1.5 to a high

of 2.6 at 12 days, but fluctuated near 1.8-2.0 through the end of the

experiment.

The rRNA/sRNA ratios for all treatments fluctuated between 2.0 and

2.5 through most of stratification, with few exceptions. During early

germination, the ratios ranged from 2.5 to 3.0. This would imply that

greater synthesis of rRNA or more catabolism of sRNA was occurring after

stratification.

The incorporation of radioactivity into RNA fractions is compiled

in Table 8. The counts per minute for the 25S and 18S subunits of rRNA

represent the sums of the 3 central 2-mm sections of gel which correspond

to the respective ultraviolet absorption peaks on the electrophoreto-

grams (Fig. 32). The counts for sRNA represent the sums of 5 central

2-mm sections of gel. The rRNA data are simply the sums of the activity

of its subunits. Some spillover of counts occurred at 22, 24, and 28

days due to the increased amounts of RNA, but the spillover was not

considered in the interest of consistency. In addition, counts could

not be determined for the rRNA of the ABA treatment at 22 days due to

destruction of rRNA which resulted from contamination of the extract.

The ratios of synthesis of the 25S and 18S subunits indicate that

RNA metabolism for all treatments was unstable during stratification,

although the ratios did increase at the last day of stratification. As

compared to the control, ABA did not inhibit synthesis of either rRNA

or sRNA during stratification. Actually, a slight enhancement of RNA



















Table 8. Incorporation of radioactive 32P04 into RNA of 'Okinawa' peach
seeds during stratification and germination.



cpm/RNA fraction
Time 25S/18S
(days) 25S 18S rRNA sRNA Ratio

--------------------------------CONTROL ----------------------


274


529
354
505
3081
3428
3015


288
186
234
970
1070
1130


----------------------------------ABA----------------------------------


733
789
842

2417
4752


1541
2860


211
429
1035
1077
797
1282


---------------------THIOUREA--------------------------------


411
141
703
1757
2482
1891


342
143
508
993
1360
1034


753
284
1211
2750
3842
2925


291
194
685
764
1405
1190


XLost rRNA sample.









2.5 10






2.0 8






1.5 6

0
0
o -


C-

1.0 4






0.5 | 2






0 0
0 0.5 I


Rf


Fig. 32. Typical electrophoretogram-histogram of polymeric RNA
fractions of 'Okinawa' peach seeds, separated on 2.4% poly-
acrylamide gels and scanned at 260 nm after electrophoresis for
75 min at 40 v. Each bar of the histogram represents the radio-
activity of the corresponding 2-mm section of gel.







79



synthesis was noted. Inhibition of RNA synthesis in relation to the

control began after germination had begun. Thiouren stimulated both

rRNA and sRNA synthesis to a slight degree on the last day of st-tttiti-

cation and after 4 days of germination, but results at the other sampling

times were consistent with those of the controls.

One of the primary objectives for the 32PO4 treatments wais to

indicate the presence of mRNA, which could not be determined by the

techniques of gel electrophoresis and spectroscopy. It is of major

interest, therefore, that none of the gels contained radioactivity in

the region between rRNA and sRNA, which is where mRNA is expected to

occur. Apparently, no mRNA synthesis occurred during the termination

of peach seed dormancy and the early stages of germination.
















DISCUSSION


Changes in total RNA and its components in 'Early Amber' peach

seeds were followed from cytokinesis to commercial fruit maturity in

order to determine the time during embryo development that major changes

in RNA concentrations occur. Quantitative changes were shown to occur,

notably a large increase in total RNA on May 11, but the fluctuations

could not be related to the size of the embryos. However, the greatest

increase occurred at the time of least increase in embryo length. Also,

the greatest increase in RNA was associated with the highest fresh

weight of seeds. Such fluctuations have been observed in other seeds,

with peaks of RNA occurring prior to maximum embryo size and subse-

quently decreasing during maturation (57, 58, 61).

Inhibitors of the nucleic acid-protein synthesizing system were

used to determine the times at which the seed would be susceptible to

inhibition and to ascertain the effects on inhibition on subsequent

germination of the seeds. FUDR, being an inhibitor of DNA synthesis

(35, 74, 83), was expected to alter the levels of RNA due to the depen-

dence of RNA synthesis on DNA. At the first 2 injections, all samples

showed either no change or decreased levels of RNA (Fig. 5). However,

the April 20 and April 27 injections of FUDR generally showed increased

RNA levels, and the May 4 injection caused decreased RNA levels.

