Gibberellic acid

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Material Information

Title:
Gibberellic acid translocation, metabolism and effects on peel quality of 'Marsh' grapefruit (Citrus paradisi Macf.)
Uncontrolled:
'Marsh' grapefruit
Citrus paradisi
Physical Description:
vii, 88 leaves : ill. ; 28 cm.
Language:
English
Creator:
Ferguson, Louise, 1947-
Publication Date:

Subjects

Subjects / Keywords:
Grapefruit   ( lcsh )
Gibberellic acid   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Includes bibliographical references (leaves 79-87).
Statement of Responsibility:
by Louise Ferguson.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000472006
notis - ACN6883
oclc - 11749100
System ID:
AA00003412:00001

Full Text








GIBBERELLIC ACID: TRANSLOCATION, METABOLISM AND
EFFECTS ON PEEL QUALITY OF
'MARSH' GRAPEFRUIT (Citrus paradisi MACF.)











By

Louise Ferguson


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



UNIVERSITY OF FLORIDA


1984















ACKNOWLEDGMENTS



I wish to thank Drs. F. S. Davies, T. A. Wheaton, and M. A. Is-

mail for support, counsel, and patience.

I wish to thank Drs. R. H. Biggs and R. C. Smith for their

cooperation.

I wish to thank the staff and faculty of the Lake Alfred Citrus

Research and Experiment Center, who made my research experience a

memorable one.

I offer special thanks to Jane Lee Stanley Van Clief for constant

encouragement, support, and friendship.















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS . ii

ABSTRACT . vi

CHAPTER I: GIBBERELLIC ACID IN GRAPEFRUIT 1

CHAPTER II: GROWTH REGULATOR AND LOW-VOLUME
IRRIGATION EFFECTS ON GRAPEFRUIT QUALITY
AND FRUIT DROP . 3

Introduction . 3
Literature Review............. 3
Effects of GA3 and 2,4-D on External Quality of
Grapefruit....... 3
Effects of GA3 and 2,4-D on Internal Quality of
Grapefruit . 4
Effects of GA3 and 2,4-D on Fruit Drop 5
Effects of Decreased Irrigation on the Efficacy of
GA3 and 2,4-D 5
Materials and Methods . 6
Results and Discussion . 8
Effects of SMC on L 8
Effects of Irrigation and GA3 and 2,4-D on Drop and
External Fruit Quality . 8
Effects of Decreased Irrigation and GA3 and 2,4-D
on Internal Fruit Quality . 11
Effects of GA3 and 2,4-D on Postfreeze Fruit Drop 14
Conclusions . 14

CHAPTER III: PREHARVEST AND POSTHARVEST GIBBERELLIC
ACID AND 2,4-DICHLOROPHENOXYACETIC ACID
APPLICATIONS FOR INCREASING STORAGE
LIFE OF GRAPEFRUIT . 15

Introduction 15
Literature Review . .16
Effects of Preharvest GA3 and 2,4-D Sprays on
External Quality of Postharvest Stored Grapefruit 16
Effects of Preharvest GA3 and 2,4-D on Internal
Quality of Postharvest Stored Grapefruit 17
Effects of Postharvest GA3 and 2,4-D Treatments on
External Quality of Postharvest Stored Grapefruit 17








Page


Effects of Postharvest GA3 and 2,4-D Treatments on
Internal Quality of Postharvest Stored Grapefruit
Materials and Methods
Results and Discussion
Effects on Color .
Effects on Peel Puncture Resistance
Effects on Decay
Effects on Internal Quality
Conclusions .

CHAPTER IV: UPTAKE, TRANSLOCATION, PERSISTENCE, AND
METABOLISM OF GIBBERELLIC ACID IN
GRAPEFRUIT

Literature Review .......
Uptake, Translocation, and Persistence of 14C-GA3
Metabolism of 14C-GA3 ...........
Materials and Methods
Application of 14C-GA3 to Attached Fruit and Leaves
Application of 14C-GA3 to Detached Fruit
Extraction of Radioactivity
High-Performance Liquid Chromatography of Leaf,
Albedo, and Flavedo Extracts
B-D-glucosidase Hydrolysis of Radioactive Fractions
N-butanol Partition of Radioactive Fractions .
Results and Discussion
Uptake, Translocation, and Persistence of Peel-
Applied 14C-GA3 .
Uptake, Translocation, and Persistence of Leaf-
Applied 14C-GA3 .
Separation of Extracted Radioactivity by High-
Performance Liquid Chromatography
B-D-glucosidase Hydrolysis of Radioactive Fractions
N-butanol of Radioactive Fractions .......
Conclusions . .

CHAPTER V: CONCLUSIONS ............


APPENDIX: GROWTH REGULATOR AND NUTRITIONAL
EFFECTS ON GRAPEFRUIT COLOR AND
STORAGE QUALITY .

Literature Review .
Effects of Nitrogen and Gibberellins on External
Grapefruit Quality .
Effects of Nitrogen and Gibberellins on Internal
Grapefruit Quality .
Materials and Methods .
Results and Discussion .
Peel Color of Tree-Stored Fruit......
Internal Quality of Tree-Stored Fruit .
Peel Color of Cold-Storage Fruit .


65

65

S 65

S66
S66
S67
S 67
S 70
72







Page

Internal Quality of Cold-Storage Fruit 72
Decay During Cold Storage . 72
Conclusions . 78

LITERATURE CITED . 79

BIOGRAPHICAL SKETCH . 88













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



GIBBERELLIC ACID: TRANSLOCATION, METABOLISM AND
EFFECTS ON PEEL QUALITY OF
'MARSH' GRAPEFRUIT (Citrus paradise MACF.)

By

Louise Ferguson

August 1984

Chairman: Dr. F. S. Davies
Major Department: Horticultural Science

Gibberellic acid maintains 'Marsh' grapefruit (Citrus paradise

Macf.) peel quality by delaying senescent peel color development and

loss of peel firmness. Combined with 2,4-dichlorophenoxyacetic acid

(2,4-D), which prevents preharvest fruit drop, the two extend the

grapefruit harvest season. It was not known if decreased irrigation

affects this treatment. It also was not known if postharvest application

of this treatment would produce the same effects. In addition, GA3

uptake, translocation and metabolism had not been examined. This

dissertation was undertaken to answer these questions.

'Marsh' grapefruit on rough lemon (Citrus jambhiri Lush.) root-

stock received irrigated and unirrigated treatments. Half of each

irrigation treatment received a GA3 and 2,4-D spray at colorbreak.

Preharvest sprays of GA3 and 2,4-D extended the grapefruit har-

vest season by increasing fruit removal and rind puncture force, delay-

ing development of senescent color, and decreasing late-season and







postfreeze fruit drop. Although soil moisture content in the top 0.9 m

of unirrigated blocks was reduced by approximately 40%, leaf water

potentials of these trees and performance of GA3 and 2,4-D were unaf-

fected.

Experiments were carried out comparing the effects of a preharvest

GA3 and 2,4-D spray, a postharvest GA3 and 2,4-D dip, and combined

spray and dip treatments on peel quality and decay during storage.

Fruit harvested in January, March, and May were stored for 12 weeks

at 15.50C. The GA3 and 2,4-D treated fruit had less senescent color

development, loss of puncture strength, and decay than controls. The

three treatments were equally effective for January and March harvests,

while preharvest and combined treatments were more effective than a

postharvest treatment in May.
14
Applied C-GA3 was absorbed by peels and leaves of attached

fruit within 1 hour, translocated from leaves to peel and the reverse

within 4 to 8 hours, and persisted in leaves and peel in measurable

amounts for 8 weeks. Accumulation was higher in peels than in leaves

no matter where the 14C-GA3 was applied. Approximately half the
14
absorbed 1C-GA3 remained in the applied form and approximately half

was converted to a water-soluble form within 96 hours. Recovery was
14
less, but metabolism was similar, when 1C-GA3 was applied to detached

fruit.














CHAPTER I

GIBBERELLIC ACID IN GRAPEFRUIT



Gibberellic acid (GA3) effectively delays normal peel senescence of

citrus (16,37,59,63,70,71). The combination of GA3 with 2,4-dichloro-

phenoxyacetic acid (2,4-D), which delays preharvest fruit drop (46,99)

and deterioration in storage (89), provides a means of extending the

grapefruit harvest season (2,25,35) and postharvest storage life (1).

Inconsistent treatment effects may result from a number of envi-

ronmental factors, including temperature and tree water status. Insuf-

ficient irrigation (35), or heavy precipitation late in the harvest season

(F.S. Davies and M.A. Ismail, personal communication, 1979) has been

observed to decrease the efficacy of GA3 and 2,4-D in preventing fruit

drop and maintaining peel quality. The experimental label for

Pro-Gibb@, a commercial GA3 formulation, currently advises that results

may vary depending on environmental conditions. The first objective of

this dissertation was to determine if a reduction in soil moisture content

following GA3 and 2,4-D application reduced the efficacy of these

materials in extending the harvest season.

Preharvest sprays of GA3 and 2,4-D decrease storage losses of

grapefruit (1). Postharvest dips of GA3 have produced similar results

with 'Shamouti' oranges (37). Postharvest 2,4-D dips also maintain peel

quality and decrease decay of grapefruit in storage (89,97). Combining

GA3 and 2,4-D preharvest or postharvest does not alter their efficacy
3j








(2,25,35). The cost of application is lower postharvest than pre-

harvest; however, GA3 and 2,4-D are not registered for postharvest

use on grapefruit. The second objective of this dissertation was to

determine whether a preharvest or postharvest application of GA3 and

2,4-D was better for maintaining grapefruit quality in storage.
14
Applications of 1C-GA3 to 'Shamouti' orange peel indicate that it

is taken up by flavedo, persists there in diminishing amounts for 100

days, and appears to remain in the applied form (38,39). Beyond this,
14
little else is known about the fate of 1C-GA3 applied to citrus peel.

