Effects of ethylene and its action inhibitor (1-Methylcyclopropene) for regulating ripening and extending the postharves...

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Effects of ethylene and its action inhibitor (1-Methylcyclopropene) for regulating ripening and extending the postharvest life of avocado
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xiii, 171 leaves : ill. ; 29 cm.
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Jeong, Jiwon
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Avocado -- Postharvest technology   ( lcsh )
Plant regulators -- Physiological effect   ( lcsh )
Plant growth inhibiting substances -- Physiological effect   ( lcsh )
Ethylene -- Physiological effect   ( lcsh )
Horticultural Science thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2001.
Bibliography:
Includes bibliographical references (leaves 158-170).
Statement of Responsibility:
by Jiwon Jeong.
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Printout.
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Vita.

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EFFECTS OF ETHYLENE AND ITS ACTION INHIBITOR
(1-METHYLCYCLOPROPENE) FOR REGULATING RIPENING AND EXTENDING
THE POSTHARVEST LIFE OF AVOCADO

















By

JIWON JEONG


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


2001













ACKNOWLEDGMENTS

I would like to thank Dr. Donald Huber for engaging me in postharvest

physiology research and for his teaching and guidance. I am also grateful to Dr. Steven

Sargent, Dr. Charles Sims, Dr. Jonathan Crane, and Dr. Rebecca Darnell who have all

given me timely advice.

I thank James Lee for all of his teaching in the laboratory and for his efficient

management of the laboratory supplies. Very special thanks go to my friends at the

Horticultural Science Department who shared with my life as a graduate student.

This dissertation is dedicated with gratitude and love to my parents and family for

allowing me to walk my path in life. I thank my wife for her patience and encouragement

along the way.














TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ............................................................................................ ii

LIST OF TABLES ........................................... ................................................................ v

LIST OF FIGURES ............................................................. ....................................... viii

LIST OF ABBREVIATIONS ..................................................................................... xi

ABSTRACT ................................................................................................................. xii

CHAPTERS

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

2 LITERATURE REVIEW ..........................................................................................3

Introduction ...................................................................................................... ............. 3
A avocado ................................................................................................ ............. 3
Fruit Development ................................................................................................ 4
Avocado Fruit Ripening............................................................................................ 4
Climacteric Characteristics ................................................. ............................... 5
Fruit Softening ...................................................................................................... 5
Cell wall changes .............................................................................................. 6
Cell wall hydrolases........................................................................... ........... .. 6
Ripening-induced compositional changes ....................................... .......... ............. 9
Modification of Ripening.... ........... .................... .................................................... 10
E thylene .............................................................................................................. 10
Gaseous Plant Growth Regulator........................................ ............................. 11
Ethylene Biosynthetic Pathway ..................................... ................................... 11
Ethylene Perception and Signal Transduction............................................. ........ ..... 11
Practical Uses of Ethylene ..................................................................................... 12
E thylene A antagonists ................................................................................................ 13
1 -Methylcyclopropene (1-MCP)................................. ............................................ 15
Inhibitor of Ethylene Responses ............................................................................ 15
Treatment of Plants ............................................................................................ 17
Effect of concentrations, exposure times, and temperatures of 1-MCP ............... 17
Effect on respiration and ethylene production of 1-MCP................................ 19
Other responses of 1-MCP.................................................................................. 19








3 POSTHARVEST ETHYLENE TREATMENT FOR UNIFORM RIPENING OF
WEST INDIAN-TYPE AVOCADO FRUIT....................................................................21

Introduction .................................................................................................................. 2 1
M materials and M ethods.................................................................................................. 22
R esu lts ........................................................................................................................... 2 7
Discussion .. ........................ .. ................................................................. ....... 44
Summary .............................................................................................................................................. 46

4 INFLUENCE OF 1-METHYLCYCLOPROPENE (1-MCP) ON RIPENING AND
CELL WALL MATRIX POLYSACCHARIDES OF AVOCADO FRUIT ....................48

Introduction ................................................................................................... ......... 48
M materials and M ethods................................................................................................. 50
R esults................................................................................................................... 57
D iscu ssion ..................................................................................................................... 6 5
Sum m ary ................................................................................................................ 7 1

5 THE EFFECTS OF 1-METHYLCYCLOPROPENE (1-MCP) AND WAXING FOR
REGULATING THE RIPENING AND EXTENDING THE STORAGE LIFE OF
AVOCADO FRUIT........................... ..................................82

Intro du action ................................................................................................................... 82
M materials and M ethods............................................................................................ 83
R esu lts .............................. ...................................................................................... .. 87
D discussion ....................... .................. ...................................................................... ...... 93
Sum m ary ................................................................................................................ 97

6 INFLUENCE OF ETHYLENE AND ITS ACTION INHIBITOR (1-MCP) ON
RIPENING AND CELL WALL MATRIX POLYSACCHARIDES OF AVOCADO
F R U IT ................................................................................................................ .............. 10 3

Intro d u ctio n ................................................................................................................. 10 3
M materials and M ethods................................................................................................ 104
R esu lts ......................................................................................................................... 1 13
D discussion ........................................................... ...... .. ..... ... ................................. .. 132
S u m m ary ......................................................................................... ........................... 138

7 SUMMARY AND CONCLUSIONS ........................................................ 153

R EFE R EN C E S ........................................................................................................... 158

B IO G RA PH IC A L SK ETC H ........................................................................................... 171













LIST OF TABLES


Table Page

3-1. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
early-harvest (06/29/98) gassed immediately for 48 h at 20'C and transferred
to 10"C or stored at 10"C for 7 d then gassed for 48 h at 20C and then
transferred to 10C................................. ....... ................ ............28

3-2. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
mid-harvest (07/13/98) gassed immediately for 48 h at 20C and transferred
to 12'C or stored at 12'C for 7 d then gassed for 48 h at 20'C and then
transferred to 12C.............................................................. ..29

3-3. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
late-harvest (07/27/98) gassed immediately for 48 h at 20C and transferred
to 13C or stored at 13C for 7 d then gassed for 48 h at 20C and then
transferred to 13C ......................................... ............. ....... ......30

3-4. Fruit quality evaluation for 'Booth 7' avocados from mid-harvest (10/03/98)
stored for 7 d at 12"C and transferred to 20'C with different ethylene
treatm ents ............................................. ....... ..... .... ................33

3-5. Fruit quality evaluation for 'Booth 7' avocados from mid-harvest (10/03/98)
stored for 14 d at 12C with different ethylene treatments..................................34

3-6. Fruit quality evaluation for 'Booth 7' avocados from late-harvest (10/17/98)
stored for 7 d at 12C and transferred to 20C with different ethylene
treatments.................................................. ..35

3-7. Fruit quality evaluation for 'Booth 7' avocados from late-harvest (10/17/98)
stored for 14 d at 12C with different ethylene treatments..................................36

3-8. Fruit quality evaluation for 'Monroe' avocados from early-harvest (11/20/98)
stored for 7 d at 13"C and transferred to 20'C with different ethylene
treatm ents ..........................................................................................38

3-9. Fruit quality evaluation for 'Monroe' avocados from early-harvest (11/20/98)
stored for 14 d at 13C with different ethylene treatments.................................39







3-10. Fruit quality evaluation for 'Monroe' avocados from mid-harvest (12/04/98)
stored for 7 d at 13"C and transferred to 20C with different ethylene
treatm ents............................................................ .......................... 40

3-11. Fruit quality evaluation for 'Monroe' avocados from mid-harvest (12/04/98)
stored for 14 d at 13"C with different ethylene treatments.........................41

3-12. Fruit quality evaluation for 'Monroe' avocados from late-harvest (12/18/98)
stored for 7 d at 13"C and transferred to 20C with different ethylene
treatments ............................................ .... ................... .... 42

3-13. Fruit quality evaluation for 'Monroe' avocados from late-harvest (12/18/98)
stored for 14 d at 13"C with different ethylene treatments...............................43

4-1. Days to peak and maximum amount CO2 and C2H4 production for 'Simmonds'
avocados stored at 200C with 1-MCP treatments. Fruit were treated with two
1-MCP concentrations (0.45 and 0.09 iL L1') and three exposure periods (6, 12,
and 24 h)................... ...................................... ..................................59

4-2. Peel color of 'Simmonds' avocados stored at 200C with 1-MCP treatments ...........60

5-1. Days to peak and maximum amount CO2 and C2H4 production for 'Tower II'
avocados treated with 1-MCP (0.9 uL L'' for 12 h at 200C) and/or wax and
stored at 200C ...... .... ...................... .. ... ......................... ........89

5-2. Days to peak and maximum amount CO2 and C2H4 production for 'Booth 7'
avocados stored at 130C after wax treatment with or without 1-MCP
(0.9 pL L-1 for 12 h at 200C)................................... ........................... 91

5-3. Peel color of 'Tower II' avocados treated with 1-MCP (0.9 PL L'' for 12 h
at 200C) and/or wax and stored at 200C................................. ....................92

6-1. Peel color of 'Booth 7' avocados stored at 130C after 1-MCP treatment
(0.9 uL L-1 for 12 h at 200C) and then transferred into 200C (85% RH).
Some fruit were treated with C2H4 (100 pL L' for 12 h at 200C) before
transfer to 200C ............ ................... ....................... .......... .. 119

6-2. Sugar composition of water-soluble UA released from EIS prepared from
avocado stored at 130C for 12 d and then transferred to 200C............................125

6-3. Sugar composition of water-soluble UA from EIS prepared from avocado
treated with 1-MCP or 1-MCP & C2H4 .................................................... 126

6-4. Sugar composition of CDTA-soluble UA from EIS prepared from avocado
stored at 130C for 12 d and then transferred to 200C.................................... 127







6-5. Sugar composition of CDTA-soluble UA from EIS prepared from avocado
treated with 1-M CP or 1-M CP & C2H4...................................................... 129

6-6. Sugar composition of 4 M alkali-soluble hemicellulose released from EIS
prepared from avocado stored at 13 C for 12 d and then transferred to
200C .......................................................... ......... 130

6-7. Sugar composition of 4 M alkali-soluble hemicellulose from EIS prepared
from avocado treated with 1-MCP or 1-MCP & C2H4.................................131













LIST OF FIGURES


Figure Page

4-1. Fruit firmness (Newton) and weight loss (%) of'Simmonds' avocados stored
at 200C with different 1-M CP treatments..................................................... 73

4-2. Effect of 1-MCP (0.45 pL L-1 for 24 h) on PG and PME activities of avocados
stored at 200C ..................................... .................. .. ....................74

4-3. Effect of 1-MCP (0.45 pL L-1 for 24 h) on Cx-cellulase and a- and P-galactosidase
activities of avocados stored at 200C............................................................ 75

4-4. Effect of 1-MCP (0.45 pL L-' for 24 h) on the amount of EIS in the mesocarp
tissue and on the changes in water-, CDTA-soluble UA, and total UA in EIS
from avocados stored at 200C................................................................... 76

4-5. Molecular mass profiles of water-soluble polyuronides from EIS prepared
from avocado treated with 1-MCP (o) and without 1-MCP () ..........................77

4-6. Molecular mass profiles of CDTA-soluble polyuronides from EIS prepared
from avocado treated with 1-MCP (o) and without 1-MCP (*)........................78

4-7. Effect of 1-MCP (0.45 tL L'' for 24 h) on the changes in the extractable
amount of 4 M alkali-soluble hemicellulose in EIS from avocados stored
at 200C .................................. ............... ............. ............ .... 79

4-8. Molecular mass profiles of 4 M alkali-soluble hemicellulose from EIS prepared
from avocado treated with 1-MCP (o) and without 1-MCP (*)..........................80

4-9. Molecular mass profiles of xyloglucan in 4 M alkali-soluble hemicellulose
from EIS prepared from avocado treated with 1-MCP (o) and without
1-M CP (*) .................... .. ........................... .......... ..................... .. 81

5-1. Fruit firmness (N) and weight loss (%) of 'Tower II' avocados treated with
1-MCP (0.9 uL L' for 12 h at 200C) and/or wax and stored at 200C...................99

5-2. Fruit firmness (N) and weight loss (%) of 'Booth 7' avocados stored at 13C
after wax treatment with (o) or without (e) 1-MCP (0.9 PL L'' for 12 h at
200C) ................ .............................. .......... ................. 100


viii








5-3. PG activity of 'Tower II' avocados treated with 1-MCP (0.9 PL L' for 12 h
at 200C) and/or wax and stored at 200C.....................................................101

5-4. Effect of 1-MCP on PG activity of 'Booth 7' avocados stored at 130C after
wax treatment with (o) or without (e) 1-MCP (0.9 gL L' for 12 h at 200C).........102

6-1. Fruit firmness (N) and weight loss (%) of 'Monroe' avocados gassed
immediately with C2H4 (100 pL L-') for 12 h at 200C, then either stored
at 13C or continuously treated with 1-MCP (4.5 uL L') for 24 h at 200C
and then transferred to 130C.............................................. .................141

6-2. Fruit firmness (N) and weight loss (%) of 'Monroe' avocados treated with
1-MCP (4.5 pLL L' for 24 h at 200C) and stored at 13C ..................................142

6-3. Fruit firmness (N) of 'Booth 7' avocados treated with 1-MCP (0.9 pL LU' for
12 h at 200C), stored at 130C for 19 d, and then transferred in 20C.....................143

6-4. Weight loss (%) of 'Booth 7' avocados treated with 1-MCP (0.9 PL L'' for
12 h at 200C), stored at 130C for 19 d, and then transferred in 20C.................. 144

6-5. Carbon dioxide (mg-kg-1 h'1) and ethylene (tL-kg-'h'1) production of 'Monroe'
avocados gassed immediately with C2H4 (100 uL L'1) for 12 h at 200C, then
either stored at 130C or continuously treated with 1-MCP (4.5 L L'') for 24 h
at 200C and then transferred to 130C......................................... ......... 145

6-6. Carbon dioxide (mg-kg'1 'h1) and ethylene (pL-kg'1-h-') production of 'Monroe'
avocados stored at 130C after 1-MCP treatment (4.5 uL L-1 for 24 h at 200C)..........146

6-7. Ethylene production (iL-kg'lh'1) of 'Booth 7' avocados treated with 1-MCP
(0.9 pL L^for 12 h at 200C), stored at 130C for 19 d, and then transferred
in 20C ............ ....................................... ................. 147

6-8. PG and PME activities of'Booth 7' avocados treated with 1-MCP (0.9 pL L'
for 12 h at 200C), stored at 130C for 19 d, and then transferred in 20C..............148

6-9. Cx-cellulase and a- and P-galactosidase activities of 'Booth 7' avocados treated
with 1-MCP (0.9 iL L'' for 12 h at 200C), stored at 130C for 19 d, and then
transferred in 20 C ......................... ........ ............................ .......... 149

6-10. The changes on the amount of EIS in the mesocarp tissue and on the changes
in water-, CDTA-soluble UA, and total UA in EIS from 'Booth 7' avocados
treated with 1-MCP (0.9 pL L-1 for 12 h at 200C), stored at 130C for 19 d,
and then transferred in 20 C .............................................................. 150







6-11. Molecular mass profiles of water-soluble polyuronides from EIS prepared
from 'Booth 7' avocado.................................................................... 151

6-12. Molecular mass profiles of CDTA-soluble polyuronides from EIS prepared
from 'Booth 7' avocado ......................................... ............. ...............152













LIST OF ABBREVIATIONS


a-Gal a-galactosidase
p-Gal P-galactosidase
CDTA trans-I ,2-cyclohexylenediamine-N,N,N',N'-tetraacetic acid
CMC carboxymethylcellulose
DMC dry matter content
DP degree of polymerization
EIS ethanol insoluble solids
1-MCP 1 -methylcyclopropene
PG polygalacturonase
PME pectinmethylesterase
RH relative humidity
UA uronic acid
XG xyloglucan













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

EFFECTS OF ETHYLENE AND ITS ACTION INHIBITOR
(1-METHYLCYCLOPROPENE) FOR REGULATING RIPENING AND EXTENDING
THE POSTHARVEST LIFE OF AVOCADO

By

Jiwon Jeong

December 2001

Chairman: Donald J. Huber
Major Department: Horticultural Science

Ethylene treatment of detached mature avocado fruit promoted the onset of

ripening. Avocado fruits from immediate ethylene treatment had more uniform ripening

and better pulp quality than control fruit (not exposed to ethylene), although ethylene

treatment did not affect fruit quality as determined by dry matter content and oil content.