ACTD is a highly specific inhibitor of RNA synthesis in that it

binds to DNA, requiring guanine in a helical structure, to suppress the

formation of all cellular RNA fractions (46, 67, 94). Its effects are

80










variable, however, as a reflection of different plant tissues' suscepti-

bility to concentration (21, 65, 69, 78) and due to the fact that mas-

sive inhibition can occur without concommitant inhibition of growth

processes requiring RNA (66, 68, 70). The greatest decrease of RNA as

a result of ACTD treatment was shown to occur at the time of greatest

fruit abscission, so it is probable that a portion of the sample con-

sisted of senescing or dead embryos. The greatest RNA increases occurred

at the fifth sampling of each of the first 3 injections and at the last

sampling of the April 20 injection.

CHI suppresses protein synthesis by preventing the transfer of

amino acid from the aminoacyl-tRNA complex to protein (46). It is

effective at low concentrations and it is fast-acting (67). CHI was

the most effective of the inhibitors in this experiment, causing the

most consistent decreases and the highest single increase in RNA (Fig.

5). As with the ACTD treatment, the only increases in RNA during the

first 2 injections occurred at the fifth sampling on May 6, and the

greatest decrease occurred in the May 1 sample of the first injection,

the latter of which was probably due to fruit abscission.

The differences and inconsistencies in RNA levels may be attrib-

utable to several factors. Among these are: small sample size, single

concentration and quantity of each inhibitor, increasing fruit size and

fresh weight variations of the seeds, time of injection in relation to

embryo growth, method of application, and unknown inherent biochemical

reactions within the developing seeds. Fractionation technique was not

considered a problem, as the RNA fractions described on the electro-

phoretograms in this and the other experiment involving RNA determin-

ations are in agreement with the RNA fractions obtained from green and










non-green tissues of other plants by the same and different extraction

techniques (56, 76, 77, 113, 114).

The observed RNA increases as the result of inhibitor application

may have been only apparent increases. Inasmuch as the growth of the

embryo depends upon the digestion of nucellus and endosperm and the

utilization of materials contained therein, inhibition of a part of the

nucleic acid-protein synthesizing mechanism could lead to altered RNA

degradation in the supportive tissues--which would be reflected as an

apparent increase in RNA. For example, if the synthesis of ribonuclease

were inhibited, an accumulation of RNA would result. Inhibition could

occur at any level between DNA and protein synthesis.

The 25S/18S ratios of the rRNA subunits are considered indicative

of the metabolism of RNA (56, 113). A ratio of 1.8-2.0 is indicative of

metabolic stability of RNA (114) and is often characteristic of the

resting state of plant tissue (77). Ratios outside this range would

indicate greater synthesis and/or degradation of one of the subunits of

rRNA. Although there appeared to be 3 distinct levels of RNA activity

during the developmental period studied, they could not be correlated

with growth activity, embryo size, or seed weight. The ratios approached

the stable range as the embryos attained full size.

The ratios within the inhibitor treatments generally increased

toward the end of the season and as injection neared the end of the

season. Injections early in the developmental period usually resulted

in lower ratios than in the controls, but injections after April 20

caused much higher ratios. Consequently, preferentially reduced syn-

thesis, probably of the 25S subunit, was indicated at the early stages,

and decreased degradation was indicated at the last 3 injections.










The ability of the embryos to grow in vitro during development was

apparently a function of size and stage of development, and was somewhat

reduced by the inhibitors (Table 3). The best growth and development

was achieved by embryos taken on May 11 and May 16. Final size of the

embryos was not attained until May 21. However, some growth and devel-

opment were noted throughout, so the embryos showed no signs of dormancy

during the developmental period studied. The seed coats, however, which

are involved in peach seed dormancy, were not present in culture.

Even at final size, the embryos were not mature. This is partly

substantiated by the fact that the embryos of short-cycle peaches (those

requiring only about 70-75 days from bloom to maturity) generally do not

mature by the time that the fruit is mature. Moreover, fruit of the

controls and treatments which were not sampled remained on the trees for

several days beyond fruit maturity, at which time they were collected

and the seeds were after-ripened. None of the seeds were viable, as

evidenced by the failure of any to germinate.

Haber ct al. (48) observed that gamma-radiation stopped DNA syn-

thesis and thus prevented cell division of lettuce seeds, but that GA

caused the irradiated seeds to germinate. Lang and Nitsan (74) sug-

gested that DNA synthesis is essential for the gibberellin-regulated

elongation of plant cells. Primarily, seed germination is initially

the result of cell elongation without concurrent cell division.

The data presented for dry seed irradiation showed that up to 200

kR permitted good peach seed germination (Tables 5 and 6). However,

subsequent seedling growth was inhibited. At dosages above 200 kR, no

germination occurred, probably because the embryos were completely

killed. These results may be contrasted to the work referred to above,










which involved application of 1300 kR gamma-radiation to lettuce seeds.

Although no cytological examinations were made, cell division and

DNA synthesis were assumed to be inhibited at dosages above 5 kR.