The third objective of this dissertation was to characterize uptake,

translocation, persistence, and metabolism of 14C-GA3 by grapefruit

leaves and fruit.

Determining the first two objectives (the effects of decreased soil

moisture content on the efficacy of preharvest applications of GA3 and

2,4-D in extending the harvest season, and the better time to apply

GA3 and 2,4-D for extending storage life) will enable more economical
14
use of GA3. Determination of the third objective, the fate of 1C-GA3

applied to grapefruit peel and leaves, will provide some fundamental

physiological evidence for the observed effects of GA3 on grapefruit

peel quality.












CHAPTER II

GROWTH REGULATOR AND LOW-VOLUME IRRIGATION EFFECTS ON
GRAPEFRUIT QUALITY AND FRUIT DROP



Introduction

The grapefruit harvest season has been extended in South Africa

(35), Central Florida (2), and Australia (25) by applying dilute sprays

of GA3 and 2,4-D at colorbreak. The GA3 delayed peel color develop-

ment (2,25,35,59) and loss of peel firmness (2,25), while 2,4-D

decreased the characteristic late-season fruit drop (2,25,35,46). The

combination maintained acceptable peel quality as late as June under

Florida conditions (2). However, not all GA3 and 2,4-D sprays have

produced consistent results. These inconsistent treatment effects may

be the result of environmental factors, including temperature and tree

water status.



Literature Review

Effects of GA3 and 2,4-D on External Quality of Grapefruit

The external quality of grapefruit is determined by color, firm-

ness, and presence of blemishes. As peel senesces, it progresses from

pale yellow to orange-yellow due to chlorophyll loss (18), is easily

punctured and deformed due to loss of firmness, and is more suscepti-

ble to decay pathogens. Sprays of GA3 and 2,4-D applied at the time

of colorbreak, mid-November to December, delay orange-yellow color

development (2,18,25,35,59,61), probably by retarding chloroplast to







chromoplast conversion in citrus peel (18,43,105). Sprays of GA3

applied when grapefruit are less than 10 mm in size and at higher

concentrations than the usual 10-25 ppm GA3 (17) cause regreening of

grapefruit, as do nitrogen nutritional treatments (39). However, re-

greening is usually not a problem with grapefruit (18). Sprays of GA3

and 2,4-D also delay loss of peel firmness (2,25). Applied alone, GA3

produces a more compact albedo (16,18,69,71) and decreases peel blem-

ishes (16,71). Alone, 2,4-D decreases corky spot of grapefruit if

applied within 2 months of anthesis (40). These effects are enhanced

by inclusion of potassium or ammonium nitrate in preharvest sprays

(4,28,71), possibly through enhancement of endogenous GA3 production

(69). (See Appendix I.)



Effects of GA3 and 2,4-D on Internal Quality of Grapefruit

Preharvest colorbreak sprays of GA3 and 2,4-D have not been

demonstrated to produce consistent, significant changes in internal

quality of grapefruit. Grapefruit internal quality is determined by total

soluble solids (TSS), percentage of acid, TSS/acid ratio, and percent-

age of juice. Reports of the effects of colorbreak sprays of GA3 and

2,4-D on these indices are conflicting and inconsistent (2,16,17,25,35,

59,99,100). The most frequent reports are of a slight increase in the

percentage of juice and a slight delay in the decrease in the percentage

of acid late in the season (15). The general consensus is that differ-

ences, if created, are slight. Another index of grapefruit internal

quality is seed sprouting, which occurs after March and renders fruit

unmarketable. Ali Dinar et al. (2) reported that colorbreak sprays of

GA3, 2,4-D, and the combination decreased seed sprouting. However,







Albrigo et al. (1) were unable to corroborate this finding. A last

index of internal grapefruit quality is granulation, which occurs late in

the harvest season and increases with time. There are no reports that

GA3 and 2,4-D decrease this problem.



Effects of GA3 and 2,4-D on Fruit Drop

One of the first uses of growth regulators was to prevent pre-

harvest fruit drop of grapefruit (100). Numerous reports indicate that

colorbreak sprays of 2,4-D at 8-20 ppm delayed late-season grapefruit

drop (2,16,25,32,35,69). In contrast, Kokkalos (59) did not reduce

late-season fruit drop in 2 of 4 years by using 2,4-D. Explant work

has shown that 2,4-D delays abscission by delaying the rise of cellu-

lases and polygalacturonases in the abscission zone, thereby delaying

separation of calyx from fruit (41). Goldschmidt (69) suggested that

2,4-D is an antagonist of ethylene. Alone, GA3 has never been demon-

strated to decrease fruit drop.



Effects of Decreased Irrigation
on the Efficacy of GA3 and 2,4-D

Gilfillan et al. (35) observed that insufficient irrigation late in the

harvest season decreased the efficacy of GA3 and 2,4-D in preventing

fruit drop and maintaining peel quality. F.S. Davies and M.A. Ismail

(personal communication, 1979) observed no effect of GA3 and 2,4-D on

peel firmness during a May and June when precipitation was higher

than average. Kokkalos (53) reported GA3 and 2,4-D to be ineffective

in preventing fruit drop in Cyprus. The label for Pro-Gibb a com-

mercial GA3 product, currently advises, "Results may vary .

depending on environmental conditions."







The objective of this experiment was to determine if a reduction in

soil moisture following GA3 and 2,4-D application reduces the efficacy of

these regulators in extending the harvest season of 'Marsh' grapefruit

under Florida conditions.



Materials and Methods

Two 210-tree blocks of 35- to 40-year-old 'Marsh' grapefruit

(Citrus paradise Macf.) trees on rough lemon (Citrus jambhiri Lush.)

rootstock from a grove located near Lake Alfred, Florida, were used

sequentially during the 1980-81 and 1981-82 seasons. Trees were

hedged north-south, spacing was 4.5x9.1 m, and soil type was Astatula

fine sand. Microsprinklers delivering 80 liters per hour were located

between alternate trees. Fertilizer, pesticide, and irrigation practices

were consistent among treatments and typical of groves in Central

Florida.

Forty completely randomized single-tree plots and 20 completely

randomized two-tree plots were used during 1980-81 and 1981-82,

respectively. Microsprinklers of 20 of the single-tree plots and 10 of

the two-tree plots as well as those of immediately surrounding trees

were capped 1 week prior to GA3 and 2,4-D application. Half the

number of irrigated and unirrigated trees were sprayed after colorbreak

on November 12, 1980, and December 14, 1981, with 50 liters of an

aqueous solution of 20 mg per liter each GA3 and 2,4-D and 0.025%

Triton X-77. The four treatments, irrigated sprayed and unsprayed

and unirrigated sprayed and unsprayed, were analyzed as a 2x2

factorial experiment.







The soil moisture content (SMC) was determined using a Troxler

neutron scattering device (Pacheco, CA). Three measurements were

made monthly at 0.45- and 0.90-m depths for one location per tree and

averaged to yield the SMC of the top 0.90 m of soil. Leaf water poten-

tials (t)L) of six randomly selected leaves from equally spaced canopy

positions of each tree were determined using a pressure chamber (91).

Leaves remained on damp paper towels in sealed plastic bags in an ice

chest until immediately before measurement. Leaf samples were collected

at 4 AM and 11 AM on the same day that SMC measurements were

taken. Sampling times were selected to reflect periods of minimum and

maximum water stress, respectively (102).

Fruit drop was determined monthly by counting and removing all

fruit within the dripline. Twenty randomly selected fruit per tree were

collected monthly to determine fruit removal force (FRF), peel color,

rind puncture force (RPF), and internal quality. The FRF was deter-

mined by an Ametek hand pull tester (Landsdale, PA), peel color by a

Hunter Color Difference Meter (Fairfax, VA), and RPF by an Instron

penetrometer with a 0.63-cm radius flat head moving at 20 cm per

minute (Canton, MA). The Hunter Color Difference Meter measures

chromaticity dimensions of colors and expresses them as "a/b" ratios;

"a" measures redness when positive and greenness when negative; "b"

measures yellowness when positive and blueness when negative. The

ratio indicates proportions of the above colors. A pale yellow grape-

fruit typically has an "a/b" ratio ranging from -0.1 to 0.2. Internal

quality was evaluated by automated systems analysis giving total soluble

solids (TSS) by the specific gravity method, percentage of acid, TSS/

acid ratio, percentage of juice, and weight per fruit including peel







(23). An additional sample of 20 fruit per tree was collected to deter-

mine the extent of seed sprouting. 'Marsh' grapefruit trees in this

study bore an average of 1500 fruit. The gradual loss of 350 fruit to

sampling and 180 fruit to drop over 6 months should not have affected

the quality of the remaining fruit (79).



Results and Discussion

Effects of SMC on L

The SMC was significantly lowered in the top 0.90 m of soil in all

unirrigated plots on all sampling dates in both seasons; yet the 4$L

values of irrigated and unirrigated trees were comparable at 4 AM and

11 AM (Fig. 2-1). Davies et al. (21) reported a similar lack of correla-

tion between iL value and SMC for 'Orlando' tangelo trees in the field.

They hypothesized that tree capacitance and ability to control water

loss via stomatal closure are factors that affect the reliability of SMC as

an indicator of a tree's water status. Their results and those given

here are consistent with grower observations that large, well-

established trees, particularly those on rough lemon rootstock (12),

generally do not have increased yields in response to supplemental

irrigation under Florida conditions (A.J. Rose, personal communication,

1982).