The 1-Methylcyclopropene (1-MCP) treatment delayed ripening of avocado fruit,

characterized by a significant reduction in the rate of fruit softening and in the timing and

intensity of the ethylene and respiratory climacterics. Avocado treated with 1-MCP also

showed significantly less weight loss and retained more green color than control fruit (not

exposed to 1-MCP).

Inhibition of polygalacturonase (PG) activity was the strongest response to

1 -MCP tind activity showed little or no recovery over the storage period. Consistent with

the marked suppression of PG levels in 1-MCP-treated avocado fruit, the solubilization

and degradation of polyuronides was significantly delayed and reduced in 1-MCP-treated







fruit. 1-MCP treatment did not significantly affect quantity or composition of the neutral

sugar of 4 M alkali-soluble hemicellulose during ripening. The 1-MCP treatment,

however, significantly reduced molecular mass downshifts in 4 M alkali-soluble

hemicelluloses and xyloglucan.

In addition to its effect on PG, 1-MCP treatment significantly delayed the

activities of Cx-cellulase, pectinmethylesterase, and total extractable a- and

P-galactosidase during avocado fruit ripening. Selected cell wall enzymes (PG,

3-galactosidase, and Cx-cellulase) were upregulated by ethylene. Exogenous ethylene

treatment before or after 1-MCP treatment did not influence fruit firmness, weight loss,

respiration, or C2H4 production, implying that ethylene response was completely

suppressed by 1-MCP.

Combined 1-MCP and wax treatment exerted maximal delay of avocado fruit

ripening by reducing the rate of fruit softening and water loss. Waxing also helped to

retain better green peel color and depressed ethylene production of avocado fruit.

Inhibition of ethylene action with 1-MCP during the early stages of the avocado

climacteric produces changes in subsequent ripening behavior. The 1-MCP blocks C2H4

action and delays C2H4-dependent ripening of avocado fruit.


xlll












CHAPTER 1
INTRODUCTION


The onset of ripening in avocado fruit is marked by a variety of biochemical

changes including a large increase in ethylene production and respiration, texture

changes, and development of flavor components (Seymour and Tucker, 1993). Fruit

softening is a major aspect of the ripening syndrome in many fruits and is considered to

be a consequence of cell wall degradation. The cell wall structure of fruit tissues has long

been a subject of interest because changes in cell wall composition and rigidity greatly

affect the firmness of the whole fruit. Such organizational changes are an integral part of

the endogenously controlled fruit-ripening process. A number of enzymes have been

implicated in cell wall metabolism in ripening fruit, including polygalacturonase,

pectinmethylesterase, Cx-cellulase, and a- and P-galactosidases.

The importance of ethylene in regulating fruit ripening has been clearly

demonstrated from analyses of fruits exhibiting suppressed ethylene biosynthesis or

action. The ethylene action inhibitor, 1-methylcyclopropene (1-MCP), has been shown to

block ethylene receptors, preventing ethylene effects in plant tissues for extended periods

(Sisler and Serek, 1997; Sisler et al., 1996a; Sisler et al., 1996b) and has provided a facile

approach for examining relationships between ethylene and fruit ripening in a range of

climacteric fruits (Abdi et al., 1998; Fan and Mattheis, 1999; Feng et al., 2000; Golding

et al., 1998; Golding et al., 1999; Lelievre et al., 1997b; Nakatsuka et al., 1997; Watkins

et al., 2000).









The objectives of the research described herein were the following: 1) to examine

the effects of postharvest application of ethylene on avocado ripening uniformity and

fruit quality; 2) to characterize physiological and biochemical responses of avocado fruit

to 1-MCP treatment and to evaluate its ability as a postharvest tool for regulating the

ripening of avocado fruit; 3) to investigate the effects of 1-MCP and waxing on ripening

characteristics in avocado fruit; and 4) to investigate the influence of 1-MCP and

ethylene on the rate of fruit softening related to changes in the activities of selected cell

wall enzymes, structural carbohydrates, and cell wall neutral sugar composition.

The overall hypothesis being tested is that ethylene will increase ripening rate,

while 1-MCP will decrease ripening rate in avocado fruit.












CHAPTER 2
LITERATURE REVIEW


Introduction

Avocado

The avocado (Persea americana Mill.) belongs to the Lauraceae family. The

avocado is now grown throughout most of the tropics and subtropics, but appears to have

originated in Central America (Bergh, 1976). Three races of avocados are known: 1)

Mexican (the most cold-tolerant fruit and generally the highest oil content); 2) West

Indian (the most cold-sensitive fruit and generally the lowest oil content); and 3)

Guatemalan, which has properties midway between the other two (Ryall and Pentzer,

1982; Seymour and Tucker, 1993). Avocados are sometimes described as tropical,

subtropical, and semitropical fruits on the basis of increasing cold hardiness and general

climactic adaptation (Bergh, 1976; Knight, 1980; Seymour and Tucker, 1993). Fruit

characteristics including size and skin texture vary considerably among the strains

(Knight, 1980).

Over 40 cultivars of avocados are grown commercially in Florida. They comprise

three general groups: cultivars of the West Indian race, cultivars of the Guatemalan race,

and "hybrid" cultivars which are mostly crosses of West Indian and Guatemalan races

(Hatton and Campbell, 1960; Hatton et al., 1964). West Indian avocados are grown

commercially in the continental United States only in southern Florida, where they

mature during the summer and early fall. The Guatemalan varieties, in contrast, mature







during the fall and winter. "Hybrid" varieties constitute nearly 90 percent of the Florida

avocados, and they mature during the fall and winter (Hatton and Campbell, 1960).

Fruit Development

The avocado fruit is classified botanically as a berry comprising seed and

pericarp, which is separated into rind (exocarp), flesh (mesocarp), and the thin, papery

layer next to the seed coat endocarpp) (Biale and Young, 1971; Seymour and Tucker,

1993). Fruit growth in avocado follows a pattern similar to that in other fruits, with rapid

cell division in the early stages after anthesis and pollination; however, cell division in

avocado continues through full maturity, and avocado fruit tend to continue growing

while attached to the tree (Schroeder, 1953; Seymour and Tucker, 1993; Valmayor,

1967).

Avocado Fruit Ripening

The onset of ripening in avocado fruit is marked by a variety of biochemical

changes including a large increase in ethylene production and respiration, texture

changes, and development of flavor components (Seymour and Tucker, 1993). Avocado

is one of the most rapidly ripening fruits (Seymour and Tucker, 1993). Eaks (1980)

reported that mature avocado fruit ripen in 6 to 12 days at 200C depending on

physiological maturity. The best ripening temperatures are between 15.5 and 240C, and

ethylene may be used to stimulate the onset of ripening (Handenburg et al., 1986).

Unlike most other fruits, avocado fruit do not normally ripen while attached to the

tree, even though full horticultural maturity may have been reached (Schroeder, 1953;

Seymour and Tucker, 1993). The precise nature of this ripening inhibition has not been

identified although the most prevalent viewpoint has been that an inhibitory substance is

transmitted from the tree to the fruit (Seymour and Tucker, 1993).







Climacteric Characteristics

The avocado is a climacteric fruit, and the onset of ripening is associated with a

sharp increase in respiration and ethylene production (Seymour and Tucker, 1993). The

respiratory climacteric in avocado fruit is initiated only after harvest (Leopold and

Kriedemann, 1975). The respiration rate of avocado typically declines after harvest, and

this transient, low respiration rate defines a lag between harvest and ripening. The sharp

increase in climacteric ethylene production is considered to be the initiator of changes in

color, texture, aroma, flavor, and other biochemical and physiological attributes (Brady,

1987; Oetiker and Yang, 1995). The climacteric increase in ethylene production is also

associated with hastened ripening of fruits. The time of onset of ethylene production in

avocado appears to be carefully controlled. Harvested avocado fruit respond to

exogenous ethylene or propylene by inducing endogenous ethylene production (Eaks,

1980).

Fruit Softening

Fruit softening is a major aspect of the ripening syndrome in many fruits and is

considered to be a consequence of cell wall degradation. The biochemical basis of

textural changes in avocado and other fruits is still incompletely understood, but probably

involves changes in the structural properties of the cell walls. The cell wall structure of

fruit tissues has long been a subject of interest because the changes in cell wall

composition and rigidity greatly affect the firmness of the whole fruit. Such

organizational changes are an integral part of the endogenously controlled fruit-ripening

process.







Cell wall changes

Fruit textural changes are due to the modification of various cell wall

components, including crystalline cellulose microfibrils, hemicelluloses, pectins, and

structural proteins (McNeil et al., 1984). The primary cell wall of fruits undergoes

structural and compositional changes as the fruits soften during ripening (Brady, 1987).

Typically, pectins are solublized and sequentially disassembled as characterized by

increased depolymerization of various pectin classes (Carrington et al., 1993; Redgwell et

al., 1992; Rose et al., 1998), and hemicelluloses are also depolymerized (Huber, 1983a).

The loss of cell wall neutral sugars, particularly galactose and/or arabinose, is closely

associated with pectin depolymerization. Hemicelluloses, notably xyloglucans, are also

modified (Rose et al., 1998).

Cell wall hydrolases

The sequential disassembly of cell wall polysaccharides during ripening may

involve disruption of both covalent and non-covalent interactions as the result of the

action of cell wall enzymes (Fisher and Bennett, 1991) and non-enzymic proteins such as

expansins. During fruit ripening, a number of qualitatively new proteins are synthesized,

some of which are cell wall-directed hydrolytic enzymes. Hydrolases temporally

associated with fruit ripening and textural changes include polygalacturonase (PG, E.C.

3.2.1.15), pectinmethylesterase (PME, E.C. 3.2.1.11), Cx-cellulase (Cx, E.C. 3.2.1.4), and

a- and P-galactosidases. In avocado fruit, both polygalacturonase and Cx-cellulase

activities have been reported to increase during ripening, whereas pectinmethylesterase

activity declines during the same period (Awad and Young, 1979; Pesis et al., 1978a;

Raymond and Phaff, 1965).







Polygalacturonase. The polygalacturonase (PG) enzymes (particularly the endo

form) have been the subject of most studies focused on the enzymology of fruit softening.

Endo-PG cleaves a-1,4-linked galacturonosyl linkages in pectin and is involved in the

degradation of cell wall polyuronides during ripening (Fisher and Bennett, 1991; Huber,

1983b). The contribution of endo-PG activity to fruit softening (particularly in tomato)

has been questioned, even though PG mRNA, PG protein, and PG activity increase

dramatically with tomato fruit ripening (Biggs and Handa, 1988; Brady et al., 1982;

Dellapenna et al., 1986). Insertion of a gene construct coding for antisense PG mRNA did

not prevent softening or polyuronide depolymerization observed during normal tomato

ripening (Smith et al., 1988). Moreover, insertion of a promoter and the gene coding for

PG into the rin (ripening inhibitor) tomato mutant caused the fruit to undergo

polyuronide solubilization and depolymerization, but not softening (Giovannoni et al.,

1989). Avocado fruit PG has been purified by Raymond and Phaff (1965) and shows the

characteristics of an endoenzyme with a pH optimum of 5.5 in sodium acetate buffer.

Polygalacturonase activity is typically not detected in avocado fruit at the pre-ripe

(preclimacteric) stage, but it increases dramatically during the climacteric and

postclimacteric stages (Awad and Young, 1979; Huber and O'Donoghue, 1993).

Polygalacturonase activity in ripening avocados has been reported to increase

approximately three days after the first appearance of Cx-cellulase activity (Awad and

Young, 1979). These observations suggest that PG is a dominant factor in the extensive

degradation of polyuronides during avocado fruit ripening (Hobson, 1962).

Pectinmethylesterase. Pectinmethylesterase (PME) catalyzes the removal of

methoxyl groups of esterified pectins and has been implicated in cell wall softening even







though its contribution to the process is not entirely clear (Huber, 1983b).

Pectinmethylesterase is responsible for the deesterification of pectin required before

polygalacturonase-mediated depolymerization of pectins during ripening (Awad and

Young, 1980; Wakabayashi et al., 2000). The presence of PME in avocado fruits and its

rapid decline in activity after harvest are well documented (Awad and Young, 1979;

Awad and Young, 1980; Zauberman and Schiffmann-Nadel, 1972).

Cx-cellulase. Cellulase traditionally has been identified by its ability to reduce the

viscosity of carboxymethylcellulose. In avocado fruit, the enzyme is an endohydrolase

and is specific for P-1,4-linked glucans (Hatfield and Nevins, 1986). Cx-cellulase first

appears at the onset of avocado fruit ripening (Christofferson et al., 1984) and increases

dramatically as the fruit soften and ripen (Awad, 1977; Awad and Young, 1979; Pesis et

al., 1978b). Avocado cellulase, however, does not produce reducing groups related to

cellulose degradation after incubation with isolated cell walls of unripe or ripe avocado

tissue (Hatfield and Nevins, 1986).

Alpha- and beta-galactosidase. The involvement of the purported avocado

a-galactosidase in pectin degradation and/or fruit softening is unclear. Alpha

(1--4)-linkage galactosyl residues are known to exist in rhamnogalacturonan II;

however, this pectic polysaccharide is a relatively minor component of the cell wall and

is thought not to play a structural role (O'Neil et al., 1990).

Beta-galactosidase is widely distributed in various plant tissues, including

developing fruits (Bartley, 1974; Bums, 1990; Gross et al., 1986). Beta-galactosidase

appears early in fruit ripening and before the appearance of PG (Pressey, 1983; Watkins

et al., 1988). Studies on tomato (Watkins et al., 1988), mango and papaya (Lazan and Ali,







1993), apple (Bartley, 1974), kiwifruit (Wegrzyn and MacRae, 1992), and hot pepper

(Gross et al., 1986) have reported dramatic increases in the activity of p-galactosidase

during ripening. One tomato P-galactosidase isozyme that increased during ripening

exhibited the ability to hydrolyze galactan-rich polysaccharides derived from tomato cell

walls (Pressey, 1983). Pressey (1983) suggested that P-galactosidase is involved in the

degradation of tomato cell wall galactans and fruit softening. Net cell wall galactosyl

content decreases with the ripening of many fruits (Gross and Sams, 1984); and the

amount of free, monomeric galactose in tomato fruit increases during ripening (Gross,

1983). These findings suggest a possible role for P-galactosidases in the modification of

cell wall components in ripening fruits.

Ripening-induced compositional changes

Cell-wall polymer alteration during fruit ripening influences the final texture of

the ripe fruit. Increased solubilization and depolymerization of pectic polysaccharides

have been observed during the ripening of many fruit types, such as tomato (Huber,

1983a), mango (Roe and Bruemmer, 1981), pear (Ahmed and Labavitch, 1980), and

muskmelon (McCollum et al., 1989). Typically, the molecular weight of the chelator-

soluble polyuronides decreases during ripening. Exceptions have been noted with apple

(Knee, 1978) and strawberry (Huber, 1984). Neither of these fruits contains active

endo-PG. In avocado fruit, the solubility of polyuronides increases substantially during

ripening, concomitant with marked downshifts in molecular mass (O'Donoghue and

Huber, 1992).

Although changes in the total amount of extractable hemicelluloses are not

usually observed, ripening is often characterized by a lowering of the molecular weight of

these polymers (Huber, 1984; McCollum et al., 1989; Tong and Gross, 1988). During







ripening of avocado, hemicelluloses exhibit a reduction in large molecular mass polymers

and a proportional increase in lower molecular mass polymers (O'Donoghue and Huber,

1992).

Another facet of cell wall degradation during fruit ripening is the loss of non-

cellulosic neutral sugars, presumably solublilized by the action of cell wall glycosidases

or exohydrolases. The most predominant change in neutral sugar composition of alcohol-

insoluble solids during the ripening of a number of fruit types was the loss of galactose,

arabinose, and/or xylose (Gross and Sams, 1984).