Primarily, irradiated seedlings were rather lacking in growth. Above

50 kR, no epicotyl growth or root proliferation was noted, even after an

additional period of observation. At dosages below 50 kR, the seedlings

tended to form terminal rosettes, and dieback of the apices occurred.

Moreover, the height of the irradiated seedlings did not equal that of

controls, and the number of leaves, while fewer than the controls, were

essentially the same or less than the number of embryonic leaves shown

to be present in peach embryos (80).

Fairly extensive root systems developed on seedlings which received

less than 30 kR. This indicates that cell division in the roots was not

inhibited at dosages below 30 kR. Too, dosages above 50 kR did not

inhibit radicle protrusion, even though the shoot was killed. Thus, it

would appear that the root portion of the embryo is less sensitive to

irradiation damage than is the shoot. However, root growth beyond mere

protrusion was inhibited above 50 kR and root elongation was inhibited

at dosages above 200 kR.

Data for irradiation of seeds at different stages of stratification

were presented for which it was shown that germination of imbibed seeds

was not affected by dosages of 20 kR, but that the time of irradiation

altered germination (Fig. 13). Germination for all dosages decreased

linearly from irradiation at the start of stratification to irradiation

at the end of stratification.

Emergence data revealed a considerable difference in irradiation

dosage response, inasmuch as at dosages above 3 kR, seedlings failed to











emerge due to dead epicotyls (Fig. 14). It is apparent that imbibed

seeds are more susceptible to irradiation damage than dry seeds.

However, of the seedlings which did emerge, dosage had only a slight

effect on emergence rate (Fig. 15).

Data for subsequent growth of the emerged seedlings indicated

that irradiation at the end of stratification was best. However, the

responses of the 0 kR seeds, which were controls, generally followed the

responses of the other treatments. Because those seeds were controls,

however, time of irradiation should have caused no difference in

growth. However, considerable differences were noted between the seeds

irradiated at 0 and 21 days. Attempts to understand this led to the

discovery of an apparent malfunction of the refrigeration unit during

stratification, so the growth data were invalidated.

FUDR, ACTD, and CHI were applied to stratifying seeds to determine

if their effects on nucleic acid and protein synthesis would be reflected

in the termination of dormancy and subsequent growth. However, the

concentrations tested did not appreciably affect either germination,

emergence, or emergence rate. Some differences were noted in final

seedling weight and height, but only at certain times during stratifi-

cation. However, injection alone was responsible for some differences

in response (Fig. 27). This could be attributed to the fact that

puncturing the seeds with the microsyringe needle facilitated the entry

of microorganisms which may have reduced the responses somewhat.

Thus, it would appear that these inhibitors are without effect on

the termination of dormancy of peach seeds under the conditions of these

experiments. However, it should be emphasized that different concen-

trations and quantities of inhibitors may elicit quite different










responses. This is particularly true for the method of application,

inasmuch as all 3 inhibitors are also antibiotics. The invasion of the

seeds by microorganisms through the injection puncture may have caused

utilization of the inhibitors against them. Too, active RNA synthesis

is necessary for cell elongation (4, 45, 65, 67), so the fact that

germination occurred would indicate that the inhibitors were not stopping

RNA synthesis. As was previously discussed, however, massive inhibition

of RNA synthesis can occur without concommitant inhibition of the RNA-

dependent growth processes.

Considering that RNA metabolism would be necessary for the processes

occurring during stratification which lead to the termination of seed

dormancy, data were presented for changes in net RNA and for RNA syn-

thesis during stratification and the early stages of germination. The

concentration of RNA fluctuated somewhat until 8 days, gradually declined

to 16 days, and increased linearly through the end of the experiment.

The same general pattern was evident for the components of total RNA and

for seeds treated with ABA and thiourea. It is interesting that

'Okinawa' seeds require less than 400 hr (15-16 days) of chilling to

break dormancy (9). Consequently, the increase in total RNA after 16

days would appear to indicate that dormancy had been broken and that RNA

synthesis was initiated for the germination and growth processes.

The 25S/18S ratios during stratification gradually decreased from

an initial value of about 2.0 (Fig. 31). It is interesting that the

value was 2.0, as it was previously mentioned that a ratio near 2.0 is

indicative of RNA stability and is characteristic of resting tissues.

At 16 days, the ratio was again near 2.0, which indicates that dormancy

was terminated and that seed germination and embryo development were











held in check by the low temperature. The change from initial stability

and subsequent return to normal at 16 days indicate the involvement of

RNA in the processes which terminate dormancy during chilling.

Limited synthesis of all fractions of RNA occurred during stratifi-

cation (Table 8). It is likely that synthesis was held in check to some

degree by low temperature, though other factors may have been involved.