Effects of Irrigation and GA. and 2,4-D
on Drop and External Fruit Quality

No differences were observed in irrigated compared with unirri-

gated trees in fruit drop, FRF, RPF, or color, although SMC differed

significantly throughout both seasons. Spray applications of GA3 and

2,4-D, however, significantly decreased late-season and total fruit drop












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and delayed loss of FRF and RPF and development of overmature peel

color both seasons (Fig. 2-2). Gilfillan et al. (35) observed that

inadequate irrigation resulting in wilting decreased the efficacy of GA3

and 2,4-D in preventing fruit drop and peel puffiness. However, they

did not measure SMC or VL. Citrus trees growing in arid climates have

shallower, less extensive root systems than trees in moderately high-

rainfall areas with deep sandy soils (20). These data indicate that GA3

and 2,4-D applications in areas with the latter characteristics should

produce favorable responses, regardless of irrigation practices. These

findings are consistent with previous reports that GA3 and 2,4-D

extend the grapefruit harvest season in Florida (2), South Africa (35),

and Australia (25).



Effects of Decreased Irrigation and GAg
and 2,4-D on Internal Fruit Quality

The TSS, percentage of acid, TSS/acid ratio, percentage of juice,

weight per fruit, and incidence of seed sprouting were unaffected by

GA3 and 2,4-D application or irrigation treatments. Previous workers

have reported only slight, inconsistent effects of GA3 and 2,4-D on

internal quality of grapefruit (2,25,35,59). Ali Dinar et al. (2)

reported a significant decrease in seed sprouting during one season in

Florida, a result not corroborated here or by Albrigo et al. (1). Ali

Dinar et al. (2) did not clearly demonstrate whether it was GA3 or

2,4-D or the combination that decreased sprouting, as all three

decreased sprouting somewhat.











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Effects of GAn and 2,4-D on Postfreeze Fruit Drop

Grove air temperatures were between -2.20 and -5.50C for 10

hours on January 12, 1981, and between -3.90 and -5.50C for 6 hours

on January 13, 1981. Temperatures remained between -2.20 and -5.50C

for 10 hours on January 12, 1982. Severe fruit drop generally occurs

when fruit are exposed to such temperature extremes; however,

January fruit drop was significantly lower for GA3 and 2,4-D treated

trees, particularly after the more severe freeze of 1981 (Fig. 2-2).

The difference in fruit-drop rates of treated and untreated trees was

again insignificant in February, suggesting that the difference in both

January was a result of the freezes.



Conclusions

These experiments support previous reports of the efficacy of GA3

and 2,4-D in extending the grapefruit harvest season by improving peel

color and firmness and decreasing late-season fruit drop. Furthermore,

under Central Florida conditions, presence or absence of low-volume

irrigation did not alter the efficacy of GA3 and 2,4-D in extending the

harvest season of large, mature grapefruit trees. Applications of GA3

and 2,4-D reduced postfreeze fruit drop.













CHAPTER III

PREHARVEST AND POSTHARVEST GIBBERELLIC ACID AND
2,4-DICHLOROPHENOXYACETIC ACID APPLICATIONS FOR
INCREASING STORAGE LIFE OF GRAPEFRUIT



Introduction
White 'Marsh' seedless grapefruit (Citrus paradise Macf.) has a

long, September to May harvest season, is a nonclimacteric fruit with a

low respiration rate and minimal starch reserves, and is unresponsive to

controlled atmosphere storage (42). For nearby markets the fruit can

be stored on the tree. Distant markets such as Japan, however,

require postharvest storage during transport. Therefore, any prehar-

vest or postharvest treatment that extends postharvest life is desirable.

Currently, GA3 and 2,4-D treatments, applied preharvest or

postharvest, show promise for extending grapefruit postharvest storage

life. Preharvest and postharvest GA3 treatments have the same effect;

both delay overripe color development and loss of peel firmness. Pre-

harvest 2,4-D also delays color development and loss of firmness some-

what, but primarily delays preharvest abscission. Postharvest 2,4-D

indirectly decreases growth of some fungal pathogens by rendering the

host grapefruit less vulnerable to pathogen entrance, particularly

around and under the calyx where Alternaria citri spores exist in

abundance (34,90). This is important because citrus fruits almost

invariably succumb to fungal invasion before physiological breakdown

renders them unmarketable (42).







Response of grapefruit to any preharvest or postharvest treatment

for extending postharvest storage life could vary depending upon when

during the season and time of day fruit is harvested (103), rootstock

(88), tree condition, cultural and harvesting practices, and intentional

or unintentional postharvest treatment (42).



Literature Review

Effects of Preharvest GA3 and 2,4-D Sprays
on External Quality of Postharvest Stored Grapefruit

Much work has been done on the effects of preharvest GA3 and

2,4-D sprays on the preharvest quality (2,25,35,59), but there are few

reports on the effects of these materials on postharvest quality. Pre-

harvest GA3 and 2,4-D sprays, applied at a rate of 10-20 ppm for GA3

and 20-40 ppm for 2,4-D, have been reported to delay postharvest

overripe color development and loss of firmness in California (15),

Florida (1), and Australia (34,36). Similar results have been obtained

with 'Shamouti' oranges in Israel (70). In contrast, Fucik (33) in

Texas did not observe preharvest GA3 and 2,4-D sprays to maintain

peel firmness of 'Ruby Red' grapefruit in storage, even though treated

fruit had firmer peels than controls at harvest. However, he applied

GA3 at only 1 ppm, whereas it was applied at 10-20 ppm in most other

studies (1,15,33,34,36).

Grapefruit invariably succumb to fungal invasion before physiologi-

cal breakdown renders them unmarketable (42). Therefore, postharvest

storage life generally depends on an interaction between physiological

and pathological factors. Preharvest sprays of GA3 and 2,4-D (33) and

2,4-D alone maintain the calyx in a more juvenile state, thus hindering

the entrance of Alternaria citri spores, which cause stem-end rot.







Bevington (10) in Australia reported less green mold (Penicillium

digitatum) wastage of navels in cold storage when they had been

treated preharvest with GA3.



Effects of Preharvest GA3 and 2,4-D on
Internal Quality of Postharvest Stored Grapefruit

Except for slight increases in percentage of juice, the internal

quality of 'Marsh' grapefruit does not change greatly with extended

storage (86). Gallasch (34) reported a decrease in percentage of acid

of stored grapefruit that had been treated preharvest with GA3 alone.

Monselise and Sasson (70) contradicted this with a report of increased

percentage of acid in stored 'Shamouti' oranges that had been treated

preharvest with GA3 and 2,4-D. They attributed this increase to

2,4-D, as GA3 alone did not produce this result.

There are no reports of preharvest GA3 sprays and 2,4-D sprays

reducing the incidence of seed sprouting and granulation in grapefruit

stored postharvest.



Effects of Postharvest GA3 and 2,4-D Treatments
on External Quality of Postharvest Stored Grapefruit

There are no reports on the use of postharvest GA3 or GA3 and

2.4-D treatments for grapefruit. Postharvest applications of GA3 main-

tained 'Shamouti' orange (37) and lemon (69) peel in a more juvenile

state during storage. El-Nabawy et al. (24) reported that postharvest

GA3 dips retarded color development, decreased weight loss, and

decreased the percentage of discards in stored 'Valencia' oranges.

Postharvest 2,4-D treatments decreased weight loss and percentage of

discards in stored 'Valencia' oranges (24), maintained the calyx in a







juvenile state in 'Marsh' grapefruit (90), 'Kinnow' mandarin, and

'Eureka' lemons (29), and decreased losses due to Alternaria in 'Marsh'

grapefruit (89,90). Application rates were higher than those of pre-

harvest sprays, ranging from 100 to 2000 ppm for GA3 and from 500 to

2000 ppm for 2,4-D (15,24,29,89,90).



Effects of Postharvest GA3 and 2,4-D Treatments
on Internal Quality of Postharvest Stored Grapefruit

Internal quality of 'Marsh' grapefruit is not altered greatly in

extended cold storage (86) except for a slight increase in the percent-

age of juice. Postharvest GA3 and 2,4-D treatments do not produce

any consistent, significant changes (15,68). El-Nabawy et al. (24)

reported slight increases in TSS and decreases in percentage of acid of

stored 'Valencia' oranges that were treated postharvest with 2,4-D or

GA3'

There are no reports of postharvest GA3 and 2,4-D treatments,

alone or combined, reducing seed sprouting or granulation in grapefruit

stored postharvest.

The objective of these experiments was to compare the ability of

preharvest and postharvest GA3 and 2,4-D treatments to maintain

grapefruit quality in storage.



Materials and Methods

Two sets of 40 'Marsh' white seedless grapefruit (Citrus paradise

Macf.) trees on rough lemon (C. jambhiri Lush.) rootstock from

the same grove were used successively during the 1980-81 and 1981-

82 seasons. Trees were growing in Astatula fine sand, were hedged

north-south in 4.5x9. 1-m spacing, and had low-volume undertree






irrigation. Normal grove production practices were followed. The 40

trees were divided into a 20-tree preharvest control treatment and a

20-tree preharvest spray treatment. Completely randomized single-tree

plots were used during 1980-81 and two-tree plots during 1981-82.

When all fruit were slightly past colorbreak on December 12, 1980, and

November 11, 1981, approximately 50 liters of an aqueous combination of

GA3 (20 ppm), 2,4-D (20 ppm), and X-77 surfactant (0.025 v/v) were

sprayed on each preharvest treatment tree. Control trees were not

sprayed.