Modification of Ripening

Avocado fruit ripening and softening can be delayed by low-temperature storage

(Bower and Cutting, 1988). Ripening is also delayed by storing the fruit under low 02 (2

to 5%) and high CO2 (3 to 10%) conditions (Biale and Young, 1971; Kader, 1992;

Seymour and Tucker, 1993). Another factor that may regulate the onset of ripening is the

internal calcium concentration. Avocado fruit with low levels of calcium ripen more

rapidly than those with higher levels of calcium (Eaks, 1985); and avocado ripening can

be delayed by vacuum infiltration of calcium (Wills and Tirmanzi, 1982).

Ethylene

Gaseous Plant Growth Regulator

Ethylene is a well-known plant hormone that controls many plant responses,

including growth, senescence, ripening, abscission, and seed germination (Abeles et al.,

1992). The hormone is a natural product of metabolism and plants respond to both

endogenous and exogenous ethylene (Abeles et al., 1992). The sharp increase in

climacteric ethylene production at the onset of ripening is considered to be the initiator of







changes in color, texture, aroma and flavor, and other biochemical and physiological

attributes (Brady, 1987; Oetiker and Yang, 1995).

Ethylene Biosynthetic Pathway

The ethylene biosynthetic pathway is established as methionine -+

S-adenosylmethionine (SAM) -+ 1-aminocyclopropane-l-carboxylic acid (ACC) -+

ethylene (Yang and Hoffman, 1984). The conversion of SAM to ACC is catalyzed by

ACC synthase (ACS), whereas the subsequent oxidation of ACC to ethylene is catalyzed

by ACC oxidase (ACO). In climacteric fruit, the transition to autocatalytic ethylene

production appears to be due to series of events in which tissue-specific ACS and ACO

genes are developmentally up-regulated. Both ACS and ACO are encoded by multigene

families and are limiting in preclimacteric fruit but are greatly induced during ripening

(Oetiker and Yang, 1995; Yueming and Jiarui, 2000).

Ethylene Perception and Signal Transduction

A current model of the ethylene signal transduction pathway was formulated on

the basis of cloned Arabidopsis genes (Chang and Shockey, 1999; Hua et al., 1995).

Ethylene is perceived by a family of histidine kinase-like receptor homodimers: ETR1,

ERS 1, ETR2, EIN4, and ERS2 in Arabidopsis. The ETR1 (ethylene-resistant) gene, the

first ethylene receptor gene cloned (Chang et al., 1993), encodes a membrane-spanning

protein that consists of an amino terminal domain, a putative histidine kinase domain, and

a receiver domain (Chang et al., 1993; Chang and Meyerowitz, 1995). The ERS (ethylene

response sensor), which lacks the receiver domain, was later isolated by cross

hybridization with ETR1 (Hua et al., 1995). The membrane-localized ethylene-binding

sites require a copper cofactor, and proper receptor function relies on a copper transporter







(Rodriguez et al., 1999). A tomato homologue of ETRI, called NR, has been cloned from

the ripening-impaired tomato mutant Never ripe (Nr) (Wilkinson et al., 1995). Never ripe

mutants are affected in ethylene perception and show no accumulation of Nr mRNA

(Payton et al., 1996). Never ripe mutants synthesize reduced quantities of ethylene

compared to wild-type fruit and retain residual ethylene responsiveness (Lanahan et al.,

1994). The identification of the Nr tomato ripening mutant as an ethylene receptor in

combination with application of the generation of transgenic fruit with reduced ethylene

production has provided substantial evidence that ethylene receptors regulate a defined

set of genes which are expressed during fruit ripening.

A raf-like kinase, CTR1, acts downstream of the ethylene receptors and appears to

be a negative regulator of ethylene responses as a loss of gene function results in

constitutive ethylene responses (Kieber et al., 1993). In the absence of ethylene, the

receptors repress responses possibly through direct activation of the downstream negative

regulator, CTR1 (Clark et al., 1998; Hua and Meyerowitz, 1998). Binding of ethylene, on

the other hand, inhibits receptor activation of CTR1 (Hua and Meyerowitz, 1998).

Downstream EIN2 is a structurally novel protein containing an integral membrane

domain (Alonso et al., 1999; Chang and Shockey, 1999). The EIN3 family of DNA-

binding proteins regulates transcription in response to ethylene, and an immediate target

of EIN3 is a DNA-binding protein of the EREBP (ethylene-responsive element binding

protein) family, which relates to the AP2 family of transcription factors (Chang and

Shockey, 1999).

Practical Uses of Ethylene

Ethylene affects the physiology of various fruits in storage. Classic examples of

the use of ethylene are its commercial application to ripen bananas and tomatoes and to







degree citrus fruits. It has been established that ethylene treatment of detached mature

avocado fruit promotes the onset of ripening (Biale, 1960; Eaks, 1966; Eaks, 1978; Eaks,

1980; Erickson and Yamashita, 1964; Gazit and Blumenfeld, 1970; Zauberman and

Fuchs, 1973). Treatment of mature 'Hass' avocados with 100 IL L-' ethylene for 24 h

after harvest hastens the onset of ripening, making them ready to eat in 3 or 4 days (Eaks,

1966). The response of the fruit to ethylene applications also depends on its maturity.

Fruit of more advanced maturity respond much more rapidly to ethylene treatment than

less mature fruits (Biale, 1960; Burg and Burg, 1965; Eaks, 1966; Hansen and Blanpied,

1968).

Ethylene Antagonists

Ethylene responses can be controlled by regulating ethylene's production or

action. The use of inhibitors of ethylene production is of limited value since they will not

protect against the effects of exogenous ethylene. Ethylene action inhibitors are

considered preferable for use in agriculture because they protect against both endogenous

and exogenous sources of ethylene.

Silver thiosulfate (STS) is a known inhibitor of ethylene action (Veen, 1983).

Silver thiosulfate probably acts through the irreversible interaction of silver ions with

ethylene binding sites (Sisler et al., 1986; Veen, 1983), and its effect is considered to be

non-competitive (Beyer, 1976). Silver thiosulfate has been used with much success on cut

flowers and potted plants (Veen, 1983). Although STS is widely used to reduce ethylene

action, environmental concerns have restricted its application in some countries (Serek

and Reid. 1993).

The search for inhibitors of ethylene action has concentrated on ethylene

analogues. In studies examining the nature of ethylene binding and the nature of the







binding site, a number of organic molecules that appear to block the ethylene receptor for

extended periods have been identified (Sisler and Blankenship, 1993a; Sisler et al., 1986;

Sisler et al., 1993). The 2,5-Norbornadiene (NBD), a competitive inhibitor of ethylene

binding (Sisler et al., 1986), was one of the first cyclic olefins to be widely used in

studies of ethylene action; however, its effectiveness requires continued exposure

because binding is competitive. Furthermore, NBD is toxic and exhibits an offensive

odor, which limits its commercial usefulness (Sisler and Serek, 1999). Sisler's group

(Sisler and Blankenship (1993a) also reported the ethylene antagonism of

diazocyclopentadiene (DACP), a putative photoaffinity label for the ethylene-binding

site. The photoactive DACP molecule binds at the ethylene binding site and then

becomes covalently attached when photoactivated (Serek et al., 1994a).

Diazocyclopentadiene's effectiveness in inhibiting ethylene action is a result of a

permanent attachment to the binding site (Serek et al., 1994b). Diazocyclopentadiene

inhibited the effects of ethylene in fruits of banana (Musa sp.), kiwifruit (Actinidia

deliciosa), persimmon (Diospyros kaki), tomato (Sisler and Blankenship, 1993b; Sisler

and Lallu, 1994), and several ornamentals including carnation, geranium (Pelargonium

zonale), and rose (Rosa hybrida) (Serek et al., 1993; Serek et al., 1994b). The

commercial usefulness of DACP is limited because of its explosiveness at high

concentrations (Sisler and Serek, 1997).

The synthetic cyclopropenes including cyclopropene (CP), 1-methylcyclopropene

(1-MCP), and 3.3-dimethylcyclopropene (3.3-DMCP), bind to the ethylene receptor and

prevent the physiological action of ethylene for extended periods in a number of plants

(Sisler and Serek, 1997; Sisler et al., 1996a; Sisler et al., 1996b). All of these are gaseous







at room temperature and have no obvious odor at the concentrations needed to protect

against the effects of ethylene (Sisler and Serek, 1997). Among recently developed

inhibitors of ethylene responses, 1-MCP has proven to be the most useful. It is more

stable than CP, is active at very low concentrations, and provides protection for a longer

time than 3,3-DMCP (Sisler and Serek, 1997). Banana and tomato fruit treated with CP

or 1-MCP at 24C remained insensitive to ethylene for 12 days and then ripened

normally. When treated with 3,3-DMCP, fruit remained insensitive for 7 days (Sisler and

Serek, 1997; Sisler et al., 1996a).


1-Methylcyclopropene (1-MCP)

Inhibitor of Ethylene Responses

The 1-methylcyclopropene (1-MCP) binds ethylene receptors and prevents

ethylene effects in plant tissues for extended periods (Sisler and Serek, 1997; Sisler et al.,

1996a; Sisler et al., 1996b). The 1-MCP is a planar molecule with a methyl group

attached at the double bond. It is a highly strained olefin, and binds in an apparently

irreversible manner to the ethylene receptor. The 1-MCP, a nontoxic and relatively

simple organic compound, inhibits ethylene action when plants are treated at

concentrations as low as 0.5 nl.-1' (Sisler and Serek, 1997).

The 1-MCP is a very effective alternative to STS as a pretreatment for ethylene-

sensitive flowers (Serek et al., 1994a; Serek et al., 1995a). At very low concentrations,

1-MCP is as effective as STS, not only in preventing the effects of exogenous ethylene,

but also in delaying senescence of flowers whose natural senescence is mediated by a rise

in endogenous ethylene production (Serek et al., 1995a). The 1-MCP has substantially







improved properties over DACP (Serek et al., 1994a). The effective concentration of 1-

MCP is more than 10-fold lower than that for DACP (Sisler et al., 1996b).

The 1-MCP competes with ethylene for the ethylene receptor. Yueming et al.

(1999) reported the affinity of 1-MCP for ethylene-binding sites using Lineweaver-Burk

plots (Whitaker, 1972). The Km for 1-MCP was lower than that estimated for ethylene.

It was suggested that 1-MCP has relatively greater affinity than ethylene for the ethylene-

binding sites and thus it was concluded that inhibition by 1-MCP is noncompetitive.

After ethylene treatment, much of the ethylene diffuses rapidly from the receptor,

whereas 1-MCP remains bound for long periods (many days) after 1-MCP treatment

(Sisler and Serek, 1997). While 1-MCP is bound, ethylene cannot bind. In ripening fruit,

even high concentrations of ethylene (e.g., 1000 pL L1') do not initiate a response after

1-MCP is bound at the receptors (Sisler and Serek, 1999). However, plants treated with

1-MCP do eventually develop sensitivity to ethylene (Sisler and Serek, 1999). It is

possible that new receptors are synthesized or that 1-MCP dissociates from the receptors

(Sisler and Serek, 1999).

The 1-MCP acts by binding to ethylene-receptors so that ethylene cannot elicit

subsequent signal transduction and transcriptional activation (Sisler and Blankenship,

1996). The 1-MCP presumably competes with ethylene for a metal in the ethylene

receptor, preventing ethylene from binding in treated tissues. As it binds to the receptor

so strongly, perhaps 1-MCP remains bound to the metal in the receptor, and the formation

of an active complex is not completed (Sisler and Serek, 1997).







Treatment of Plants with 1-MCP

Effect of concentration, exposure time, and temperature

The 1-MCP is effective in inhibiting ethylene action in various plant tissues at nL

L-' levels. There are large differences in the 1-MCP concentrations required to inactivate

ethylene responses in different plant species and tissues, and the 1-MCP concentrations

required to protect plants against ethylene depend on the time of exposure. With longer

exposure periods, lower 1-MCP concentrations are effective (Sisler et al., 1996b). The 1-

MCP concentrations required to protect plants against ethylene also depend on the

temperature of exposure. At lower temperatures, higher concentrations of 1-MCP are

required for complete protection of carnation flowers against ethylene (Sisler et al.,

1996b), suggesting that attachment to the receptor is less than at higher temperatures

(Sisler and Serek, 1997).

The 1-MCP substantially extends the display life of various cut flowers and potted

plants (Porat et al., 1995; Serek et al., 1994a; Serek et al., 1994b). The 1-MCP

concentrations as low as 0.5 nL L-' were sufficient to protect carnation (Dianthus

caryophyllus) flowers for several days against ethylene (Sisler and Serek, 1997).

Treatment with 40 nL LU' of 1-MCP for 6 h was required for complete inhibition of

ethylene effects in pea seedlings (Sisler and Serek, 1997). Pretreatment with low

concentrations (1 to 20 nL Li' range) of 1-MCP for 6 h eliminated the ethylene-induced

abscission or wilting of alstromeria, begonia, carnation, matthiola, phalaenopsis, and rose

(Porat et al., 1995; Serek et al., 1994a; Serek et al., 1995a). The 1-MCP at 20 nL L'

prevented ethylene-induced bud, flower, and leaf abscission; and flower senescence

without phytotoxic symptoms (Serek et al., 1994a). Ornamentals treated with 0.5 nL L'







1-MCP did not respond to ethylene even at concentrations as high as 1000 nL LU' (Serek

et al., 1994a; Sisler et al., 1995).

With fruits, 1-MCP tends to behave differently depending on the fruit type. 1-

MCP delays ripening and improves storage quality of climacteric fruits such as pears

(Pyrus cominuni. L. cv. Passe-Crassane) (Lelievre et al., 1997b), bananas (Musa sp.)

(Golding et al., 1998; Golding et al., 1999; Sisler and Serek, 1997), plums (Prunus

salicina Lindl)(Abdi et al., 1998), tomatoes (Lycopersicon esculentum Mill) (Nakatsuka

et al., 1997; Sisler and Serek, 1997), apples (Malus sylvestris L.) (Fan and Mattheis,

1999; Watkins et al., 2000), and avocado (Persea Americana Mill) (Feng et al., 2000). In

tomato fruits obtained commercially at the mature green stage, 1-MCP treatment at 7 nL

L' for 24 h was needed for complete protection against ethylene (Sisler and Serek, 1997).

1-MCP treatment at 0.5 nL L-' for 24 h protected banana against exogenous ethylene

(Sisler and Serek, 1997). 1-MCP treatment of apricot fruit at 1 LL L-' for 4 h at 200C

delayed the onset of ethylene production and reduced the respiration rate (Fang et al.,

2000). This delay was sufficient to extend the storage life of apricot by delaying loss of

firmness and titratable acidity as well as color changes associated with ripening.

In strawberry, a non-climacteric fruit, treatment with 1-MCP at 5 to 15 nL LU

extended the storage life by =35% at 200C and 150% at 50C. At higher 1-MCP

concentrations (500 nL L'1), however, there was an accelerated softening due mostly to

the onset of rotting with a 30% to 60% decrease in storage life at both 20 and 5C (Ku et

al., 1999).

The storage life of broccoli increased with increasing 1-MCP concentration and

exposure time at both 5 and 200C (Ku and Wills, 1999). Broccoli treated with 1-MCP







(1 gL L') at 200C exhibited a significantly longer storage life than that treated with 1-

MCP at 5C (Ku and Wills, 1999).

Effect on respiration and ethylene production

The 1-MCP significantly delays the onset of increased ethylene production and

respiration in climacteric fruits (Fan and Mattheis, 1999; Fang et al., 2000; Golding et al.,

1998; Golding et al., 1999). The 1-MCP blocked ethylene and suppressed respiration in

apple fruit (Fan and Mattheis, 1999). Similarly 1-MCP treatment of apricot fruit at 1 L

L-1 for 4 h at 20C delayed the onset of ethylene production and reduced respiration rate

(Fang et al., 2000). Treatment of carnation flowers with 1-MCP prevented much of the

climacteric rise in ethylene production (Sisler et al., 1996b). In contrast, 1-MCP

treatment of grapefruit significantly enhanced ethylene production (Mullins et al., 2000),

and it has also been reported with mature green banana treated with 1-MCP (45 tL L'1

for 1 h) 6 or 12 h after propylene treatment (500 tL L'') (Golding et al., 1998).