One other factor is that RNA synthesis per se is not necessary to

terminate dormancy. It is noteworthy that ABA enhanced synthesis during

stratification, as it has been reported to inhibit RNA synthesis in

seeds (73, 119, 123). It is also noteworthy that greater rRNA synthesis

occurred, and that the 25S/18S ratios of synthesis during stratification

were near unity. This latter fact reveals that changes in the ratios of

rRNA represent greater degradation of only 1 subunit, probably 25S.

No radioactivity was present in the region of the gel corresponding

to mRNA during stratification or early germination. Consequently, it

would appear that mRNA synthesis is not required for the termination of

seed dormancy or for the early stages of germination of peach seeds.

If synthesis is not required, it follows that there must be sufficient

mRNA present, i.e., long-lived mRNA. This is in agreement with the

results showing the existence of long-lived or stable mRNA (17, 31, 128).

Developing 'Early Amber' seeds contained more total RNA than did

mature 'Okinawa' seeds. However, 'Okinawa' contained a greater portion

of rRNA, but 'Early Amber' contained more than twice as much sRNA per

gram.


1

















SUMMARY AND CONCLUSIONS


Quantitative changes in polymeric RNA fractions occurred in 'Early

Amber' peach seeds during the developmental period from cytokinesis to

fruit maturity. Fluctuations which occurred could not be related to

changes in embryo length or seed weight. FUDR, ACTD, and CHI applied

at intervals during development altered the levels of RNA, but the

changes were not consistent. CHI caused the greatest changes in RNA

levels, and April 20 was the most effective time to apply the chemicals.

The inhibitors reduced the growth of excised embryos cultured in vitro.

Peach seed germination was shown to be independent of DNA synthesis

and concurrent cell division under the conditions described. Although

cytological examinations of irradiated seeds were not performed, it was

inferred that cell division and DNA synthesis were inhibited on the

basis of subsequent growth. Roots were less sensitive to irradiation

damage than shoots, and imbibed seeds were more sensitive than dry

seeds. There was a negative linear relationship between germination

and irradiation prior to, during, and after stratification. The results

of time of irradiation on subsequent seedling growth were inconclusive

due to an equipment malfunction.

Treatment with FUDR, ACTD, and CHI at various times during strati-

fication slightly affected seed germination and subsequent growth.

There were statistical differences in germination, seedling weight, and

shoot height. Due to the effect of injection, the presence of micro-

organisms, and the occurrence of a highly significant statistical inter-











action between chemical treatment and time of application, the results

were considered inconsequential.

RNA levels of 'Okinawa' seeds declined slightly during stratifi-

cation. Both ABA and thiourea applied prior to stratification slightly

enhanced RNA synthesis. There was no synthesis of mRNA during either

stratification or germination.

Although the role of nucleic acid metabolism in peach seed dormancy

was not elucidated, several pertinent conclusions were made. No

definite relationship between nucleic acid levels and dormancy was

shown. The termination of peach seed dormancy apparently does not

require DNA synthesis or cell division, on the basis of seed irradiation

and FUDR treatment. RNA synthesis does not appear to be required to

terminate dormancy, as shown by ACTD treatment, seed irradiation, and

changes in RNA levels during chilling.

Protein synthesis may not be required, in light of CHI treatment

and the other treatments described in these studies.

There is apparently a stable or long-lived mRNA in peach seeds.

















APPENDIX: BUD-BREAK STUDY


Based on the contention that the mechanism of dormancy is quite

similar in both seeds and buds of the same plant, dormant 'Okinawa'

potted seedlings which had not been subjected to chilling temperatures

were treated with various thiol and related compounds, the effects of

which were known on seed dormancy. The treatments included thiourea,

urea, thioacetamide, acetamide, and l-allyl-2-thiourea at concentra-

tions of 101, 10-2, and 10-3 M. In addition, 4 x 10-1 M l-allyl-2-

thiourea was applied. Plants used for controls were similarly treated

with distilled water. Each solution contained 0.01% Triton X-100.

Five replications of 2 plants each were used.

Non-branched plants of 40-45 cm, each having about 20 axillary buds,

were selected for treatment. After the removal of the leaves, the plants

were inverted and immersed to the soil line for 10 sec in the appropriate

solution. The numbers of axillary buds having broken under greenhouse

conditions were cumulatively recorded at 10, 17, and 24 days after

treatment (Table 9).

Only 10-1 M thiourea and 10-1 M l-allyl-2-thiourea were statisti-

cally superior to the water control in the stimulation of bud-break.

The stimulation by these 2 treatments was evident early in the experi-

ment, inasmuch as little change occurred after 10 days. Neither urea,

acetamide, nor thioacetamide at the concentrations tested caused any

change from the control or from each other during the experiment.

Both 101 and 10-2 M thiourea were more effective than 10-3 M, but




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