On January 26, March 23, and May 18, 1981, and January 18,

March 15, and May 10, 1982, approximately 60 fruit per tree were

randomly harvested from the four quadrants of each tree. Blemished

fruit were removed, and the remainder was washed on a packinghouse

line and air dried. Initial seed sprouting counts were done on 20 fruit

per tree. An additional 20 fruit per tree were used to obtain initial

peel color measurement by the Hunter Color Difference Meter (47), peel

puncture strength by the Instron penetrometer (48), and juice quality

by automated systems analysis (23). Peel color was tested on one

location per fruit and peel firmness on four. The remaining fruit were

combined within each of the two preharvest treatments, control and

preharvest spray, and then divided into two equal lots within these

treatments. One lot from each field treatment was dipped for 1 minute

in an aqueous combination of GA3 (100 ppm), 2,4-D (500 ppm), and

X-77 surfactant (0.025% v/v). These preharvest and postharvest

treatments produced four storage treatments: a control treatment

without preharvest spray or postharvest dip, a treatment with

postharvest dip only, a treatment with preharvest spray only, and a







treatment with both spray and dip. All fruit received 1000 ppm

thiabendazole (TBZ), were dried and waxed on a packinghouse line,

were packed into 20-kg cartons (four replications per storage treat-

ment), and were stored at 15.50C and 95% relative humidity (67) for 12

weeks. Color readings on 20 randomly selected fruit per carton and

decay checks on all fruit were done weekly. At 12 weeks, peel punc-

ture resistance testing and juice analysis were repeated on two 20-fruit

samples per treatment and seed sprouting was counted on 20 fruit per

treatment.

The Hunter Color Difference Meter measures chromaticity dimen-

sions of colors and expresses them as "a/b" ratios; "a" measures red-

ness when positive and greenness when negative, and "b" measures

yellowness when positive and blueness when negative. The ratio indi-

cates proportions of the above colors (47). A pale yellow, marketable

grapefruit has an "a/b" ratio ranging from -0.1 to 0.2.

The Instron penetrometer determines peel puncture resistance in

newtons (N) required to puncture a 0.63 cm hole in the peel using a

flat head moving at 20 cm per min. N is a unit of force independent of

mass and is not comparable to the mass unit, kg.

Data were analyzed using analysis of variance and Duncan's

multiple range test.



Results and Discussion

Effects on Color

The GA3 and 2,4-D delayed overripe peel color development when

applied before or after harvest (Fig. 3-1). Fruit from trees treated

preharvest had significantly lower "a/b" ratios than controls on all six






















































Fig. 3-1. Comparison of preharvest, postharvest, and combined pre-
harvest and postharvest GA3 and 2,4-D treatments on peel color
at harvest (bottom of bar) and after 12 weeks of storage (top of
bar). Treatment means within a harvest date at bottom (harvest)
and top (after storage) separated by Duncan's multiple range test,
P = <0.05. Means are an average of 80 values.




















ORANGE 0.5


0,4



0.3


0.2


0.1


YELLOW


-0.1


GREEN -0.2



a/b RATIO


ORANGE 0.5



0.4


0.3



0.2


0.1


YELLOW


GREEN -0.1


CONTROL

POSTHARVEST

PREHARVEST

COMBINED

a


b b


b b


b b

JAN 26-APRIL 27


1980-1981

MAR 23-JUNE 15 MAY 18-AUG 10


b b


a a


b b


b b


JAN 26-APRIL 27 MAR 15-JUNE 7

1981-1982


MAY 10-AUU ;


""' ~^ "'^ '







harvest dates, confirming an earlier Florida report (2). Fruit that had

received any of the GA3 and 2,4-D treatments still had significantly

lower "a/b" ratios than controls after 12 weeks in storage. All treat-

ments were equally effective from January to March in delaying overripe

peel color, but in May, combined preharvest and postharvest treatment

was significantly more effective than postharvest, but not preharvest,

treatment. Therefore, prior to April, there was no advantage to com-

bined preharvest and postharvest treatment, as both delayed color

development equally well alone. Preharvest or combination preharvest

and postharvest treatment was best for fruit harvested in May, as

either produced significantly less overripe color development. If only a

single GA3 and 2,4-D application is possible, preharvest is preferable,

as it consistently produced a lower "a/b" ratio than postharvest appli-

cations (2,25,35,59).



Effects on Peel Puncture Resistance

The GA3 and 2,4-D maintained peel puncture resistance when

applied either preharvest or postharvest (Fig. 3-2). Fruit from trees

that were treated preharvest had significantly higher puncture resis-

tance than controls, confirming earlier data reported by Ali Dinar et al.

(2). However, for harvested fruit they reported puncture resistance

from 6.14 to 8.90 N compared with the 13.75 to 21.50 N here. Our

values agree with two other Florida reports (F.S. Davies and M.A.

Ismail, 1979, and A. J. Rose, 1979, both personal communications) and

one Australian report (25). Fruit treated before or after harvest, or

at both times, had consistently higher puncture resistance after 12

weeks in storage than the controls did. All treatments were equally






















































Fig. 3-2. Comparison of preharvest, postharvest, and combined pre-
harvest and postharvest GA3 and 2,4-D treatments on peel punc-
ture resistance (higher values = higher resistance) at harvest (top
of bars) and after 12 weeks of storage (bottom of bars). Treat-
ment means within a harvest date at top (at harvest) and at
bottom (after storage) separated by Duncan's multiple range test,
P = <0.05. Means are an average of 80 values.















































PUNCTURE
RESISTANCE


CONTROL

POSTHARV

PREHARVE

COMBINED


'EST

ST


b b
a a




b b


b b






a a
b



a


1980-1981

JAN 26-APRIL 27 MAR 23-JUNE 15


b b


b b




a ad




a


MAY 18-AUG 10


b b


b b



a a



Sc


JAN 18-APRIL 12


MAH 15-JUNE 12

1981-1982

DATES OF STORAGE


MAY 10-AUG 2


... ...- ._ ....- ._ ..... .^ ...i---


=







effective for January and March harvests, but for May harvests, com-

bined preharvest and postharvest treatment was significantly better

than postharvest treatment. Therefore, through March, there was no

advantage to combined preharvest and postharvest treatment, as a

single preharvest or postharvest treatment maintained peel puncture

resistance as well. After March, preharvest or combined preharvest

and postharvest treatment was more effective than a postharvest treat-

ment. However, if only a single GA3 or 2,4-D treatment is possible,

preharvest treatment is preferable, as it also decreases late-season fruit

drop losses (2,25, 32,35).



Effects on Decay

The GA3 and 2,4-D treatments decreased decay in storage (Fig.

3-3). There were no significant differences between preharvest, post-

harvest, and combined preharvest and postharvest applications; all

resulted in significantly less decay than in untreated control fruit.

Onset of decay was 1 to 2 weeks earlier and stem-end rot was more

frequent in control fruit. These data agree with earlier reports of

2,4-D's ability to decrease grapefruit decay, particularly stem-end rot,

when applied postharvest (89,90,98). Preharvest or postharvest appli-

cations of GA3 have been reported to delay peel senescence, thereby

maintaining peel quality in storage (69). The results here suggest that

these decreases in decay are partially a result of better peel condition,

rendering the fruit less susceptible to injury and therefore to invasion

by fungal pathogens. This would explain why preharvest and post-

harvest treatments function equally well.























































Fig. 3-3. Comparison of preharvest, postharvest, and combined pre-
harvest and postharvest GA3 and 2,4-D treatments on decay after
12 weeks in storage. Treatment means within a storage period
separated by Duncan's multiple range test, P = <0.05. Means are
an average of 80 values.



















% OF TOTAL








25%



20%


15%


10%


5%



DECAY





25%


20%


15%


10%


CONTROL
POSTHARVEST
PREHARVEST
COMBINED


1980-1981


1981-1982

DATES OF STORAGE







Effects on Internal Quality

Juice content, total soluble solids (TSS), percentage of acid,

TSS/of acid ratio, and individual fruit weights did not display any

significant, consistent differences due to treatment either at harvest or

after 12 weeks in storage (data not shown). This is consistent with

earlier reports for preharvest (2,25,35,59) and postharvest (15,24,68)

treatments. Seed sprouting was unaffected by all treatments during

either season. This disagrees with an earlier Florida report (2) and

agrees with another (1).



Conclusions

These results confirm earlier reports that preharvest GA3 and

2,4-D sprays maintain grapefruit peel quality on the tree (2,25,35) and

in storage (1) without affecting internal quality (2,25,35). Preharvest

GA3 and 2,4-D application was the best treatment under most circum-

stances. Overripe color development and loss of puncture resistance

were retarded more effectively through storage when combined GA3 and

2,4-D was applied preharvest. This is consistent with Goldschmidt and

Eilati's report of GA3 delaying color development more effectively in

unharvested than harvested 'Shamouti' oranges (37). Preharvest GA3

and 2,4-D produced better peel quality in tree-stored fruit and delayed

abscission (2,25,32,35), thereby extending the harvest season. Post-

harvest GA3 and 2,4-D dips, although less expensive than preharvest

sprays and equally effective in maintaining peel quality and decreasing

decay of stored fruit harvested through March, will not enable exten-

sion of the harvest season. Applying both a preharvest spray and a

postharvest dip was advantageous only for fruit harvested in May.

Otherwise, a preharvest spray or a postharvest dip had equal effects.




30


Despite GA3's effectiveness in slowing peel senescence in grape-

fruit, seed germination and granulation in late season fruit still remain

a problem. Both result in development of off flavors and lower grade

of fresh market and processing fruit.













CHAPTER IV

UPTAKE, TRANSLOCATION, PERSISTENCE, AND METABOLISM
OF GIBBERELLIC ACID IN GRAPEFRUIT



Literature Review

Uptake, Translocation, and Persistence of 14C-GA3

Little is known about uptake and transport of gibberellins in

grapefruit. Gibberellin uptake and transport studies using excised

sections of coleoptiles, stems, or petioles indicate that gibberellins are

readily absorbed, their movement is passive and nonpolar in xylem and

phloem at rates of 5-25 mm per 12-hour period, and interchange occurs

between xylem and phloem (7,11,13,66,72,113). These results suggest

that gibberellins are transported with carbohydrates. Acropetal and

basipetal polar movement of gibberellins occurs in coleus petioles (52),

stems (53), and subapical root sections of several higher plants (3,7,

53,72,78). However, these may not be examples of true polar move-

ment, but may simply be movement toward a growth center (78,87,108).