Other responses of 1-MCP

The 1-MCP delays symptoms of senescence (such as electrolyte leakage and lipid

fluidity) in Petunia (Serek et al., 1995b). The 1-MCP retards storage-induced leaf

yellowing, but reduces rooting ability of stored cuttings (Muller et al., 1997).

The 1-MCP treatment enhanced xylanase-induced ethylene production in tomato

(Anderson et al., 1996) and inhibited ethylene epinasty in tomato plants (Cardinale et al.,

1995). In 'Passe-Crassane' pears, 1-MCP treatment resulted in reduced accumulation of

ACC oxidase transcripts and ethylene production during chilling (Lelievre et al., 1997b).

Apricot fruit treated with 1 pL L'' 1-MCP at for 4 h at 200C showed a reduction in

titratable acidity loss during storage at 0 or 200C and delayed production of volatile

alcohol and esters during ripening at 200C (Fang et al., 2000). The 1-MCP (0.45 mmol-m







3 for 12 h) inhibited color changes in apple fruit (Fan and Mattheis, 1999). While 1-MCP

(45 pL L for 1 h) delayed ripening of bananas, it also caused uneven peel degreening

and suppressed volatile aroma production (Golding et al., 1998). The 1-MCP (50 100

nL L'1 for 1 h) delayed degreening of oranges but increased incidence of chilling injury,

rot development and off-flavors (Porat et al., 1999).

The 1-MCP extended the postharvest life of strawberries mainly through a delay

in the development of rotting, but with the beneficial response obtained over a narrow

concentration range (Ku et al., 1999). Since deterioration was primarily related to decay,

high concentrations of 1-MCP may interfere with a natural defense system in strawberry

fruit (Ku et al., 1999).












CHAPTER 3
POSTHARVEST ETHYLENE TREATMENT FOR UNIFORM RIPENING OF WEST
INDIAN AND WEST INDIAN-GUATEMALAN HYBRID AVOCADO FRUIT

Introduction

Ethylene plays a vital role in the ripening of climacteric fruits, and whether

applied exogenously or produced naturally, initiates ripening and softening. The avocado

(Persea americana Mill.) is one of the most rapidly ripening of fruits, often completing

ripening within 5 to 7 d following harvest (Seymour and Tucker, 1993).

Ethylene treatment of detached mature avocado fruit promotes the onset of

ripening (Eaks, 1966, 1980; Gazit and Blumenfeld, 1970; Zauberman and Fuchs, 1973;

Zauberman et al., 1988). Treatment of mature 'Hass' avocados with 100 PL L'' ethylene

for 24 h after harvest hastens the onset of ripening, making them ready to eat in 3 or 4 d

(Eaks, 1966). A number of fruits, notably banana (Inaba and Nakamura, 1986), tomato

(Jahn, 1975), pear (Chen et al., 1996), and mango (Barmore, 1975), are commercially

harvested prior to the onset of ripening and treated with ethylene gas to compress the

ripening period and allow uniform ripening among fruits.

Avocados, especially West Indian cultivars or their hybrids, have potential to be

marketed as a premium-quality product. As with all avocado types, they do not ripen until

harvested. However, this convenience creates difficulty in marketing avocados with

uniform ripeness since the timing of ripening initiation can vary widely within fruit lots,

depending upon the stage of maturation at harvest and the variety. However, the

influence of ethylene on the ripening of these cultivars has not previously been studied,







unlike Guatemalan types, Mexican types, or their hybrids. Programmed ripening would

give shippers the ability to ship high quality, uniformly ripe avocados and lead to the

implementation of a premium-quality avocado program.

The objective of this research was to determine the effects of temperature and

duration of ethylene gassing on fruit quality, time to ripen, ripening uniformity, and shelf

life of early-season ('Simmonds'), mid-season ('Booth 7') and late-season ('Monroe')

varieties. Hypothesis: Treatment of avocado fruit with ethylene will assist in initiating

uniform ripening and maintaining fruit quality.


Materials and Methods

A series of experiments were performed during the 1998-99 production season to

determine the effects of postharvest application of ethylene gas on avocado ripening and

fruit quality. These experiments were tested on early-season ('Simmonds'), mid-season

('Booth 7'), and late-season ('Monroe') cultivars.

Plant Materials

Early-season ('Simmonds') cultivar

'Simmonds' is a West Indian avocado cultivar and is chilling sensitive (Crane et

al., 1996; Hatton and Reeder, 1965). Mature avocado fruit were obtained from a

commercial grower in Homestead, Florida, packed in fiberboard cartons, and transported

to the Postharvest Horticulture Laboratory in Gainesville within 24 h after harvest.

Individual experiments were carried out for fruit picked at early (29 June 1998), mid- (13

July 1998), and late (27 July 1998) harvest dates. Fruit were selected for uniformity of

size (average weight, early-harvest date 476.3 g, mid-harvest date 436.6 g, and late-

harvest date 422.4 g) and shape (average diameter at equatorial region, early-harvest date




23


9.4 cm, mid-harvest date 9.1 cm, and late-harvest date 9.1 cm), and then were surface

sterilized in a 15% (90 mM NaOCI) commercial bleach solution, rinsed, and dried.

Mid-season ('Booth 7') cultivar

'Booth 7' is a "hybrid" cultivar, a cross of West Indian and Guatemalan races

(Hatton, 1960; Hatton, 1964). Individual experiments were carried out for fruit picked at

mid- (3 Oct 1998) and late (17 Oct 1998) harvest dates. Fruit were selected for uniformity

of size (average weight, mid-harvest date 453.6 g and late-harvest date 411 .lg) and shape

(average diameter at equatorial region, mid-harvest date 9.1 cm and late-harvest date 8.9

cm).

Late-season ('Monroe') cultivar

'Monroe' is a "hybrid" cultivar, which is a cross of West Indian and Guatemalan

strains (Hatton, 1960; Hatton, 1964). Individual experiments were carried out for fruit

picked at early (20 Nov 1998), mid- (4 Dec 1998), and late (18 Dec 1998) harvest dates.

Fruit were selected for uniformity of size (average weight, early-harvest date 799.5 g,

mid-harvest date 771.1 g, and late-harvest date 703.1 g) and shape (average diameter at

equatorial region, early-harvest date 10.7 cm, mid-harvest date 10.4 cm, and late-harvest

date 10.4 cm).

Experimental Procedures

Early-season ('Simmonds') cultivar

For the immediate ethylene gassing treatment, 24 fruit were placed in a chamber

(174 L) and treated with flow-through air or 100 pL L'' ethylene at a flow rate of 4000

mL min-'. CO2 levels in the treatment chambers did not exceed 0.05%. Ethylene was

applied to fruits at 200C and 85% relative humidity (RH) for 48 h. Following the ethylene

treatment, fruit were transferred to air at 10 or 120C (85% RH) for early (29 June 1998)







and mid- (13 July 1998) harvest dates, respectively, and stored for 5 or 12 days. For late

(27 July 1998) harvest dates, ethylene-treated fruit were transferred to air at 130C (85%

RH), and stored for 5 or 9 d. After 5 d storage, fruits were transferred to 200C (85% RH)

and evaluated on a daily basis for fruit quality until fully ripe. Full-ripe stage was defined

as the point at which fruits softened to 10 to 15 N of firmness values, considered too soft

for commercial handling by author. For the delayed ethylene treatment, ethylene was

applied to fruits at 200C and 85% relative humidity (RH) for 48 h after 7 d at 10, 12, or

13C (85% RH) for early, mid-, and late harvest dates, respectively. Following the

ethylene treatment, fruit were transferred to air at 10 or 120C (85% RH) for early and

mid-harvest dates, respectively, and stored until day 12. For late harvest dates, ethylene-

treated fruit were transferred to air at 130C (85% RH) and stored until day 9. Control fruit

(not exposed to ethylene) were maintained under identical storage conditions.

Mid-season ('Booth 7') cultivar

Forty-five fruit were placed in a chamber (174 L) and immediately treated with

flow-through air or 100 pAT-1 ethylene at a flow rate of 4000 mL min'. CO2 levels in the

treatment chambers did not exceed 0.05%. Ethylene treatment was performed for two

exposure periods (12 or 24 h) at two temperatures (12 or 200C) and 85% relative

humidity (RH). Following the ethylene treatments, fruit were transferred to air at 12C

(85% RH). Control fruit (not exposed to ethylene) were maintained under identical

storage conditions. After 7 d storage, fruits from each treatment were transferred to 200C

(85% RH) and evaluated on a daily basis for fruit quality until fully ripe.







Late-season ('Monroe') cultivar

Forty-five fruit were placed in a chamber (174 L) and immediately treated with

flow-through air or 100 tL L'1 ethylene at a flow rate of 4000 mL min-'. Ethylene

treatment was performed for two exposure periods (12 or 24 h) at two temperatures (13

or 200C) and 85% relative humidity (RH). Following the ethylene treatments, all fruits

were transferred to storage rooms at 130C (85% RH). Control fruit (not exposed to

ethylene) were maintained under identical storage conditions. After 7 d, fruits from each

treatment were transferred to 200C (85% RH) and evaluated on a daily basis for fruit

quality until the full-ripe stage. Fruit quality was assessed on the basis of fruit firmness,

peel color, pulp dry matter and oil content, and incidence of decay. Oil content was

determined at harvest and at the full-ripe stage. Incidence of decay was measured as the

percentage of fruit exhibiting decay at the stem-end or on the peel.

Fruit Firmness

Firmness was determined on whole, unpeeled fruits using an Instron Universal

Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat-plate probe (5 cm

in diameter) and 50 kg load cell. After establishing zero force contact between the probe

and the equatorial region of the fruit, the probe was driven with a crosshead speed of 10

mm min-'. The force was recorded at 2.5 mm deformation and was determined at two

equidistant points on the equatorial region of each fruit.

Peel Color

Each fruit was marked at the equatorial region (2 regions per fruit), and color at

the same location was recorded every other day as L*, hue angle, and chroma value with

a Minolta Chroma Meter (CR-2000, Minolta Camera Co Ltd., Japan). The chroma meter







was calibrated with a white standard tile. The color was reported as hue angle (H), with

a value of 900 representing a totally yellow color and 1800 a totally green color. The

results are presented as lightness (L*), chroma (C*), and hue angle (H). The chroma and

hue angle were calculated from the measured a* and b* values using the formulas C* =

(a*2+b*2) 1/2 and H = arc tangent (b*/a*) (McGuire, 1992).

Pulp Dry Matter Content

Each fruit was cut into longitudinal slices, and the peel and seed discarded. A pulp

sample (5 g) from the longitudinal slice was weighed, dried at 600C in an oven for 48 h,

and then re-weighed. Dry matter content was determined by the following equation:

Pulp dry matter content (%) = (M/Md X 100,

Where M, is the initial weight of the fresh sample and M is the final weight of

dried sample.

Oil Content

Oil content was determined for the mid- and late-season cultivars using

modifications to the procedure of Folch et al (1957). Mesocarp tissue (0.5 g) taken from

the equator of each fruit was homogenized with 15 ml of chloroform:methanol (2:1, v/v)

for 1.5 min using a Polytron (Kinematica Gmbh Karens, Lunzern, Switzerland) (at speed

setting #7). The samples were filtered through GF/C filter paper (Whatman) and re-

extracted with 15 mL of chloroform:methanol (2:1, v/v). Twenty-five percent of the total

volume of 0.88 % KCl was added in the samples, and they were transferred to a

separatory funnel. The lower, lipid-containing layer was collected and washed with 25%

of total volume of distilled H20. The samples were separated from distilled H20, dried

with anhydrous sodium sulfate for 12 h, and filtered into tared test tubes. The solution







was evaporated with an Evapomix (Buchler Instruments, Inc.) maintained below 50"C for

12 h. The tubes were again weighed for estimation of total oil content.

Statistical Analysis

The experiments were laid out in a completely randomized design. Statistical

procedures were performed using the PC-SAS software package (SAS-Insititute, 1985).

Data were subjected to ANOVA using the General Linear Model (Minitab, State College,

PA). Differences between means were determined using Duncan's multiple range test.


Results

Early-Season ('Simmonds') Cultivar

Fruit firmness

Initial firmness of early-, mid-, and late-harvest fruit averaged 107.4 9.5, 103.1

+ 15.5, and 93.1 + 18.0 N, respectively (Tables 3-1, 3-2, and 3-3). By comparison,

avocado fruit at the full-ripe stage typically exhibit firmness values in the range of 10 to

15 N. In early-harvest fruit, although firmness declined significantly during storage,

control and ethylene-treated fruit exhibited no significant differences in average firmness

values after 5 d at 100C followed by 3 d at 200C or 12 d at 100C (Table 3-1). After 12 d at

100C, avocado fruit from the immediate ethylene treatment showed less variability in

firmness than those from control and delayed ethylene treatment. In mid-harvest fruit,

firmness declined dramatically during storage for 5 and 12 d at 120C, and significant (p <

0.05) differences between control and ethylene-treated fruits were evident (Table 3-2).

Control fruit stored for 5 d at 120C were significantly firmer (31.4 5.5 N, data not

shown) than ethylene-treated fruit (14.9 2.6 N) and required 2 d at 200C for all fruits to

reach the full-ripe stage (10 15 N). After 12 d at 120C, fruits from both immediate and







delayed ethylene treatments were softer and showed more uniformity in firmness values

than control fruit stored under similar condition.

Table 3-1. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
early-harvest (06/29/98) gassed immediately for 48 h at 20C and transferred to 10C or
stored at 10C for 7 d then gassed for 48 h at 20C and then transferred to 10C. Values
followed by the same letter do not differ significantly according to Duncan's Multiple
Range Test (p < 0.05). Data are means standard deviation of 6 independent samples.

Treatment Days to Fruit Decay Dry L* Chroma Hue
fully ripe firmness incidence matter value value angle
(N) (%) (%) (Ho)

At harvest -- 107.4 -- 12.9 40.9 24.5 125.7
S9.5 1.0 1.8 2.2 1.8

Control 5 d at
(noC2H4 100C 14.0 0 11.8 43.5 24.2 125.2
gassing) & 3 d at 1.7 a a a a
20C

Immediate 5 d at
C2H4 10"C 14.3 0 11.8 44.2 26.6 123.9
gassing & 3 d at 2.3 a a a a
20C
Control
(noC2H4 12 d at 13.5 0 12.9 49.5 25.8 117.8
gassing) 10C 2.8 a a b a

Immediate
C2H4 12 d at 13.3 0 11.6 46.2 33.4 119.6
gassing 10"C 1.3 b b a a

Delayed
C2H4 12 d at 11.5 0 11.8 46.3 23.9 119.1
gassing 100C 2.3 b b b a


In late-harvest fruit, firmness declined during storage for 5 and 9 d at 130C (Table

3-3). Control and ethylene-treated fruit exhibited significant (P < 0.05) differences in

average firmness values for fruit stored for 5 and 9 d at 130C. As with control fruit from

the mid-harvest date, control fruit stored for 5 d at 130C were significantly firmer (19.0 +







3.4 N, data not shown) than ethylene-treated fruit (10.8 2.0 N) and required 2 d 20C

for all fruits to reach the full-ripe stage (10 to 15 N). After 9 d at 130C, fruit from

immediate ethylene treatment were significantly softer and showed more uniformity in

firmness values than fruit from delayed gassing treatment and air-treated fruit stored

under similar conditions (Table 3-3).

Table 3-2. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
mid-harvest (07/13/98) gassed immediately for 48 h at 20C and transferred to 12C or
stored at 12C for 7 d then gassed for 48 h at 20C and then transferred to 12C. Values
followed by the same letter do not differ significantly according to Duncan's Multiple
Range Test (p < 0.05). Data are means standard deviation of 6 independent samples.

Treatment Days to Fruit Decay Dry L* Chroma Hue
fully ripe firmness incidence matter value value angle
(N) (%) (%) (Ho)

At harvest -- 103.1 -- 13.2 40.9 26.1 125.0
15.5 1.0 +1.4 2.9 1.9

Control 5 d at
(no C2H4 12'C 11.9 0 12.3 49.3 24.4 119.4
gassing) & 2 d at 1.8 a a a a
20C

Immediate
C2H4 5 d at 14.9 0 12.1 47.6 25.1 119.7
gassing 12C 2.6 a a a a

Control
(no C2H4 12 d at 13.4 16.7 12.2 50.6 28.9 116.1
gassing) 12C 2.7 a c b a

Immediate
C2H4 12 d at 10.1 16.7 10.9 71.5 46.8 91.7
gassing 12C 1.8 b a a c

Delayed
C2H4 12 d at 10.0 16.7 11.8 53.1 31.7 112.0
gassing 120C 1.4 a b b b







Peel color and decay incidence

At harvest, the avocado peel was moderately green (hue angle= 125.7, 125.0, and

122.0 in early-, mid-, and late-harvest fruit, respectively, where pure yellow = 90 and pure

green = 180) (Tables 3-1, 3-2, and 3-3). Changes in hue angle constituted the major

alteration of color coordinates of fruit. The decline in hue angle represented the change

from green to yellow.

Table 3-3. Fruit quality evaluation at the full-ripe stage for 'Simmonds' avocados from
late-harvest (07/27/98) gassed immediately for 48 h at 20C and transferred to 13"C or
stored at 13C for 7 d then gassed for 48 h at 20"C and then transferred to 13C. Values
followed by the same letter do not differ significantly according to Duncan's Multiple
Range Test (p < 0.05). Data are means standard deviation of 6 independent samples.

Treatment Days to Fruit Decay Dry L* Chroma Hue
fully ripe firmness incidence matter value value angle
(N) (%) (%) (Ho)

At harvest -- 93.1 -- 12.3 42.7 19.2 122.0
18.0 +0.7 1.5 2.0 0.9

Control 5 d at
(noC2H4 13C & 2 12.9 16.7 12.5 49.5 38.0 118.2
gassing) d at 200C 2.5 a a a a

Immediate
C2H4 5 d at 10.8 0 12.3 48.3 34.9 119.5
gassing 13C 2.0 a a a a
Control
(noC2H4 9 d at 10.2 25.0 12.2 49.3 36.6 118.5
gassing) 13C 2.6 a b b a

Immediate
C2H4 9 d at 8.7 16.7 12.3 50.9 41.7 114.7
gassing 13C 1.2 a ab a b

Delayed
C2H4 9 d at 10.5 16.7 12.7 52.9 42.4 113.2
gassing 130C 3.1 a a a b







In early-harvest fruit, no significant differences in the hue angle of peel color

among treatments were observed due to ethylene treatment after 5 d at 100C followed by

3 d at 200C or 12 d at 10C (Table 3-1). There were, however, significant differences in

the L value (L*) and chroma value (C) of the peel color among fruit from all treatments

after 12 d at 100C. In mid-harvest fruit, no significant differences in peel color were

observed between control and ethylene-treated fruits after 5 d at 120C (Table 3-2). There

were, however, significant (P <0.05) differences in the hue angle, L value (L*), and

chroma value (C) of the peel color among fruit from all treatments after 12 d at 120C.

Fruit from the immediate ethylene treatment had the lowest hue angle (91.7), with the

highest L* value (71.5) and chroma value (46.8) (Table 3-2). In late-harvest fruit, no

significant differences in peel color were observed between control and ethylene-treated

fruits after 5 days at 130C (Table 3-3). There were significant (P <0.05) differences in the

hue angle, L value (L*), and chroma value (C) of the peel color among fruit from all

treatments after 9 d at 120C. Control fruit had the highest hue angle (118.5) (Table 3-2).

Fruit stored continuously for 5 d at 10, 12, or 130C exhibited no decay symptoms

except for control fruit (16.7%) from late-harvest (Tables 3-1, 3-2, and 3-3). Early

harvest fruits from all treatments did not show decay at the full-ripe stage (10 to 15 N)

(Table 3-1). However, mid-harvest fruit from all treatments showed decay (16.7%) after

12 d storage at 120C (Table 3-2). In late-harvest fruit, control fruit exhibited decay

(16.7%) after 5 d storage at 130C followed by 2 d at 200C, and showed the highest decay

(25%) after 9 d storage at 130C (Table 3-3).

Pulp dry matter content

Initial pulp dry matter content (DMC) of early-, mid-, and late-harvest fruit

averaged 12.9, 13.2, and 12.3%, respectively (Tables 3-1, 3-2, and 3-3). In early-harvest







fruit, there were no significant differences in DMC among treatments for avocados after 5

d at 100C followed by 3 d at 200C (Table 3-1). During storage, DMC of fruit from all

treatments decreased compared with DMC of freshly harvested fruit. Ethylene-treated

fruit lost significantly more dry matter (lower DMC) than control fruit, after 12 d storage

at 100C. There were no significant differences in DMC among treatments for mid- and

late-avocados at the full ripe stage (10 to 15 N) (Tables 3-2 and 3-3).

Mid-Season ('Booth 7') Cultivar

Fruit firmness

Initial firmness of mid- and late-harvest fruit averaged 179.9 24.5 and 140.6

32.4 N, respectively. In mid-harvest fruit, firmness of fruit declined significantly during

storage at 120C, and fruit from all treatments exhibited significant (P < 0.05) differences

in average firmness values after 7 d at 120C (Table 3-4). Fruit treated with ethylene for

24 h at 200C were softer (20.9 3.8 N) than those from control and other ethylene

treatments and had less variability in firmness. Fruit from all treatments after 7 d at 120C

were somewhat firm (over 21 N) and required 3 d at 200C to reach the full-ripe stage (10

to 15 N). Fruit from all treatments reached full-ripe firmness after 14 d at 12C (Table 3-

5). In late-harvest fruit, fruit from all treatments exhibited significant (P < 0.05)

differences in average firmness values after 7 d at 120C (Table 3-6). Late-harvest fruit

treated with ethylene for 24 h at 200C were softer (18.6 3.6 N) than those from control

and other ethylene treatments. Fruit from all treatments after 7 d at 120C were somewhat

firm (over 19 N) and required 3 d at 200C to reach the full-ripe stage. Fruit from all

treatments reached full-ripe firmness after 14 d at 12C (Table 3-7). Fruits treated with

ethylene for 24 h at 12C and for 12 h at 200C showed less variability in firmness,

although the differences were not statistically significant (Table 3-7).












Table 3-4. Fruit quality evaluation for 'Booth 7' avocados from mid-harvest (10/03/98) stored for 7 d at 12"C and transferred to 20C
with different ethylene treatments. Data are means standard deviation of 6 independent samples.

Storage time and temperature
Initial ethylene
treatment 7 d at 12'C 7 d at 12'C + 3 d at 20"C

Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle
firmness incidence matter firmness incidence matter content
(C) (h) (N) (%) (%) (H) (N) (%) (%) (%) (H)

12 0 46.613.5 0 14.6 122.3 10.71.6 0 14.4 3.8 120.1

12 12 54.215.5 0 14.4 123.7 10.31.0 0 14.5 4.8 120.2

12 24 60.614.6 0 14.2 124.8 12.71.5 0 12.6 4.5 121.7

20 12 75.930.8 0 14.5 124.4 12.53.3 0 13.7 4.3 122.5

20 24 20.93.8 0 14.6 121.4 10.70.8 16.7 13.0 3.6 117.8







Table 3-5. Fruit quality evaluation for 'Booth 7' avocados from mid-harvest (10/03/98)
stored for 14 d at 12'C with different ethylene treatments. Values followed by the same
letter do not differ significantly according to Duncan's Multiple Range Test (p < 0.05).
Data are means standard deviation of 6 independent samples.

Initial ethylene Storage time and temperature
treatment 14d at 12C
Temp Time Fruit Decay Dry Oil Hue angle
firmness incidence matter content
(C) (h) (N) (%) (%) (%) (H)
12 0 11.61.7 0 13.8 4.5 120.7ab
12 12 10.82.2 0 14.7 4.6 122.3a
12 24 11.42.0 0 13.8 3.5 120.5ab
20 12 13.01.9 0 14.1 3.3 120.8ab
20 24 11.1+2.7 0 13.5 4.1 118.5b

Peel color and decay incidence

At harvest, the avocado peel was moderately green (hue angle=125.9 and 124.8 of

mid- and late-harvest fruit, respectively). In mid-harvest fruit, fruit treated with ethylene

for 24 h at 120C and for 12 h at 200C retained more green color after 7 d storage at 120C

or 7 d at 120C followed by 3 d at 200C, although the differences were not statistically

significant (Table 3-4). After 7 d at 120C followed by 3 d at 200C or 14 d storage at 120C,

the peel of fruit treated with ethylene for 24 h at 200C showed more loss in greenness

(Tables 3-4 and 3-5). In late-harvest fruit, there were no significant differences in peel

color among treatments after 7 or 14 d storage at 120C (Tables 3-6 and 3-7). Fruit treated

with ethylene for 24 h at 200C showed significant loss in greenness after 7 d at 120C

followed by 3 d at 200C (Table 3-6).











Table 3-6. Fruit quality evaluation for 'Booth 7' avocados from late-harvest (10/17/98) stored for 7 d at 12C and transferred to 20"C
with different ethylene treatments. Values followed by the same letter do not differ significantly according to Duncan's Multiple
Range Test (p < 0.05). Data are means standard deviation of 6 independent samples.

Storage time and temperature
Initial ethylene
treatment 7 d at 12'C 7 d at 12"C + 3 d at 20C

Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle
firmness incidence matter firmness incidence matter content
(C) (h) (N) (%) (%) (H) (N) (%) (%) (%) (H)

12 0 29.77.2 0 15.8 123.1 11.72.3 0 15.3 5.3 118.0a

12 12 29.410.7 0 15.1 124.0 11.22.4 0 14.7 5.3 120.9a

12 24 22.13.0 0 14.4 123.9 11.01.1 0 15.4 4.9 120.3a

20 12 23.87.0 0 15.7 122.7 10.21.5 0 15.0 5.4 118.1a

20 24 18.63.6 0 15.2 121.0 10.21.8 0 14.7 5.0 114.2b







Table 3-7. Fruit quality evaluation for 'Booth 7' avocados from late-harvest (10/17/98)
stored for 14 d at 12C with different ethylene treatments. Data are means standard
deviation of 6 independent samples.

Initial ethylene Storage time and temperature
treatment 14d at 120C
Temp Time Fruit Decay Dry Oil Hue angle
firmness incidence matter content
(C) (h) (N) (%) (%) (%) (H)
12 0 11.01.9 0 14.7 5.0 121.3
12 12 10.52.0 0 15.0 5.6 120.3
12 24 10.3+1.3 0 15.4 6.1 120.3
20 12 10.91.2 0 15.2 6.4 119.6
20 24 9.52.0 0 14.8 4.9 119.2

Mid-harvest avocado fruit from all treatments stored continuously for 7 d at 120C

exhibited no decay symptoms (Table 3-4). However, following an additional 3 d at 200C,

over 16.7% of fruits from the 24-h ethylene treatment at 200C showed decay. Surface

decay of control and ethylene-treated fruits stored for 14 d at 120C was not observed

(Table 3-5). Late-harvest fruits from all treatments exhibited no decay symptoms after 7

or 14 d storage at 120C and 7 d at 120C followed by 3 d at 200C (Tables 3-6 and 3-7).

Pulp dry matter and oil contents

Initial pulp dry matter content (DMC) of mid- and late-harvest fruit averaged 15.3

and 17.4%, respectively. In mid- and late-harvest fruit, there were no significant

differences in DMC among treatments after 7 or 14 d storage at 120C and 7 d at 12C

followed by 3 d at 200C (Tables 3-4, 3-5, 3-6, and 3-7). Initial oil content (%) of mid-

and late-harvest fruit averaged 5.1 and 4.9%, respectively and showed slight change

during storage (Tables 3-4, 3-5, 3-6, and 3-7). However, control and ethylene -treated







fruit exhibited no significant differences in average oil content at the full-ripe stage (10 to

15 N).

Late-Season ('Monroe') Cultivar

Fruit firmness

Initial firmness of early-, mid-, and late-harvest fruit averaged 177 33, 176

27, and 198 26 N, respectively. In early-harvest fruit, although firmness declined

rapidly during storage, fruit from all treatments exhibited no significant differences in

average firmness values after 7 d at 130C or after 7 d at 130C followed by 4 d at 200C

(Table 3-8). After 14 d at 13C, avocados from all ethylene treatments were softer

(around 13 N) than control (not exposed to ethylene) fruit (33 N), however, ethylene-

treated fruit exhibited significantly less variability in firmness (Table 3-9). Although

control fruit reached full-ripe firmness within 2 d of transfer to 200C after 14 d at 130C,

over 50% of the fruit showed decay prior to reaching the full-ripe stage (data not shown).

In mid-harvest fruit, firmness declined dramatically during storage for 7 or 14 d at 130C,

but differences among fruit from all treatments were not evident (Tables 3-10 and 3-11).

Non-uniform ripening was noted among control fruit stored for 7 d at 130C followed by 3

d at 200C (Table 3-10). In late-harvest fruit, fruit from all treatments exhibited significant

(P < 0.05) differences in average firmness values after storage for 7 d at 130C (Table 3-

12). As with control fruit from the early- and mid-harvest dates, fruit held continuously in

air for 14 d at 130C exhibited high variability in firmness (Table 3-13). Ethylene-treated

fruit stored for 7 d at 130C followed by 3 d at 200C or 14 d at 130C were softer and

showed significantly more uniformity in firmness than control fruit stored under similar

conditions (Tables 3-12 and 3-13).












Table 3-8. Fruit quality evaluation for 'Monroe' avocados from early-harvest (11/20/98) stored for 7 d at 13C and transferred to 20C
with different ethylene treatments. Data are means standard deviation of 6 independent samples.

Storage time and temperature
Initial ethylene
treatment 7 d at 130C 7 d at 13 "C + 4 d at 20C

Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle
firmness incidence matter firmness incidence matter content
(C) (h) (N) (%) (%) (H) (N) (%) (%) (%) (H)

13 0 41.817.5 0 15.4 123.7 10.92.0 50 14.6 6.3 119.3

13 12 46.1+16.0 0 17.0 124.1 13.1+2.3 50 15.1 5.9 119.8

13 24 44.5+12.8 0 16.8 125.4 15.22.1 0 14.1 5.3 122.0

20 12 39.47.6 0 15.2 124.1 14.32.5 16.7 14.2 5.4 120.3

20 24 26.97.1 0 15.6 124.5 11.01.8 50 14.6 5.6 121.1







Table 3-9. Fruit quality evaluation for 'Monroe' avocados from early-harvest (11/20/98)
stored for 14 d at 13 C with different ethylene treatments. Data are means standard
deviation of 6 independent samples.

Initial ethylene Storage time and temperature
treatment 14d at 13C
Temp Time Fruit Decay Dry Oil Hue angle
firmness incidence matter content
(C) (h) (N) (%) (%) (%) (H)
13 0 32.918.9 16.7 15.6 123.2
13 12 12.83.5 16.7 13.9 5.0 121.4
13 24 13.30.9 16.7 14.5 6.0 121.0
20 12 12.81.5 16.7 15.4 6.8 122.5
20 24 13.51.9 16.7 13.9 4.7 120.2
Oil content was not measured because fruits were not fully ripe.

Peel color and decay incidence

At harvest, the avocado peel was moderately green (hue angle= 126.4, 124.1, and

126.5 for early-, mid-, and late-harvest fruit, respectively, where pure yellow = 90 and

pure green = 180) and showed little change during storage. At the full-ripe stage, there

were no significant differences in peel color among fruit from all treatments (Tables 3-8 to

3-13). In early-harvest, avocado fruits stored continuously for 7 d at 130C exhibited no

symptoms of decay (Table 3-8). Following an additional 4 d at 200C to reach the full-ripe

stage, however, fruit from the 24-h ethylene treatment at 130C had no incidence of decay.

Surface decay in control and ethylene-treated fruit stored for 14 d at 13 C was observed,

but was not severe (Table 3-9). In mid-harvest, fruit from the 24-h ethylene treatment at

13C had no incidence of decay after 7 d at 130C followed by 3 d at 200C and showed the

lo\\cst incidence of decay of all treatments 14 d at 130C (Tables 3-10 and 3-11).