Goldschmidt and Eilati (37) applied GA3 to the flavedo of

'Shamouti' oranges before and after harvest. Delay of peel color devel-

opment indicated that GA3 was absorbed by both attached and detached

fruits, although this effect was more marked and persistent in the

former. Delay of peel color development beyond the area of flavedo

application suggested lateral diffusive movement of the GA3. This

movement was further and more toward the stylar end in attached than

detached fruit. Goldschmidt and Galili (38) applied H-GA3 to detached
3 eace







'Shamouti' orange peels and recovered measurable amounts of radio-

activity from the flavedo after 5 days of air or ethylene storage. In a

later study (39) they applied 14C-GA3 to 'Valencia' oranges on the tree

and recovered only 2% of applied radioactivity from the flavedo after

24 hours.

Evidence for localization of GA3 following uptake and translocation

within citrus peel is indirect. Exogenously applied GA3 delays loss of

chlorophyll, RNA, and proteins (30,31) in the flavedo and maintains a

more compact structure in the albedo (69), suggesting that it or its

metabolites are localized in these areas. Some gibberellin biosynthesis

and metabolism occurs in chloroplasts and leucoplasts (72). Ohlrogge et

al. (76) and Silk and Jones (94) suggest that gibberellins are compart-

mentalized in vacuoles.

Evidence for persistence of exogenously applied GA3 in citrus peel

is both indirect and direct. Sprays of GA3 delay peel color develop-

ment and loss of rind firmness for as long as 7 months after applica-

tion. This may be an indirect indication of GA3's persistence in citrus

peel (16). Direct evidence of persistence has been demonstrated by

Jordan et al. (57), who found 0.10-0.15 ppm GA3 in lemon peel 7 days
14
after application. Goldschmidt and Galili (39) applied 1C-GA3 to

'Valencia' oranges on the tree and recovered 1% of the applied radio-

activity after 100 days. However, until the presence of endogenous

GA3 is established in citrus peel, physiochemical methods of detecting

GA3 cannot conclusively demonstrate the persistence of absorbed GA3.







Metabolism of 14C-GA,

Little is known about gibberellin metabolism in higher plants.

Glycosylation, the attachment of a glucose molecule through an ether

bond forming a water-soluble gibberellin glucoside, is common with

gibberellins in higher plants. Water-soluble, "bound," gibberellin-like

substances were discovered in higher plants in the 1960s (58,65,66,74).

Hydrolysis with acid, ficin, emulsin, papain, or B-D-glucosidase (6,7,

9,45,54,65,74,82,110) released free gibberellins and glucose. "Bound"

is now reserved for unidentified gibberellin-like substances, whereas

"conjugated" describes complexes in which both gibberellin and its

bound counterpart are known (62,92).

Conjugated gibberellins are much less active than free gibberellins

(8,93,103,109,111). The deactivation of free gibberellins by glycosyla-

tion (7,62,72), the frequency and rapidity with which it happens to

applied gibberellins (7,11,113), and its reversibility (9,75,77,92)

suggest that glycosylation is a regulatory mechanism. Possible func-

tions of deactivation by glycosylation could be to aid transport and

storage of free gibberellins (62).

Primary evidence for glycosylation as an aid to gibberellin trans-

port comes from xylem and phloem feeding studies. Water-soluble

glucosides can be transported in xylem and phloem more readily than

less soluble free gibberellins. Bowen and Wareing (11) reported that a
14
portion of 1C-GA3 applied to willow shoots was quickly converted to

conjugates once in xylem and phloem. The major fraction of gibber-

ellins in spring bleeding sap of deciduous trees was in the form of

gibberellin conjugates (92).







Primary evidence for glycosylation of gibberellins as a storage form

of free gibberellins comes from studies with seeds. Immature seeds

were injected with radiolabeled gibberellins, and relative levels of

radioactive gibberellin conjugates and free gibberellins were observed.

Barendse et al. (9) and Sembdner et al. (92) reported partial conver-

sions of free gibberellins into conjugates prior to and through maturity,

then a conversion of these conjugates into free gibberellins during

germination. Sembdner et al. (92) also observed a conversion of

applied GA3 to its conjugated form in developing bean pods. These

studies are consistent with reports by Hashimoto and Rappaport (44)

and Pegg (77), who determined levels of endogenous gibberellins in

developing seeds. Endogenous free gibberellins steadily decreased to

very low levels at maturity as endogenous conjugated gibberellins

increased. The situation reverses with germination. Pegg (77) sug-

gested that this reversal is caused by hydrolysis of conjugated gibber-

ellins. Barendse (6) observed enhanced conversion of 3H-GA3 to a
14
water-soluble compound when 1C-glucose was applied simultaneously to

Japanese morning glory plants, suggesting that GA3-glucoside formation

was enhanced in the developing seed pods when there was available

glucose to conjugate with.

Interconversions of free and conjugated gibberellins also occur in

peaches and apricots (50,51,72). Free gibberellins are most abundant

in tissue during periods of maximum growth (50,51), then decrease

sharply relative to conjugated forms as growth decreases (51,72).

These data suggest that each tissue maintains its own supply of gibber-

ellins, although other studies suggest that bound gibberellins in mature

seeds are available to other fruit tissues (49,106).







Endogenous GA3-glucosides have been found in several higher

plants (66,72,92,104,111,112). Moreover, exogenous application of
14
1C-GA3 forms a glucoside indistinguishable from the endogenous one

(7,22,92,93). Glucosides of gibberellins are difficult to separate from

acidified aqueous solutions with ethyl acetate; however, glucosides can

be partitioned from aqueous solutions with N-butanol (7,72,101).

Goldschmidt and Galili (38,39) applied radiolabeled GA3 to 'Shamouti'

and 'Valencia' orange peels. In both experiments, diethyl ether or

ethyl acetate partitions contained approximately one half of the recov-

ered radioactivity on all sampling dates. The other half remained in

the aqueous fraction. The organic fractions co-chromatographed with a

GA3 standard on silica gel H-coated plates. Thin-layer chromatography

peaks decreased in height and broadened with each successive sampling

date (1, 10, 100 days), suggesting catabolism of the 1C-GA3. Total

recovered radioactivity decreased, but the ratio of ethyl acetate and

water-soluble fractions remained relatively constant, indicating that
14
1C-GA3 glucosides recovered in the water-soluble fraction may be

slowly converted to 1C-GA3 soluble in the ethyl acetate fraction. Silk

and Jones (94) proposed a similar situation with H-GA1 in excised

lettuce hypocotyls. Alternative explanations might be that the ethyl

acetate-soluble fraction consists of free 1C-GA3 (14,22) or 1C-GA3

sequestered in an organelle (76), and as it is used or released it is

degraded into a water-soluble form. However, without N-butanol parti-

tioning to separate glucosides, or positive identification via gas chroma-

tography-mass spectroscopy, these possibilities cannot be verified.

Chapters II and III of this dissertation demonstrated the ability of

exogenously applied GA3 to maintain preharvest and postharvest peel







quality of 'Marsh' grapefruit. However, little is known about the fate

of exogenously applied 14C-GA3. Therefore, this study will determine

the uptake, translocation, and beginning metabolism of 14C-GA3 applied

to attached 'Marsh' grapefruit peel and leaves.



Materials and Methods

Application of 14C-GA3 to
Attached Fruit and Leaves

Unlabeled GA3 was obtained from Abbott Laboratories, Chicago,

Illinois (Pro-Gibb, 3.91% in isopropyl alcohol), and 14C-GA3 (1,7,12,
14
18) ( C-GA3, 14 mCi/mMol) was purchased from The Radiochemical

Centre, Amersham, England. The original ethyl acetate solution of
14
1C-GA3 was reduced to dryness under vacuum at 370C and dissolved

in 25 ml of an aqueous solution of 1.0% isopropanol, 0.025% X-77, and

0.01 ml of Pro-Gibb which produced a stock solution of 20 ppm GA3

solution containing 1.65x105 disintegrations per minute (DPM) per 200-pl

aliquot.

Sixty-six mature grapefruit with three or more subtending leaves

not more than 10 cm from the fruit were randomly selected, no more

than two per tree, from 40 container-grown 3-year-old 'Marsh' grape-

fruit (Citrus paradise Macf.) on Milam (C. jambhiri hybrid ?) rootstock.
14
Thirty-three fruit from different trees had 1C-GA3 applied to the fruit
14
surface and 33 fruit from different trees had 1C-GA3 applied to both

surfaces of the subtending leaves. Each fruit was visually divided into

four quadrants, and 50 pl of the stock solution were evenly dotted on

the surface with a microsyringe and spread about with the tip. Leaf

application was done in the same manner. Each fruit and leaf set








received a total of 1.65x10s DPM. Application was done between 10 and

11 AM in full sunlight at temperatures of 28-320C and relatively

humidities of 59-84%.



Application of 14C-GAg to Detached Fruit

Mature grapefruit were randomly harvested from 40 container-

grown 3-year-old 'Marsh' white seedless grapefruit trees on Milam

rootstock. Fruit were washed with tepid water, air dried, and divided

into three replicate sets; 14C-GA3 was then applied as previously

described. Fruit were held at 260C and 65-89% relative humidity in

fluorescent light until sampled at 0, 1, 2, 8, 24, and 48 hours. Sam-

pling was done as described below for attached fruit.