Table 3-10. Fruit quality evaluation for 'Monroe' avocados from mid-harvest (12/04/98) stored for 7 d at 13"C and transferred to 20"C
with different ethylene treatments. Data are means standard deviation of 6 independent samples.

Storage time and temperature
Initial ethylene
treatment 7 d at 13"C 7 d at 13"C + 3 d at 20C

Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle
firmness incidence matter firmness incidence matter content
(C) (h) (N) (%) (%) (H) (N) (%) (%) (%) (H)

13 0 47.3+32.6 0 16.3 125.7 19.313.3 50 16.4 6.7 122.7

13 12 36.110.7 0 17.7 125.9 15.94.3 33.3 15.2 5.3 122.3

13 24 29.34.5 0 16.2 122.7 14.94.2 0 16.1 5.5 120.3

20 12 29.620.8 0 16.5 124.0 12.62.6 16.7 17.4 5.9 119.2

20 24 27.63.9 0 17.5 124.7 15.92.6 16.7 16.1 5.9 120.0







Table 3-11. Fruit quality evaluation for 'Monroe' avocados from mid-harvest (12/04/98)
stored for 14 d at 13 C with different ethylene treatments. Values followed by the same
letter do not differ significantly according to Duncan's Multiple Range Test (p < 0.05).
Data are means standard deviation of 6 independent samples.

Initial ethylene Storage time and temperature
treatment 14d at 13C

Temp Time Fruit Decay Dry Oil Hue angle
firmness incidence matter content
(C) (h) (N) (%) (%) (%) (H)
13 0 13.83.3 33.3 16.0b 5.6 122.5
13 12 11.73.3 33.3 18.6a 7.0 121.0
13 24 14.61.9 16.7 16.0b 6.0 121.9
20 12 12.82.7 33.3 18.2a 6.6 120.5
20 24 14.34.0 33.3 17.0ab 6.0 118.7

Control fruit stored for 7 d at 130C followed by 3 d at 200C exhibited the highest decay

incidence (50%) of all treatments (Table 3-10). In late-harvest, fruit from the 24-h

ethylene treatment at 130C also had no incidence of decay after 7 d at 130C and 3 d at

200C, or after 14 d at 130C (Tables 3-12 and 3-13).

Pulp dry matter content and oil content

Initial pulp dry matter content (DMC) of early-, mid-, and late-harvest fruit

averaged 16.7, 17.9, and 16.7%, respectively. In early-harvest fruit, there were no

significant differences in DMC among treatments after 7 or 14 d storage at 130C or 7 d at

130C followed by 3 d at 200C (Tables 3-8 and 3-9). In mid-harvest fruit, there were no

significant differences in DMC among treatments for avocados after 7 d at 130C (Table

3-10)., whereas there were significant (P < 0.05) differences in DMC among treatments

after 14 d at 130C (Table 3-11).











Table 3-12. Fruit quality evaluation for 'Monroe' avocados from late-harvest (12/18/98) stored for 7 d at 13"C and transferred to 20C
with different ethylene treatments. Data are means standard deviation of 6 independent samples.

Storage time and temperature
Initial ethylene
treatment 7 d at 13"C 7 d at 13"C + 3 d at 20C

Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle
firmness incidence matter firmness incidence matter content
(C) (h) (N) (%) (%) (H) (N) (%) (%) (%) (H)

13 0 95.832.8 0 15.8 125.0 20.810.6 33.3 16.9 6.1 122.1

13 12 61.735.9 0 16.3 124.8 12.33.6 0 16.4 6.2 121.8

13 24 51.325.5 0 16.2 125.2 15.35.0 0 16.6 6.3 122.5

20 12 50.839.5 0 16.8 125.7 13.97.0 16.7 15.8 6.6 123.5

20 24 28.36.0 0 18.4 124.0 14.74.3 16.7 16.5 6.8 122.1







Table 3-13. Fruit quality evaluation for 'Monroe' avocados from late-harvest (12/18/98)
stored for 14 d at 13"C with different ethylene treatments. Data are means standard
deviation of 6 independent samples.

Initial ethylene Storage time and temperature
treatment


Fruit
firmness
(N)

42.233.0

14.52.4

14.04.6

12.02.6

12.01.9


E
inc


14 d at 13*C

)ecay Dry matter Oil
idence conte]
(%) (%) (%)

16.7 14.7 5.6

33.3 16.5 5.0

0 15.6 5.5

16.7 15.8 5.3

33.3 16.4 5.7


nt


Hue angle

(H)

121.7

120.0

118.0

118.4

119.5


Fruit from the air treatment and the 24-h ethylene treatment at 130C followed by

14 d of storage at 130C lost more dry matter (lowest DMC) than other treatments (Table

3-11). In late-harvest fruit, there were no significant differences in DMC among

treatments, although DMC of fruit from all treatments slightly decreased after 14 d at

130C compared with DMC of freshly harvested fruit (Tables 3-12 and 3-13). Initial oil

content (%) of early-, mid-, and late-harvest fruit averaged 5.4, 5.4, and 5.3%,

respectively, and did not change significantly during storage (Tables 3-8 to 3-13). There

were no significant differences in oil content among treatments after 7 d at 130C followed

by 3 d at 200C or 14 d at 130C (Tables 3-8 to 3-13).


Temp

(CC)

13

13

13

20

20


Time

(h)

0

12

24

12

24


nt


D
inc







Discussion

In the present study, several parameters (firmness, peel color, dry matter content,

oil content and decay incidence) were examined to determine the effects of postharvest

application of ethylene on ripening uniformity and fruit quality.

'Simmonds' avocado stored at 13C ripened more quickly than fruit held at 10 or

12C, and those from immediate ethylene treatment ripened more quickly and uniformly

than those with delayed gassing or air-treated (Tables 3-1 to 3-3). Uniform ripening was

defined as fruits in a given lot softening with less variability in firmness at the full-ripe

stage (10 to 15 N). In 'Booth 7' avocado, fruit from both mid- and late-harvest required 3

d at 200C following 7 d storage at 12C to reach full-ripe stage, whereas fruit were

already soft after 14 d storage at 120C. Comparing 'Monroe' fruit from the three harvest

dates, after 7 d storage at 130C, ethylene-treated avocados from the early harvest required

4 d at 20C to reach the full-ripe stage (10 to 15 N), whereas those from the middle- and

late-harvests required 3 d at 200C (Tables 3-8 to 3-13). This observation is consistent

with reports that avocado fruit at more advanced maturity at harvest respond more rapidly

to ethylene (Biale, 1960; Burg and Burg, 1965; Eaks, 1966; Hansen and Blanpied, 1968).

However, after 14 d storage at 13C, ethylene-treated fruit from the three harvest dates

were already soft. Control fruit (not exposed to ethylene) from the early- and late-

harvests stored for 14 d at 130C were firmer (32.918.9 and 42.233.0, respectively) than

ethylene-treated fruit and required 2 to 3 d at 200C for all fruit to reach the full-ripe stage.

Oil content (%) of 'Booth 7' and 'Monroe' avocados did not change significantly

during storage, in agreement with the report that oil synthesis is essentially complete

when avocado fruit are immature (Platt and Thomson, 1992) and is not metabolized







during ripening (Dolendo et al., 1966). Unlike the Mexican and Guatemalan type

avocados which have higher oil content (about 15 to 30%), estimating pulp oil using dry

matter content was not feasible with the West Indian-type due to the wide variability in

oil content between individual fruit, as noted by Hatton et al. (1964).

'Simmonds' early-season avocado fruits stored at 10 and 12C for 7 or 14 d

exhibited symptoms of chilling injury, that were characterized by black pitting lesions on

the peel upon removal from storage. After storage at 200C, the pulp showed gray-black

discoloration and blackening of the vascular bundle. Most fruits stored at 100C became

unacceptable during storage at 200C. The incidence and severity of chilling injury

increased with storage time at 200C. No specific internal symptoms of chilling injury,

such as discoloration of the flesh, were observed in any fruit during storage at 130C.

However, following storage at 200C, minor mesocarp discoloration and vascular

browning were observed at full-ripe storage (10 to 15 N). Fruit from the delayed ethylene

treatment (7 d storage at 130C followed by 2 d ethylene gassing at 200C) showed more

mesocarp discoloration and vascular browning than control fruit (7 d storage at 130C

followed by 2 d at 200C). 'Simmonds' avocados gassed immediately at 200C for 2 d and

stored at 130C for 7 d showed less mesocarp discoloration and vascular browning than

control fruit and delayed ethylene-treated fruit. Stem-end rot was observed in fully ripe

fruits after 12 d storage at 10 and 12C or after 9 d storage at 130C. In 'Booth 7'

avocados, neither specific internal symptoms of chilling injury nor discoloration of the

imcsocarp were observed during the storage at 120C. However, following ripening at

200C, minor mesocarp discoloration and localized vascular browning was observed.

Stem-end decay was also observed on some full ripe fruit, but the incidence was not







severe. In 'Monroe' avocados, no specific internal symptoms of chilling injury, such as

discoloration of the flesh (mesocarp), were observed in any fruit during storage at 130C.

However, following ripening at 200C, minor mesocarp discoloration and localized

vascular browning was observed. Decay at the stem-end and on the peel was also

observed on some full ripe fruit, but the incidence was not severe.

Therefore, treatment of avocado fruit with ethylene assisted in initiating uniform

ripening and maintaining fruit quality.


Summary

A series of experiments were performed during the 1998-99 production season to

determine the effects of postharvest application of ethylene on ripening uniformity and

fruit quality of early-season ('Simmonds'), mid-season ('Booth 7') and late-season

('Monroe') varieties. Overall results indicated that avocados from ethylene treatments

ripened more uniformly than control fruit (not exposed to ethylene). Although the peel of

ethylene-treated fruits was significantly more yellow than that of control fruits, the pulp of

preconditioned fruits had acceptable appearance and was palatable. There were no

significant differences in dry matter content or oil content of the pulp during fruit

ripening due to ethylene treatment. It was not possible to estimate pulp oil content of

West Indian type avocado fruits using dry matter content due to wide variability of oil

content between individual fruits.

From the tests with 'Simmonds' avocados, it was determined that fruits from

immediate ethylene treatment had more uniform ripening and better pulp quality than

fruit from delayed ethylene treatment. Tests with 'Booth 7' avocados revealed that best

fruit quality was obtained with immediate ethylene treatments at 200C for 12 h or at 120C







for 24 h. After subsequent storage at 120C in air for 5 or 12 d, fruit from this treatment

developed no chilling injury, remained acceptably firm and ripened normally. 'Monroe'

avocados exposed to ethylene (100 tL L'') at 130C for 24 h ripened more uniformly than

fruit treated at 200C for 12 h or control fruit. During 14 d of storage at 13C, fruit from

this treatment ripened normally while maintaining marketable fruit firmness, had good

quality, and also had lower incidence of decay. Ethylene treatment did not affect fruit

quality as determined by peel color, dry matter content, and oil content.












CHAPTER 4
INFLUENCE OF 1-METHYLCYCLOPROPENE (1-MCP) ON RIPENING AND CELL
WALL MATRIX POLYSACCHARIDES OF AVOCADO FRUIT


Introduction

The avocado (Persea americana Mill.) is a climacteric fruit that is characterized

by a surge in ethylene production at the onset of ripening. This climacteric increase in

ethylene production is associated with hastened ripening of fruits. Avocado is one of the

most rapidly ripening of fruits, often completing ripening within 5 to 7 d following

harvest (Seymour and Tucker, 1993).

The importance of ethylene in regulating fruit ripening has been clearly

demonstrated from analyses of fruits exhibiting suppressed ethylene biosynthesis or

action. For example, melon fruit expressing a 1-aminocyclopropeneoxidase antisense

construct retained only 0.5% of normal ethylene production (Ayub et al., 1996) and fruit

softening was completely blocked (Guis et al., 1997). Similarly, tomato fruits expressing

an antisense construct for aminocyclopropene synthase failed to ripen unless provided

with exogenous ethylene (Oeller et al., 1991). The identification of the Nr tomato

ripening mutant gene as an ethylene receptor (Lanahan et al., 1994; Wilkinson et al.,

1995) has provided evidence that ethylene receptors regulate a defined set of genes which

are expressed during fruit ripening. Nr mutants are affected in ethylene perception

(Payton et al., 1996) and impaired in ethylene response (Lanahan et al., 1994).

In addition to the use of fruit lines with suppressed ethylene synthesis or

perception, the application of compounds that block ethylene action (Sisler and Serek,







1997; Yueming and Jiarui, 2000) has provided a facile approach for examining

relationships between ethylene, fruit ripening, and senescence in a range of horticultural

commodities. 2,5-Norbomadiene (NBD) was perhaps the first widely employed cyclic

olefin utilized for its ability to compete with ethylene at the receptor level (Sisler et al.,

1986); however, the effectiveness of NBD, a toxic and offensive smelling compound

(Sisler and Serek, 1999) required the continued presence of the gas. Sisler and

Blankenship (1993a) also reported the ethylene antagonism of diazocyclopentadiene

(DACP), a photoaffinity label for the ethylene-binding site. DACP's effectiveness at

inhibiting ethylene action is a result of a permanent attachment to the binding site (Serek

et al., 1994a). It is, however, explosive in high concentrations, which limits its

commercial usefulness (Sisler and Serek, 1997).

Recently, 1-methylcyclopropene (1-MCP), a synthetic cyclopropene, has been

shown to block ethylene receptors, preventing ethylene effects in plant tissues for

extended periods (Sisler and Serek, 1997; Sisler et al., 1996a; Sisler et al., 1996b). This

material is nontoxic, odorless, and effective when plants are treated at concentrations as

low as 0.5 nL L'' (Sisler and Serek, 1997). Although 1-MCP binding to the ethylene

receptor sites is irreversible, it appears that synthesis of new receptors can allow eventual

recovery of the ripening process (Sisler et al., 1996a).

1-MCP has been shown to delay fruit ripening and improve storage quality of

climacteric fruits including pears (Pyrus communis L. cv. Passe-Crassane) (Lelievre et

al., 1997b), bananas (Musa sp.) (Golding et al., 1998; Golding et al., 1999; Sisler and

Serek, 1997), plums (Prunus salicina Lindl) (Abdi et al., 1998), tomatoes (Lycopersicon

e.sculentuII i Mill) (Nakatsuka et al., 1997; Sisler and Serek, 1997), apples (Malus







sylvestris L.) (Fan and Mattheis, 1999; Watkins et al., 2000), and avocados (Persea

AmILricma Mill) (Feng et al., 2000). 1-MCP, therefore, has provided a valuable tool to

investigate ethylene metabolism in ripening climacteric fruit (Nakatsuka et al., 1997) and

has the potential to extend the storage life of horticultural products.

The objectives of the present research were to characterize physiological and

biochemical responses of avocado fruit to different concentrations and exposure periods

of 1-MCP treatment during avocado fruit ripening and to evaluate its ability as a

postharvest tool for regulating the ripening of avocado fruit. We also used application of

1-MCP to identify and differentiate some ripening processes that are dependent on

ethylene action. Hypothesis: Treatment of avocado fruit with 1-MCP will reduce the rate

of ripening and extend the postharvest life of avocado while ensuring high quality.


Materials and Methods

Plant Materials

'Simmonds', an early season cultivar, was selected for this experiment. It is a

West Indian type avocado and is chilling sensitive (Crane et al., 1996; Hatton and

Reeder, 1965). Recommended storage temperature is 130C to avoid chilling injury

(Seymour and Tucker, 1993). Mature avocado fruit were obtained from a commercial

grower in Homestead, Florida, packed in fiberboard cartons, and transported to the

Postharvest Horticulture Laboratory in Gainesville within 24 h of harvest. Fruit were

selected for uniformity of weight (757 31 g) and shape (diameter at equatorial region,

10.5 0.7), and then were surface sterilized in a 15% (90 mM NaOCI) commercial

bleach solution, rinsed, and dried.







1-MCP Treatment

Twelve fruit were placed in 18-L containers and exposed to 1-MCP by releasing

the gas from a commercial powdered formulation (Ethyblock, Floralife, Burr Ridge, IL).