Extraction of Radioactivity

Three replicates of treated fruit with untreated leaves and three

replicates of untreated fruit with treated leaves were harvested 0, 1, 2,
14
4, and 8 hours, 1 and 4 days, and 1, 2, 4, and 8 weeks after 1C-GA3
14
application. Surface 1C-GA3 was removed by washing three times with

95% ethanol. A 5-ml aliquot of the combined washes was added to 15 ml

of Aquasol II (New England Nuclear) liquid scintillation cocktail (LSC)
14
for quantification of unabsorbed 1C-GA3. Woody tissue between fruit

abscission zone and proximal end of the petiole was kept separate from

woody tissue proximal to the leaf cluster. The flavedo was removed

with a potato peeler, albedo with a sharp knife, and seeds with tweez-

ers. Juice was extracted with a Wearever mechanical juicer, homoge-

nized in a Lourdes grinder for 2 minutes, and filtered with Whatman #1

paper, and a 5-ml aliquot was combined with 15 ml of LSC. Twigs were







homogenized three times with 25 ml of 95% ethanol and filtered with

suction through Whatman #1 paper. The three twig filtrates were

combined and partitioned against 150-ml volumes of petroleum ether until

clear, reduced to 5 ml under vacuum at 370C, and combined with 15 ml

LSC. Radioactivity was recovered from albedo, flavedo, and leaves,

using a modification of the procedure developed by Wheaton and

Bausher (107) (Fig. 4-1). A polyvinylpyrrolidone (PVP) column was

added after the ion exchange column to remove phenolics that might

interfere with later bioassays (73). All samples were counted in a

Beckman LS 5800 Series tabletop counter three times for 10 minutes and

the values averaged. Efficiency was approximately 90%, quench negli-

gible and disregarded, and a 38 counts per minute (CPM) background

subtracted. All recoveries were corrected to percentage of DPM

applied.



High-Performance Liquid Chromatography
of Leaf, Albedo, and Flavedo Extracts

Separation of metabolites in leaf, flavedo, and albedo extracts was

done with a Waters Associates modular high-performance liquid chroma-

tography (HPLC) system (107). Column was a Waters u-Bondapak

phenyl (300x3.9 mm) reversed-phase analytical column held at 300C with

C18 precolumn. Injector was U6K connected to twin Waters pumps, all

controlled by a Model 660 solvent programmer. Solvent for pump A was

0.2% ammonium acetate (NH4Ac) at pH 5.6, made with 2 ml of acetic acid

(HAc) in 1000 ml of deionized H20. The pH was adjusted with dilute

ammonium hydroxide (NH4OH), and the final solution was filtered with

suction and agitation through a 0.45 pm Gelman Acopor membrane filter.

The solvent for pump B was 50% pump A solvent and 50%, 95% ethanol






















C QC

m u


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filtered together as described. Pumps were programmed to deliver a

linear gradient progressing from 100% solvent A to 100% solvent B over

50 minutes. The flow rate was 1.5 ml per minute. Each 2.0-ml sample

injection was repeated three times. Fractions were collected every

minute in vials containing 10 ml of LSC. Only fractions 1-20 were

collected, as preliminary trials recovered little radioactivity in fractions

21-50. Fractions 21-50 were collected in bulk, reduced to dryness in

vacuo at 370C, dissolved in 10 ml of LSC, and read as a single sample.



B-D-glucosidase Hydrolysis of Radioactive Fractions

Fractions containing radioactivity were collected, reduced at 370C

repeatedly with 95% ethanol to remove HPLC solvent salts, and dissolved

in 1 ml of 100 mM PO4 buffer at pH 6.5. Fractions were combined with

1 ml of purified B-D-glucosidase (Sigma Ltd.) at 5.7 units per milliliter

and incubated in a 37C water bath for 3 hours. Samples were then

reduced at 370C, dissolved in 80% methanol, and centrifuged at 4200 xg

for 15 minutes to remove denatured enzyme. The supernatant was

reduced to dryness in vacuo at 370C, redissolved in 1 ml HPLC solvent

A, passed through a Millipore AP prefilter and 0.2 Gelman Metricel

membrane in a syringe, and rechromatographed to determine if hydroly-

sis altered retention time.



N-butanol Partition of Radioactive Fractions

Fractions containing radioactivity were collected, dried repeatedly

at 370C with 95% ethanol to remove residual ammonium acetate salts from

the HPLC solvents, and dissolved in 5 ml of 50% methanol. This was

reduced at 37C to approximately 2 ml, adjusted to pH 2.5 with dilute








HC1, and partitioned as described by Russell (85). Partition phases

were combined with 10 ml of LSC. The partitioning procedure sepa-

rated free gibberellins from their ether and ester glucosides and the

polar metabolites (Fig. 4-2).



Results and Discussion

Uptake, Translocation, and
Persistence of Peel-Applied 14C-GA3

Uptake of peel-applied 14C-GA3 began immediately after applica-

tion, was most rapid within 1 hour, and slowed between 1 and 2 hours

after application (Fig. 4-3). The amount of radioactivity decreased in

the peel during the next 8 weeks. Radioactivity was present in twigs

within 4 hours, reaching subtending leaves between 4 and 8 hours past

application. Radioactivity remained fairly constant, increasing in twigs

through 4 weeks. Leaves accumulated radioactivity through 1 week
14
with a subsequent decrease through 8 weeks. Residual 1C-GA3 on the

peel surface decreased sharply within 1 hour of application, remained

fairly stable through 4 days, then decreased steadily through 8 weeks.

No radioactivity was recovered from seeds or fruit pulp.
14
Uptake of peel-applied 1C-GA3 to detached fruit was comparable

to that of attached fruit (Fig. 4-4). Uptake began within 1 hour of

application and continued for 8 hours. The radioactivity recovered

decreased from 8 through 48 hours. No radioactivity was recovered

from seeds or juice.































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Uptake, Translocation, and
Persistence of Leaf-Applied 14C-GA3
14
Leaves began absorbing leaf-applied 1C-GA3 immediately, reaching

a maximum level within 2 hours (Fig. 4-5). The amount of label

decreased steadily over the next 8 weeks. Radioactivity was present in

twigs within 2 hours and persisted over 4 weeks. Radioactivity was

extracted from the peel within 8 hours. It reached a maximum at 2
14
weeks and then diminished slowly over 8 weeks. Residual 1C-GA3 on

the leaf surface decreased sharply for the first 2 hours and steadily

thereafter. No radioactivity was recovered from seeds or pulp.
14
Both leaves and peel absorbed 1C-GA3 within 1 hour, but peel

absorbed more in a shorter time and accumulated a greater amount
14
whether the C-GA3 was peel- or leaf-applied. This apparently

greater uptake may be the result of better uptake by the peel or less

translocation away from the peel than from the leaves. The greater

accumulation may be the result of photosynthate translocation from leaf

to peel.

The recovery of radioactivity from peel tissues reported here is

eightfold that previously reported for citrus peel application (39). The

total radioactivity recovered within 8 hours was 73.7% of peel-applied

and 80.2% of leaf-applied .radioactivity. Radioactivity recovered from

detached fruit was less: a total of 58.8% 8 hours after application.

Possible explanations for these losses include incomplete extraction,

metabolism to other compounds or oxidation to CO2, and translocation to

other tissues. The first possibility cannot account for all the losses;

the extraction procedure used had a 91.2% efficiency, determined using
14
a 1C-GA3 standard (Fig. 4-1). Davies and Rappaport (22) demon-

strated that GA3 is rapidly metabolized in higher plants, which could











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account for some losses; however, the most likely cause of these losses

is translocation to tissues not sampled. Translocation of radioactivity

begins within 2 hours and is not limited to fruit, subtending leaves,

and twigs. Low levels of radioactivity were measured in twigs proximal

to sampled leaves within 2 to 4 hours of application (data not shown).

However, translocation cannot account for the poor recovery levels in

detached fruit. Possibly detached fruit metabolize absorbed GA3 more

rapidly; Goldschmidt and Galili (37) reported that the biological effects

of GA3 on detached 'Shamouti' oranges were less marked and persistent

in detached than in attached fruit.

The translocation of radioactivity reported here agrees with the

report by Goldschmidt and Eilati (37) suggesting diffusive movement of

GA3 in the flavedo. However, they observed only flavedo, which lacks

vascular tissue. Vascular tissue does exist in the albedo, where radio-

activity was recovered in the present study. Therefore, vascular

transport of absorbed radioactivity once it diffuses from the flavedo to

the albedo would be a reasonable assumption. Translocation of gibber-

ellins via the vascular system has also been reported to occur in other

higher plants (13,66)

The long persistence of radioactivity reported here is supported

by an earlier study of Goldschmidt and Galili (39). However, they
14
reported only 2% recovery of peel-applied 1C-GA3 after 24 hours and

less than 1% after 100 days, compared with the 16.7% at 24 hours and

6.7% at 56 days reported here.
14
The uptake, translocation, and persistence of 1C-GA3 by citrus

peel determined here are consistent with the observed field effects

of GA3 on grapefruit. Preharvest applications of GA3 delay color







development and loss of peel firmness, but do not affect seed sprouting

or juice quality (2,25,35,59). These effects are evident within 1 month

and may persist 6 months. Data here demonstrate that leaf or peel

application of 14C-GA3 results in rapid accumulation of radioactivity in

the peel (within 24 hours), but not in the seeds or juice, even after 8

weeks. These studies also demonstrate persistence of measurable radio-

activity in peel tissues after 8 weeks. It is not known if these levels

are physiologically active or what the radioactive compound is, but the

sites of accumulation are consistent with the effects produced by spray

treatments of unlabeled GA3 at the same concentration.



Separation of Extracted Radioactivity by
High-Performance Liquid Chromatography
14
Metabolism of applied C-GA3 in both attached and detached fruit

was quite similar. However, the time course of the former was longer.

Therefore, only the metabolism data for the attached fruit will be

discussed.

Koshioka et al. (60) reported a "double-peaking" phenomenon with

the use of a reversed-phase HPLC C18 column for gibberellin separa-

tions. They found that the degree of sample purification caused reten-

tion time shifts and recommended that C18 columns be used only for

highly purified samples. The same problem arose with the similar

reversed-phase u-Bondapak phenyl column (Waters Associates) used in

this study. The presence of leaf, flavedo, or albedo extract in samples

consistently reduced retention times by 2 minutes. This was a problem

because suspected metabolites of GA3 have been demonstrated to elute

within 1 to 4 minutes earlier than GA3 on reversed-phase columns (60).

The possibility of mistaking less purified samples for metabolites was







solved by establishing the retention time of 14C-GA3 in the presence of

albedo, flavedo, and leaf extracts.

Radioactivity recovered from flavedo, albedo, and leaves consis-

tently eluted in the same fractions (Fig. 4-6). Radioactivity in

fractions 12-15 consistently peaked in fraction 13, co-chromatographing

with the 14C-GA3 standard, and radioactivity in fractions 4-7 consis-

tently peaked in fraction 6. The only exception was albedo extracts,

which eluted almost as much radioactivity in fraction 14 as in 13. In all

tissues the ratio of the two fractions changed rapidly over the first 4

days after application, then remained approximately stable over the next

8 weeks (Fig. 4-7). Radioactivity recovered in fractions 4-7 increased

from 1.2% in a total of 30.2% applied activity recovered at 1 hr to 8.1%

of 16.9% applied activity recovered at 4 days. Therefore, the percent-

age of radioactivity recovered in fractions 12-15 decreased from over

96% of the recovered activity at 1 hour to 52% at 4 days, while the

percentage of activity recovered in fractions 4-7 increased correspond-

ingly. This pattern is consistent with reports of rapid metabolism of

absorbed gibberellins in other higher plants (14,22). As plant tissues
14
metabolize 1C-GA3, the 12-15 peak diminishes and the 4-7 peak
14
increases. The 14-GA3 is metabolized over time until a relatively

constant ratio between the two fractions is attained. Alternately,
14
1C-GA3 may be metabolized by plant tissues until it can be compart-

mentalized, possibly in vacuoles (76). Once this occurs, the 1C-GA3

is slowly released as needed and subsequently degraded, maintaining a
14
fairly constant ratio of 1C-GA3 and its metabolites (43). Studies using

radiolabeled GA3 applied to 'Shamouti' and 'Valencia' orange peel sup-

port these possibilities (38,39). In both cases an ethylacetate or acid















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diethyl ether fraction and a water-soluble fraction were extracted,

which were tentatively identified as GA3-like and metabolite fractions,

respectively.



B-D-glucosidase Hydrolysis of Radioactive Fractions

The C13 and C3 glucose ethers are transport and storage metabo-

lites of GA3 in higher plants; they are biologically inactive relative to

GA3 (7). These metabolites have slightly lower retention times than

GA3 on reversed-phase columns (60). Hydrolysis of the glucose moiety

from the GA3 increases the retention time of this fraction. Hydrolysis

of fractions 12-15 and fractions 4-7, however, produces no change in

retention times, indicating that the radioactivity recovered in fractions
14
4-7 and 12-15 is probably not the C3 or C13 ethers of 1C-GA3. This

suggests that grapefruit peel and leaves do not conjugate GA3, but

metabolize it.



N-butanol of Radioactive Fractions

Partitioning gibberellins with N-butanol is a method of separating

free gibberellins from their esters and ethers. The results of partition-

ing fractions 4-7 were inconclusive (data not shown); all recovered

radioactivity partitioned into the polar metabolite phase. Partitioning

fractions 12-15 gave 83.3% in the free gibberellin phase, 8.5% in the

polar metabolite phase, and 7.9% in the glucoside phase (Fig. 4-8).

When these phases were combined and rechromatographed, radioactivity

recovered in the polar metabolite and GA glucoside phases was

insignificant. The radioactivity in the free gibberellin phase co-
chromatographed with the 14C-GA3 standard. The rather constant
chromatographed with the C-GA3 standard. The rather constant
3














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percentage of radioactivity that partitioned into the polar metabolite and

GA glucoside phases suggests that the radioactivity recovered in these

phases may be the result of inefficient partitioning.
14
These results suggest that the 1C-GA3 accumulated in grapefruit

peel remains in the applied form, possibly sequestered in the vacuoles,

and is slowly metabolized to a more polar form.



Conclusions

These uptake, translocation, persistence, and metabolism studies

partially clarify the fate of GA3 applied to grapefruit peel and leaves.
14
Absorption of 1C-GA3 by leaves and peel began within 1 hour of

application and continued for 8 hours. Peel absorbed more radioactivity

at a faster rate than leaves. Translocation of radioactivity from leaves

to peel and the reverse began in 4 to 8 hours and continued for 4

weeks. No radioactivity was recovered from juice or seeds. Radio-

activity persisted in albedo, flavedo, and leaves for 8 weeks with the
14
highest accumulations in peel tissues. Separation of 1C-GA3 metabo-

lites by reversed-phase HPLC produced two peaks of radioactivity.

Analysis of these two peaks by B-D-glucosidase hydrolysis, N-butanol

partitioning, and co-chromatography with 14C-GA3 standards suggested
that one was 14C-GA3 and the other, metabolites.
that one was C-GAn and the other, metabolites.
3












CHAPTER V

CONCLUSIONS



There were three objectives to this dissertation. The first was to

determine if decreased irrigation affected the ability of a preharvest

GA3 and 2,4-D colorbreak spray to extend the harvest season of

'Marsh' white grapefruit. The second was to compare preharvest and

postharvest, and combined preharvest and postharvest, treatments with

GA3 and 2,4-D for ability to extend the storage life of grapefruit. The

third objective was to study the translocation and metabolism of

14C-GA3 in grapefruit leaves and fruit.

Decreased late-season irrigation did not affect the ability of pre-

harvest GA3 and 2,4-D sprays to extend the harvest season of 'Marsh'

grapefruit in Florida. The GA3 delayed overripe color development and

loss of peel firmness without affecting internal quality. The 2,4-D

decreased late-season fruit drop. When the GA3 and 2,4-D sprays were

applied 6 weeks prior to a mild freeze, they decreased postfreeze fruit

drop.

A preharvest GA3 and 2,4-D spray or postharvest GA3 and 2,4-D

dip maintained peel quality in storage equally well for fruit harvested

through mid-March. However, preharvest sprays had the added advan-

tage of extending the harvest season and providing postfreeze fruit-

drop protection. Applying both preharvest spray and postharvest dip

maintained fruit quality better only if fruit was harvested in mid-May.







Both preharvest and postharvest treatments produced the same effects:

external peel quality was maintained while internal quality was unaf-

fected. All treatments decreased decay in storage equally well on all

harvest dates.

Peel and leaves began absorbing 1C-GA3 within 1 hour of applica-

tion and continued to do so for 8 hours. Leaves absorbed 1C-GA3

more slowly than peel. Within 2-4 hours of application, radioactivity

was translocated from peel to leaves and the reverse. Radioactivity,

whether applied or translocated there, persisted in leaves and peel

tissue for 8 weeks. However, accumulation of radioactivity was always

greater in peel than in leaves. No radioactivity was recovered from

seeds or juice. Separation of peel-extracted metabolites by reversed-

phase HPLC produced two peaks of radioactivity. Analysis of these
14
peaks suggested that one was 1C-GA3 and the other, a polar

metabolite.

The results of these three studies are consistent with one another.
14
Applied 1C-GA3 was readily absorbed, accumulated in peel but not

internal tissues, and persisted for at least 8 weeks. The spray and

dip GA3 treatments produced changes in peel, but not internal, quality

that were measurable within 6 weeks and persisted as long as 5 months.

Therefore, radioactivity accumulated and persisted in the same tissues

in which GA3 sprays and dips produced their effects. It is not known

if the radioactivity extracted from peel tissues is the active form of GA3

or if the levels measured are physiologically active. However, the

steady decline of the extracted radioactivity and its possible degrada-

tion are consistent with the decreasing effects of GA3 on peel quality

over time.







The last remaining question is, How does GA3 produce its effects?

Does absorbed GA3 or its metabolites initiate a physiological process,

then undergo degradation? Or are the effects produced as long as GA3

or active metabolites persist? Evidence presented in these studies

supports both possibilities, but the latter more strongly. The effects

of GA3 on peel quality were more marked when it was applied earlier;

preharvest applications produced greater effects than postharvest

applications at lower concentrations. However, this may simply be the

result of delaying a process, peel senescence, earlier in its develop-

ment. The ability of GA3 to produce an effect, no matter when

applied, and the persistence of 14C-GA3 argue for the latter possi-

bility. This possibility is supported by the fact that peel senescence in

citrus is associated with a decrease in endogenous gibberellins. There-

fore, the data in these studies suggest that applied GA3 delays grape-

fruit peel senescence as long as the absorbed GA3 or its metabolites

persist in physiologically active levels.