The concentrations selected, 0.09 and 0.45 pL L1', were achieved through addition of or

5 mg of the powder, respectively, to 100 mL of FloraLife buffer following

manufacturer's instructions (Floralife, Ethyblock product specification sheet). Following

addition of the buffer to the 1-MCP, the beakers were transferred to the 18-L containers,

which were sealed immediately. 1-MCP treatment at each concentration was performed

for three exposure periods (6, 12, and 24 h) at 200C and 85% relative humidity (RH).

Immediately following 1-MCP treatments, the fruit were removed from the chambers and

transferred to 200C storage facilities (85% RH). Control fruit (not exposed to 1-MCP)

were maintained under identical storage conditions. Samples of fruit from each treatment

were evaluated for fruit quality on a daily basis until they reached the full-ripe stage (10

to 20 N, considered too soft for commercial handling by the author). Fruit quality was

assessed on the basis of fruit firmness, weight loss, CO2 and C2H4 production, and peel

color. Mesocarp tissue derived from the equatorial region of selected fruit was stored at

-30 o C and used for analysis of cell wall enzymes and structural polysaccharides.

Fruit Firmness

Firmness was determined on whole, unpeeled fruit using an Instron Universial

Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat-plate probe (5 cm

in diameter) and 50-kg load cell. After establishing zero force contact between the probe

and the equatorial region of the fruit, the probe was driven with a crosshead speed of 10

mm min-'. The force was recorded at 2.5 mm deformation and was determined at two







equidistant points on the equatorial region of each fruit. The same four fruit of each

treatment were measured repeatedly every other day until they reached the full-ripe stage.

Respiration and Ethylene Evolution

Respiration and ethylene production were measured every other day using the

same four fruit of each treatment. Fruit were individually sealed for 30 min in 2-L plastic

containers prior to sampling. A 0.5 mL gas sample was withdrawn by syringe through a

rubber septum, and carbon dioxide determined using a Gow-Mac gas chromatograph

(Series 580, Bridge water, NJ, USA) equipped with a thermal conductivity detector

(TCD). Ethylene was measured by injecting a 1.0 ml gas sample into a HP 5890 gas

chromatograph (Hewlett Packard, Avondale, PA, USA) equipped with a flame ionization

detector.

Peel Color

Individual fruit were marked at the equatorial region (2 regions per fruit), and

color at the same location was recorded every other day as L*, hue angle, and chroma

value with a Minolta Chroma Meter (CR-2000, Minolta Camera Co Ltd., Japan). The

chroma meter was calibrated with a white standard tile. The color was reported as hue

angle (H), with a value of 900 representing a totally yellow color and 1800 a totally

green color. The results are presented as lightness (L*), chroma (C*), and hue angle (H).

The chroma and hue angle were calculated from the measured a* and b* values using the

formulas C* = (a*2+b*2) 1/2 and H = arc tangent (b*/a*) (McGuire, 1992).

Preparation of Cell-Free Protein Extract

Partially thawed mesocarp tissue (10 g) was homogenized with 40 mL of ice-cold

95% EtOH for 1 min in an Omnimixer (Model 17150, Newtown, CT, USA) and

centrifuged at 7840 g for 10 min at 40C. The supernatant was discarded and the pellets







were resuspended in 50 mL of ice-cold 80% EtOH for 1 min and again centrifuged at

7840 g for 10 min at 40C. The pellets were transferred to 50 mL of ice-cold acetone for

10 min followed by centrifugation (7840 g, 10 min, 4C). After 2 additional acetone

washings, the pellets were suspended in 50 mL of ice-cold 80% EtOH, stirred with a

spatula, kept for 10 min in an ice-cold water bath (50C), and then centrifuged (7840 g, 10

min, 4C). The pellets were transferred to 30 ml of 10 mM Na-acetate, pH 6.0,

containing 1.8 M NaC1, for 30 min in ice-cold (50C) water bath and centrifuged. The

supernatant was analyzed for enzyme activities as described below. Protein content was

measured using the bicinchoninic method (Smith et al., 1985) with bovine serum albumin

as a standard.

Enzyme Assays

Polygalacturonase (PG, E.C. 3.2.1.15) activity was assayed reductometrically by

incubating a 100 pL aliquot of the cell-free protein extract with 500 IL (2 mg) of

polygalacturonic acid (from orange peel, Sigma Chemical Co, St. Louis, MO, USA)

dissolved in 30 mM KOAc, pH 5.5, containing 100 mM KC1. After 30 min at 340C,

uronic acid reducing groups were measured using the method of Milner and Avigad

(1967). PG activity was expressed as mol D-galacturonic acid equivalents produced per

kg protein per minute. Pectinmethylesterase (PME, E.C. 3.1.1.11) was measured using

modifications of the method of Hagerman and Austin (1986). A 0.5% (w/v) solution of

citrus pectin (Sigma Chemical Co, St. Louis, MO, USA) was prepared in 0.1 M NaCl and

adjusted to pH 7.5. A 0.01% (w/v) solution of bromothymol blue was prepared in 0.003

M potassium phosphate, pH 7.5. In a cuvette, 2.0 mL of the 0.5% citrus pectin were

mixed with 0.15 mL of bromothymol blue and 0.83 mL of water, pH 7.5. The reaction







was initiated by adding 20 pL of the cell-free protein extract adjusted to pH 7.5, and the

decrease in A620 was recorded. PME activity was expressed as AA620 per mg protein per

minute. Cx-Cellulase (endo-l,4-p-glucanase; E.C. 3.2.1.4) activity was measured

viscometrically. A 100 pL aliquot of the cell-free protein extract was added to 1.5 mL of

a 2.5% solution of carboxymethylcellulose (CMC.7HSP, Fisher Scientific Co, Fair Lawn,

NJ, USA) in 40 mM NaOAc, pH 5.0, with 0.02% NaN3, and the mixture was incubated at

room temperature for 30 min. The time required for the solution to pass through a

calibrated portion of a 1-mL pipette was recorded. Activity was expressed as % change

in viscosity per mg protein per min.

a- and p-galactosidase activities were measured using modifications of the

method of Pharr et al. (1976). p-N02-phenyl a- and P-D-galactopyranosides (Sigma

Chemical Co, St. Louis, MO, USA) were used as substrates. Substrates were prepared at

2 mg mLl' in 0.1 M NaOAc, pH 5.2. A 200 pIL aliquot of the cell-free protein extract

adjusted to pH 5.2 was added to 200 plL of substrate, and the reaction mixture incubated

at 370C for 15 min. The release of p -NO2-phenol was measured spectrometrically at 400

nm. Activity was expressed as moles of NO2-phenol equivalents released per kg protein

per minute. NO2-phenol concentration was determined using free NO2-phenol (Fisher

Scientific Co, Fair Lawn, NJ, USA).

Preparation of Ethanol-Insoluble Solids

Approximately 20 g of partially thawed mesocarp were homogenized in 80 mL of

cold 95% ethanol over ice for 3 min in a Polytron (Kinematica Gmbh Karens, Lunzern,

Switzerland) at speed setting #7. The homogenate was heated for 20 min in a boiling

water bath. The EIS were filtered under vacuum through glass fiber filters (GF/C,







Whatman) and washed with 100 mL of 95% ethanol. The EIS were transferred to 100 ml

of chloroform:methanol (1:1, v/v) and stirred for 30 min at room temperature. The EIS

were filtered under vacuum through glass fiber filters (GF/C, Whatman) and washed with

100 mL of acetone. EIS samples were dried in an oven at 400C for 5 h and stored in a

desiccator at room temperature.

Pectin Extraction and Analysis

Water and CDTA (1,2-cyclohexylenedinitrilotetraacetic acid) soluble pectins

were extracted from 30 mg of EIS incubated successively with 7 ml of distilled water and

7 mL 50 mM Na-acetate, pH 6.5, containing 50 mM CDTA. Extractions were carried out

at 34C for 4 h and quantified in terms of extractable uronic acid (UA) content. UA were

determined by the hydroxydiphenyl assay (Blumenkrantz and Asboe-Hansen, 1973) and

expressed as pg mg'1 EIS. Total uronic acids in the EIS preparations were determined

using the method of Ahmed and Labavitch (1977).

Gel Permeation Chromatography of Polyuronides

The water and CDTA-soluble uronic acids were concentrated using rotary

evaporation to a final UA concentration of approximately 500 Pg mL'1. Two-ml aliquots

(1 mg UA equivalents) were passed through a Sepharose CL-2B-300 (Sigma Chemical

Co, St. Louis, MO, USA) column (1.5 cm wide and 30 cm high) operated with 200 mM

ammonium acetate, pH 5.0 (Mort et al., 1991). Two-ml fractions were collected at a flow

rate of 40 mL h'1, and aliquots (0.5 mL) were used for the determination of UA content.

The column void (Vo) and total (VT) volumes were identified by the elution positions of

Blue Dextran (2000 kDa.) and glucose, respectively. The UA content in each column

fraction was expressed as a percentage of the total UA recovered.








Hemicellulose Extraction

Hemicelluloses were isolated using the method of Huber and Nevins (1981) as

modified by de Vetten and Huber (1990). To remove the major portion of pectin prior to

hemicellulose extraction, 200 mg of EIS were incubated in 100 mL of 40 mM Na-

phosphate, pH 6.8, in a boiling water bath for 20 min. The suspension was filtered

through Miracloth and washed with 1 L of distilled water. The residues were filtered

under vacuum through glass fiber filters (GF/C, Whatman) and washed with 200 mL of

100% acetone. The phosphate-buffer-treated EIS were dried in an oven at 400C for 5 h.

For hemicellulose extraction, 50 mg of EIS were incubated in 5 mL of 4 M KOH

including 26 mM NaBH4 for 12 h at room temperature. The suspension was filtered

through glass fiber filters. The solution was neutralized on ice with concentrated acetic

acid and then dialyzed (mwco 2000 Da.) overnight against running tap water followed by

deionized water (2 x 4 L, 12 h total). Total carbohydrates in the alkali-soluble extracts

were determined by the phenol-sulfuric acid method (Dubois et al., 1956) and expressed

as pg glucose equivalents in 200 mg EIS.

Gel Permeation Chromatography of Hemicelluloses

Two ml of the hemicellulose extracts (at approximately 1 mg mL'1 glucose

equivalents) were heated for 5 min in a boiling water bath (to disperse aggregates) before

being applied to a column (2 cm wide, 30 cm high) of Sepharose CL-6B-100 (Sigma

Chemical Co, St. Louis, MO, USA) operated with 200 mM ammonium acetate, pH 5.0, at

room temperature. Fractions of 2 mL were collected and aliquots (0.5 mL) were assayed

for total sugars (Dubois et al., 1956) and xyloglucan (Kooiman, 1960). The column Vo

and Vt were identified by the elution positions of Blue Dextran 2000 and glucose,







respectively. The column was calibrated with dextran standards of 70, 40, and 10 kDa

(Sigma, St. Louis, MO).

Statistical Analysis

The experiments were laid out in a completely randomized design. Statistical

procedures were performed using the PC-SAS software package (SAS-Insititute, 1985).

Data were subjected to ANOVA using the General Linear Model (Minitab, State College,

PA). Differences between means were determined using Duncan's multiple range test.


Results

Avocado Fruit Firmness and Weight Loss

Changes in 'Simmonds' avocado fruit firmness following 1-MCP treatment are

shown in Figure 4-1A. Control fruit softened rapidly and completed ripening (softened to

10 to 20 N) within 8 d of storage. 1-MCP at 0.09 tL L-' for 6 or 12 h had no significant

effect on fruit firmness relative to control fruit. Firmness decrease of fruit treated with

1-MCP at 0.45 tL L-' was significantly delayed and varied with time of exposure to the

gas. After 8 d of storage at 200C, fruit treated with 1-MCP at 0.45 PL LU' for 6, 12, or 24

h exhibited firmness values of 20.1, 28.5, and 41.6 N, respectively. Firmness of fruit

treated with 1-MCP at 0.45 pL L-' for 6 and 12 h reached a full-ripe firmness value (10 to

20 N) after about 10 days. Fruit treated with 0.45 pL L'' for 24 h required an additional 2

d to reach the full-ripe stage.

Weight loss trends for 'Simmonds' avocado fruit during storage at 20C are

shown in Figure 4-lB. Control fruit and fruit treated at 0.09 pL L'' for 6 or 12 h showed

no differences in the magnitude and rate of weight loss. After 8 d, at which time fruit

\ ere fully ripe, cumulative weight loss ranged between 6.5 and 7.0 %. The rate of







weight loss in fruit treated with 1-MCP at 0.45 tL L-' paralleled the effects of the gas on

firmness. After 8 d of storage, fruit treated with 1-MCP at 0.45 iL LU' for 6, 12, or 24 h

showed weight loss values of 5.0, 4.5, and 3.9 %, respectively. Final weight loss values

of fruit treated with the higher 1-MCP concentration (0.45 PL L'') were not significantly

different from control fruit and fruit treated with 1-MCP at 0.09 PL L'' for 6, 12, and 24

h. Regardless of treatment, fruit when ripe showed overall weight loss values ranging

from 6 to 7 % (Fig. 4-1B).

Respiration and Ethylene Evolution

Respiration trends in fruit treated with 1-MCP at 0.45 pL L"1 for 12 or 24 h were

atypical. Respiration in control fruit and fruit treated with 1-MCP at 0.09 pL L' for 6 or

12 h began to increase after 2 d storage at 200C (not shown) and CO2 production reached

maxima of 51.7, 55.4, and 47.5 mg kg-' h'1, respectively, after 6 to 7 d storage at 200C

(Table 4-1). CO2 production of fruit treated with 1-MCP at 0.45 PL L'1 for 6 h increased

initially after 4 d storage at 200C (not shown), reaching a maximum of 43.6 mg kg'' h'1

after 9.3 d storage at 200C (Table 4-1). There were no significant differences in the

maximum CO2 production rate between control fruit and fruit treated with 1-MCP at 0.09

pL L'1 for 6 h, but the application of 1-MCP at 0.09 pL L'' for 12 h and at 0.45 pL L-' for

6 h slightly suppressed the magnitude of the respiratory peak (Table 4-1).

The CO2 production of fruit treated with 1-MCP at 0.45 pL L'1 for 12 and 24 h

showed a delayed and attenuated climacteric pattern. Increased respiration for fruit

treated with 1-MCP at 0.45 pL LU' for 12 and 24 h was first evident at day 6 and day 10,

respectively (not shown). A distinct peak of CO2 production did not occur during the

storage period, and maximum CO2 production rates were reduced nearly 40 % compared

with all other treatments when experiments were terminated (Table 4-1).








Table 4-1. Days to peak and maximum amount CO2 and C2H4 production for 'Simmonds'
avocados stored at 200C with 1-MCP treatments. Fruit were treated with two 1-MCP
concentrations (0.45 and 0.09 uL L') and three exposure periods (6, 12, and 24 h). Initial
rates of C2H4 and CO2 production were 0.5 pL kg" h-' and 61.3 mg kg'' h'1, respectively.
Data are means standard deviation of 4 independent samples.

CO2 C2H4

Treatments Days to Maximum Days to Maximum
peak (mg kg'1 h-') peak (pL kg' h'')
Control (no 1-MCP) 6 51.7 3.9 6 124.2 39.0

1-MCP (0.09 ptL L'for 6 h) 7.3 55.4 + 9.8 6 130.3 69.0

1-MCP (0.09 utL L-'for 12 h) 6.7 47.5 + 1.6 6 103.5 37.8

1-MCP (0.45 iL LL'for 6 h) 9.3 43.6 8.3 9 117.0 40.0

1-MCP (0.45 pL L'for 12 h) 10x 25.8 17.5x 10x 45.9 64.6x

1-MCP (0.45 pL L'for 24 h) 12 30.3 + 13.5" 12 50.5 57.0'

S- Ethylene and respiratory climacteric peak did not occur during storage at 200C, and
data were measured when experiments were terminated.