APPENDIX

GROWTH REGULATOR AND NUTRITIONAL EFFECTS ON
GRAPEFRUIT COLOR AND STORAGE QUALITY



Literature Review

Effects of Nitrogen and Gibberellins
on External Grapefruit Quality

An increase in tree nitrogen within the range for high-level

production has what are usually regarded as adverse effects on the

external quality of citrus (28). In most cases nitrogen produces a

coarser, thicker peel and a firmer albedo, and delays color develop-

ment. These effects are well documented on navel, 'Valencia', pine-

apple, and 'Hamlin' oranges and grapefruit (4,5,26,28,55,56,64,71,80,

81,83,84,95). High levels of nitrogen in grapefruit produced delays in

color development and firmer peels in Arizona (28) and Florida (96)

grapefruit. Results were similar for foliar and soil-applied nitrogen

(28). The effects of nitrogen on external peel quality are similar to

those produced by GA3 (see Chapter II). The combination of nitrogen

and GA3 in a foliar spray produces greater effects than either individu-

ally; Monselise et al. (71) and Embleton et al. (26) demonstrated that

the addition of potassium nitrate or ammonium phosphate to GA3 sprays

reduced the incidence of creasing and delayed orange color development

in 'Valencia' oranges better than the nutritional or GA3 treatment alone.

Monselise suggested that nitrogen application enhances the production of

endogenous gibberellins.






Effects of Nitrogen and Gibberellins on
Internal Grapefruit Quality

The effects of nitrogen on internal quality are inconclusive and

contradictory (27,28). Nitrogen applications to grapefruit have been

reported to reduce the percentage of juice and increase the acidity and

total soluble solids (TSS) without affecting the TSS/acid ratio (26,28),

to decrease the TSS and the TSS/acid ratio (28), to have small, incon-

sisten effects on these factors (4,83,84), and to decrease the percent-

age of juice up to 2.6% of leaf dry weight of nitrogen and decrease it

less thereafter (27). The inconsistency of these effects may be a

result of when the nitrogen is applied (28). There are no reports that

soil or foliar applications of nitrogen decrease seed sprouting or section

drying in grapefruit. The effects of GA3 on internal fruit quality were

covered in Chapter II; generally, effects are small and inconsistent.

There are no reports on the effects of combined nutritional and gibber-

ellin treatments on grapefruit internal quality.

Little work has been done on the effects of nutritional sprays,

alone or combined with gibberellins, on grapefruit quality in Florida.

The objective of this study was to compare nitrogen and gibberellin

colorbreak sprays for their ability to delay color change and seed

sprouting of tree-stored and cold-storage fruit under Florida

conditions. Juice quality and decay in storage were also observed.



Materials and Methods

Thirty mature 'Marsh' grapefruit (Citrus paradisi Macf.) trees on

rough lemon (C. jambhiri Lush.) rootstock were selected from a grove

near Lucerne Park, Florida. Trees were planted at 4.5x9.1 m spacing.

Cultural practices and soil type (deep ridge sand) were typical of local

commercial groves.







Completely randomized single-tree plots were used with four treat-

ments and six replications of each. Treatments were (1) KNO3 (2%) +

NH4NO3 (2%); (2) GA4+7 (20 ppm); (3) GA3 (20 ppm); and (4) KNO3

(2%) + NH4NO3 (2%) + GA3 (20 ppm). All treatment and control water

spray contained X-77 (0.025%). Pro-Gibb was the GA3 source,

Promalin was the GA4+7 source, and both nutritional sprays contained

approximately 35% nitrogen. Approximately 50 liters of dilute spray

were applied per tree on December 5, 1979.

Twenty fruit per tree were evaluated prior to treatment and bi-

monthly January through June. Color, juice quality, and seed sprout-

ing were tested by use of the Hunter Color Difference Meter (Fairfax,

VA), automated systems analysis (23), and observation, respectively.

Eighty fruit per tree were harvested in March, washed, treated

with thiabendazole (1000 ppm) as a fungicide, waxed, packed into 20-kg

cartons at 30 fruit per carton, and stored at 15.50C and 96% relative

humidity for 14 weeks. Juice analysis, seed sprouting, and color

evaluation of samples of 20 fruit per replication were done before and

after storage. Surface decay was evaluated weekly.



Results and Discussion
Peel Color of Tree-Stored Fruit

All treatments delayed peel color change compared with controls

(C) (Fig. A-i). Combined nutritional (N) and GA3 treatment produced

the best results in March and June. This supports previous evidence

that combined nitrogen nutritional and GA3 sprays delayed color change

in 'Valencia' oranges better than either alone (19,26). GA4+7 and GA3

alone were equally effective.














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Internal Quality of Tree-Stored Fruit

There were no significant differences in internal quality prior to

treatment, and treatments had no significant effects on total soluble

solids (TSS), percentage of acid, or seed sprouting (data not shown).

Compared with controls, all treatments increased the percentage of juice

by January and by March; only trees treated with GA3, alone or com-

bined with nutritional treatment, had increased juice content. By June

only GA3-treated fruit had significantly higher juice content (Table

A-l). There were no differences among treatments. Only combined

nutritional and GA3 treatment produced a significantly higher ratio than

controls on all dates. However, there was little difference among

treatments. These results contradict previous results showing that

nitrogen sprays decreased the TSS/acid ratios in 'Valencia' oranges by

increasing the percentage of acid (19,26). Percentage of acid of all

treated fruit was not significantly lower than that of controls (data not

shown). Reports of inconsistent effects of growth regulators on inter-

nal quality of grapefruit are common in the literature (2,25,35,59).

The incidence of seed sprouting in treated fruit was equal to that of

controls, remaining below 3% through March and increasing to 15-19% in

June (data not shown). This disagrees with one report (2), which

showed that GA3 decreased seed sprouting in grapefruit, and agrees

with another (1). Perhaps seed sprouting is a function of harvest

date. The dissenting report (2) had an earlier harvest date than this

study and the other report (1).
















Table A-1. Effects on internal quality of tree-stored grapefruit.



Date Sampled

January 11 March 24 June 18

% Acid TSS/Acid % Juice TSS/Acid % Acid TSS/Acid


** ** t **

Control 53A 8.2A 53A 8.8AB 51A 10.9A

KNO3 and 55B 8.6AB 54AB 8.5A 52AB 10.9A
NH4NO3

GA4+ 55B 8.8AB 54AB 9.5BC 56AB 11.7B

GA3 56B 8.4A 55B 9.3ABC 57B 11.3A

KNO3 and 55B 9.2B 55B 10.2C 53AB 13.3B
NH4NO3
and GA3


Note: Column means with different levels are significant as
follows: **, P = <0.01; *, P = <0.50; t, P = <0.10. N = 120.








Peel Color of Cold-Storage Fruit

All treatments delayed peel color change in cold-storage fruit with

little difference among treatments (Fig. A-2). Tree-stored fruit receiv-

ing the same treatments changed color more rapidly during this time

period with one exception. Untreated control fruit in cold storage

changed color twice as rapidly as tree-stored control fruit. This sug-

gests that all the treatments are particularly effective in delaying

postharvest acceleration of color change (37) as well as delaying color

change preharvest.



Internal Quality of Cold-Storage Fruit

There were no significant differences in internal quality prior to

storage or in percentage of juice or seed sprouting after storage (data

not shown). The TSS were increased in fruit receiving combined

growth regulator and nutritional treatment, and the percentage of acid

was decreased in all treated fruit. All treated fruit therefore had

higher TSS/percentage of acid ratios than controls (Table A-2). Seed

sprouting was not different from that in controls, remaining below 4%

for all treatments. Correspondingly treated tree-stored fruit had a

much higher incidence of sprouting, 15-19%, which was perhaps due to

higher temperatures in the grove than in cold storage and to a later

harvest date (1).



Decay During Cold Storage

All growth regulator treatments significantly decreased decay in

storage, but GA4+7 and combined GA3 and nutritional treatment were

the most effective (Fig. A-3).
















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Table A-2. Effects on internal quality of cold-
storage grapefruit.



Date Sampled: June 18

TSS % Acid TSS/Acid


Control 9.1B 0.9B 10.4A

KNO3 and 8.6A 0.8A 11.6B
NH4NO3

GA4+7 9.4B 0.8A 11.4B

GA3 9.1B 0.8A 11.2B

KNO3 and 9.8C 0.8C 12.9C
NH4NO3
and GA3


Note: Column means with different letters
are significant at P = <0.01. N = 120.













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CNJ -







Conclusions

The additive effects of combined nutritional and GA3 spray were

most effective in delaying color change of tree-stored fruit March to

June. Only GA4+7 and GA3 treatments were less effective than com-

bined treatment and they were equal to each other. Only GA, was

more effective than the nutritional treatment. All four treatments were

equally effective in delaying color change of fruit in cold storage. All

four treatments produced inconsistent changes on juice quality and had

no effect on seed sprouting. These results support previous reports

(1,2) that indicate that seed sprouting may be a function of tempera-

ture and harvest date and is not affected by applied growth regulators.

Decay in storage was reduced by combined GA3 and nutritional treat-

ment, GA3 alone, and GA4+7. These results support the use of

combined gibberellin and nutritional sprays to delay color change of

tree-stored and cold-storage grapefruit and to delay decay in cold

storage.













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BIOGRAPHICAL SKETCH


Louise Ferguson was born February 20, 1947, in San Bernardino,

California. She completed her secondary education at Governor Mifflin

High School in Shillington, Pennsylvania, in 1965. She received the

degree of Bachelor of Arts (in anthropology) from Pennsylvania State

University in 1965 and the degree of Master of Science (in horticultural

science--vegetable crops) from the University of Florida in 1980.







I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Frederick S. Davies, Chairman
Associate Professor of
Horticultural Science




I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




T. Adair Wheaton
Professor of Horticultural
Science




I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




M. A. Ismail
Professor of Horticultural
Science




I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




R. Hilton Biggs
Professor of Horticultural
Science







I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Richard C. Smith
Professor of Botany




This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.


August 1984


Dean College of IAgriculture


Dean for Graduate Studies and
Research

























































UNIVERSITY OF FLORIDA

1262 08554 0697 III
3 1262 08554 0697