The time to attain the maximum ethylene production closely paralleled that for

respiratory rates in all treatments (Table 4-1). Ethylene production in control fruit and

fruit treated with 1-MCP at 0.09 uL L-' for 6 or 12 h showed characteristic climacteric

patterns during storage at 200C. Ethylene production began to increase after 2 d in

storage (not shown) and reached maximum values of 124.2, 130.3, and 103.5 pl kg'1 hl',

respectively, after 6 d storage at 200C (Table 4-1). Ethylene production of fruit treated

with 1-MCP at 0.45 pL L'' for 6 h began to rise after 4 d storage at 200C, reaching a

maximum of 117 pl kg'1 h'' after 9 d storage at 200C. There were no significant

differences in the maximum amount of ethylene production between control fruit and

fruit treated with 1-MCP at 0.09 iL L-' for 6 and 12 h and 0.45 tiL L-' for 6 h (Table 4-1).







Ethylene production in fruit treated with 1-MCP at 0.45 pL LU' for 12 or 24 h

began to increase after 6 and 10 d of storage, respectively (data not shown). As for CO2

production, a distinct peak of ethylene production was not observed during storage at

200C, and maximum ethylene production rates were reduced over 50 % compared with

all other treatments when experiments were terminated (Table 4-1).

Peel Color

The peel of avocado fruit prior to storage had a moderate green color (hue angle =

123.6, where pure yellow = 90 and pure green = 180). At the full-ripe stage, there were

significant differences in the L value (L*), chroma value (C), and hue angle of the peel

color among fruit from all treatments (Table 4-2).

Table 4-2. Peel color of 'Simmonds' avocados stored at 200C with 1-MCP treatments.
Peel color was measured at the full-ripe stage. Fruit were treated with two 1-MCP
concentrations (0.45 and 0.09 pL L1') and three exposure periods (6, 12, and 24 h).
Values followed by the same letter do not differ significantly according to Duncan's
Multiple Range Test (p < 0.05). Initial L*, chroma, and hue angle were 46.5, 24.2, and
123.6, respectively.


Treatments

Control (no 1-MCP)

1-MCP (0.09 tL L-1

1-MCP (0.09 iLL L'

1-MCP (0.45 pLL U'

1-MCP (0.45 LtL U'

1-MCP (0.45 pL L'


for 6 h)

for 12 h)

for 6 h)

for 12 h)

for 24 h)


Days to fully
ripe at 200C

8

8

8

10

10

12


46.3

47.5

49.7

46.1

45.3

44.1


,* Chroma Hue angle
(H)

be 33.2 ab 122.3 ab

ab 33.9 ab 120.8 b

a 37.4 a 120.0 b

bc 32.4 ab 121.0 ab

be 31.6 b 123.5 a

c 26.0 c 123.5 a


Changes in hue angle constituted the major alteration of color coordinates of fruit.

The decline in hue angle represented the change from green to yellow, and the increase in


~







chroma value reflected increasing intensity of yellow color. At the full-ripe stage, fruit

treated with 1-MCP at 0.45 pL L'' for 12 and 24 h retained more green color than fruit

treated with 1-MCP at 0.09 pL L'' for 6 and 12 h. Fruit treated with 1-MCP at 0.45 PL L'

' for 24 h had the lowest L* value (44.1) and chroma value (26.0), with the highest hue

angle (123.5) (Table 4-2). These data indicate that the peel of avocado fruit treated with

1-MCP at 0.45 pL LU' for 24 h retained moderate green color with low color intensity

(light) during 12 days storage at 20 oC.

Enzyme Activity

Based on the previous results employing a range of 1-MCP concentrations and

treatment durations, experiments addressing the effects of the gas on selected cell wall

enzymes and polysaccharides were performed only with fruit exposed to the higher

1-MCP level (0.45 pL L'' for 24 h). The activities of cell wall enzymes in cell-free

protein extracts of avocado fruit treated with 1-MCP at 0.45 IL L-1 for 24 h are shown in

Figures 4-2 and 4-3. The activity of polygalacturonase (PG) was very low in freshly

harvested, preclimacteric fruit, increased during the climacteric period, and continued to

increase during the postclimacteric phase (Fig. 4-2A). The pattern of PG accumulation in

'Simmonds' avocado fruit is consistent with activity levels reported for 'Fuerte' avocado

(Awad and Young, 1979; Zauberman and Schiffmann-Nadel, 1972). PG activity in

'Simmonds' fruit treated with 0.45 iL LU' 1-MCP for 24 h remained at levels comparable

to or slightly below those detected at harvest (Fig. 4-2A).

PME activity in control fruit declined from a maximum value at harvest to a

minimum levcl at the full-ripe (day 8) stage (Fig. 4-2B). The trend for I-MCP treated

fruit paralleled that for control fruit, although the decline in activity was slightly delayed.







The levels of PME in 1-MCP treated fruit after 12 d were similar to those noted for

control fruit at 8 d.

Cx-cellulase (endo-1,4-p-glucanase) activity was not detected in fruit measured

within 24 h of harvest (Fig. 4-3A). In control fruit, Cx-cellulase levels increased

significantly after day 4, reaching levels 13.4-fold higher than those noted at day 4 within

4 d. Cx-cellulase in fruit treated with 0.45 tiL L-' 1-MCP for 24 h was not detectable until

day 4. Cx-cellulase levels increased slowly after day 4 and significantly after day 8,

reaching levels 5.6-fold higher than levels at day 8 within 4 d.

Total a- and p-galactosidase activities are shown in Figures 4-3B and 4-3C. Total

a-galactosidase activity of control fruit decreased significantly during storage and

reached a minimum at day 8. The decline was less pronounced in fruit treated with 0.45

pL L-' 1-MCP for 24 h. a-galactosidase in 1-MCP-treated fruit reached a minimum at day

8, thereafter remaining constant. Total P-galactosidase activity of control fruit and fruit

treated with 0.45 p.L L' 1-MCP decreased after harvest and reached a minimum at day 4

and day 8, respectively. P-galactosidase activity in both control and 1-MCP treated fruit

remained constant through the remaining storage period at 200C.

Solubility and Molecular Mass of Avocado Polyuronides

Polyuonides solubilility was markedly altered during ripening and in response to

treatment with 1-MCP. Figure 4-4 illustrates changes in water- and CDTA-soluble UA

content, total UA content, and EIS levels for both control fruit and fruit treated with 0.45

pL L 1-MCP for 24 h. During the first 4 days of storage, total UA levels remained

nearly constant in both control and 1-MCP-treated fruit. By the full-ripe stage in control

fruit (8 d), total UA in EIS had declined by nearly 30% (Fig. 4-4A). In sharp contrast,

total UA levels in 1-MCP-treated fruit remained constant throughout the 12-day storage







period. Water-soluble UA in control fruit increased significantly after 4 d at 200C, and an

additional 2.1-fold when fruit were fully ripe (Fig. 4-4B). The levels of CDTA-soluble

UA, though much lower than water-soluble levels, decreased significantly after 4 d at

20C (Fig. 4-4C). In 1-MCP-treated fruit, water-soluble UA increased significantly after

8 days at 200C and reached levels 1.8-fold higher than those at day 0 (Fig. 4-4B). In

contrast to control fruit, the levels of CDTA-soluble UA in 1 -MCP-treated fruit remained

constant through 12 d of storage at 200C (Fig. 4-4C). By the full-ripe stage in control

fruit (8 d), EIS levels in mesocarp tissues had declined by nearly 8.3 % (Figure 4-4D). In

contrast, EIS levels in 1-MCP treated fruit remained constant throughout 12 d of storage

at 200C.

Gel permeation profiles of water-soluble polyuronides from control and 1-MCP-

treated avocado on days 0, 4, 8, and 12 are shown in Figure 4-5. At the pre-ripe stage

(Day 0, Fig. 4-5A), water-soluble polyuronides, representing approximately 20% of total

EIS UA, eluted as a polydisperse population. As ripening proceeded (Day 4 through 8;

Fig. 4-5B, 4-5C), water-soluble polyuronides of control fruit exhibited molecular mass

downshifts involving increases in the levels of intermediate and low molecular mass

polymers, the latter eluting near the total column volume. Molecular mass downshifts in

1-MCP-treated fruit were considerably more limited throughout 8 d of storage. At the

full-ripe stage (Day 12), the water-soluble polyuronides of 1-MCP-treated fruit showed

evidence of further mol mass downshifts (Fig. 4-5D); however, as evident from Figure 4-

4 (A, B) the levels of water-soluble polyuronides in 1-MCP-treated fruit represented a

proportionally much lower percentage (40%) of total EIS UA compared with the 70% for

control fruit.







The molecular mass distributions and downshifts of CDTA-soluble polyuronides

paralleled those evident for water-soluble polymers (Fig. 4-6). Mol mass downshifts

were extensive for ripe control fruit (Day 8, Fig. 4-6C) and considerably less pronounced

for ripe 1-MCP-treated fruit (12 d, Fig. 4-6D). CDTA-soluble polyuronides were a

relatively minor component of avocado polyuronides, representing approximately 7%

(control) or 9% (1-MCP treated) of total EIS UA levels.

Molecular Mass of Hemicelluloses and Xyloglucan

The extractable amount of hemicellulose was altered during fruit ripening. Figure

4-7 shows changes in the amount of 4 M alkali-soluble hemicellulose for both control and

1-MCP treated fruit. During the first 4 d of storage, there were no apparent changes in

the amount of hemicellulose in either control or 1-MCP treated fruit. After 4 d at 200C, 4

M alkali-soluble hemicellulose per mg of EIS in both control and 1-MCP treated fruit

decreased by approximately 25% within 4 or 8 d, respectively.

Gel permeation profiles of 4 M alkali-soluble hemicelluloses and xyloglucan from

control and 1-MCP-treated (0.45 lL L' 1-MCP for 24 h) avocado fruit are shown in

Figures 4-8 and 4-9. In control fruit, hemicelluloses at each developmental stage were

polydisperse and exhibited a gradual but limited molecular mass downshift during

ripening (Fig. 4-8). The changes in polymer distribution were evident as a gradual peak

sharpening, with peak elution volumes noted at 40, 42, and 44 ml for hemicelluloses from

day 0, 4, and 8 (ripe) fruit, respectively. Molecular mass downshifts were delayed in

1-MCP-treated fruit (day 0, 40 ml; day 4, 40 ml; day 8, 42 ml; day 10, 44 ml); however,

profiles from full-ripe 1-MCP-treated fruit (Day 12, Fig. 4-8D) were nearly

indistinguishable from those from full-ripe control fruit (Day 8, Fig. 4-8C).







As illustrated in Figure 4-9, changes in molecular mass of xyloglucan (XG)

paralleled those observed for total hemicelluloses, though XG-enriched polymers eluted

as larger molecular mass components of the total hemicelluloses. As noted for total

hemicelluloses, the changes in XG distribution were evident as a gradual peak sharpening

during ripening (Fig. 4-9). XG from 1-MCP-treated fruit at each developmental stage

eluted as a polydisperse population with a slight decrease in average molecular mass.

1-MCP-treated fruit at the full-ripe stage (Day 12, Fig. 4-9D) showed a slightly less

pronounced downshift in XG molecular mass elutionn volume of XG peak = 36 mL)

compared with control fruit (XG peak = 40 mL) (day 8, Fig. 4-9C).


Discussion

In the present study, several parameters (firmness, weight loss, respiration and

C2H4 production, peel color, selected cell wall enzymes activities and structural

carbohydrates) were examined to determine the efficacy of 1-MCP in delaying avocado

fruit ripening. Avocado firmness was significantly retained in response to 1-MCP

treatment, consistent with the fact that softening is one of the most ethylene-sensitive

ripening processes (Lelievre et al., 1997a). Significantly delayed softening by 1-MCP

substantiates that ethylene is involved in augmenting the activity of softening-related

metabolism. Similar effects of 1-MCP in attenuating fruit softening have been observed.

Feng et al. (2000) reported that treatments for 24 h with 30 to 70 nL L'' 1-MCP prior to

exposure to ethylene delayed ethylene-induced softening of 'Hass' avocado, a Mexican-

Guatemalan hybrid, by 10 to 12 d. In apricot, fruit treated with 1 PL L'U 1-MCP for 4 h at

20C had significantly higher firmness compared to untreated fruit after 10 d storage at

20C (Fan et al., 2000). Rupasinghe et al. (2000) found that 'McIntosh' and 'Delicious'







apples treated with 1 liL L-' 1-MCP for 18 h at 200C showed a significant delay in fruit

softening.

The concentration of and length of exposure to 1-MCP significantly influenced

the effect of the gas in delaying avocado fruit ripening. Avocado fruit treated with 0.45

pL L' 1-MCP for 24 h at 20'C required 2 to 4 more days at 200C to reach the full-ripe

stage compared with fruit treated with 0.09 or 0.45 gtL L"' 1-MCP for 6 or 12 h at 20C.

These data are consistent with those of Feng et al. (2000), who reported that increasing

the concentration of 1-MCP to 30, 50, or 70 nL L-1 caused a progressive delay in fruit

softening of 'Hass' avocado.

1-MCP significantly delayed the onset of climacteric ethylene production and

respiration in avocado fruit. The concentration of and length of exposure to 1-MCP

influenced the delaying effect of 1-MCP on ethylene production and respiration patterns

of 'Simmonds' avocado fruit. Ethylene and CO2 production did not fully recover in

avocado fruit treated with 0.45 pL L' 1-MCP for 12 or 24 h at 200C, with maximum

production rates remaining 50 % and 70 % lower, respectively, than those for all other

treatments. Delayed climacteric ethylene production and respiration has also been

reported for apricot (Fan et al., 2000), apple (Fan et al., 1999), banana (Golding et al.,

1998; Golding et al., 1999), and avocado (cv. Hass) (Feng et al. 2000) fruits.

The concentration of 1-MCP required to impart ethylene-insensitivity depends on

the length of exposure to the gas. With longer exposure to 1-MCP, lower concentrations

are required (Sisler et al., 1996a). The levels of 1-MCP required to protect plants from

ethylene effects also depends on the exposure temperature. Ku and Wills (1999) found

that the storage life of broccoli increased with an increase in the concentration and







exposure period of 1-MCP treatment at 5 and 200C and also reported that broccoli treated

with 1 [iL L' 1-MCP at 200C exhibited a significantly longer storage life than that treated

at 50C. We have observed that 1-MCP treatment at 200C had more effect on delaying

avocado fruit ripening than at 12 C (Jeong, unpublished). In carnation, the effectiveness

of 1-MCP is 4-fold higher at room temperature (240C) than at 4C (Sisler and Serek,

1997). Sisler and Serek (1997) have suggested that 1-MCP more rapidly and efficiently

associates with receptors at higher temperatures.

Further increases in the concentration of 1-MCP (e.g., 2.3 or 4.5 PL L'' for 24 h)

did not result in a further delay in ripening compared with treatment at 0.45 iL L'' 1-

MCP for 24 h (data not shown). This indicates that treatment with 0.45 pgl1' 1-MCP for

24 h is sufficient to exert maximal delay of avocado ripening. The concentration of 1-

MCP necessary for maximum response varies from crop to crop, and with maturation,

temperature, and duration of exposure (Rupasinghe et al., 2000). Although relatively low

concentrations of 1-MCP were effective in suppressing ethylene effects in carnation

flowers (0.5 nL L', Sisler, 1996a; Sisler, 1997), ethylene-induced abscission, and flower

senescence for potted flowering plants (20 nL L' 1, Serek et al., 1994b), and ripening of

banana fruit (0.7 nL L'', Sisler and Serek, 1997), the levels necessary to delay avocado

ripening (0.45 utL L'') are comparable to those found effective for delaying ripening of

apricot (1 [il'l', Fan et al., 2000), and 'McIntosh' and 'Delicious' apples (1 pL L',

Rupasinghe et al. 2000). In an analysis of the absorption capacity of tomato and avocado

fruits for 1-MCP, an equivalent mass of avocado tissue absorbed 2 to 3-fold higher levels

of 1-MCP gas (Jeong, unpublished). The greater amount of 1-MCP required to block

C2H4 action in avocado could be due to the high oil content in these fruit, which could act







as a competitive reservoir for 1-MCP. The gradual (but incomplete) recovery of C2H4

production in 0.45 tL L'' 1-MCP-treated avocados during storage at 200C suggests either

the synthesis of new receptor proteins, metabolism of the 1-MCP receptor-protein

complex, or dissociation of 1-MCP from the receptor sites (Sisler and Serek, 1999; Sisler

et al., 1996a).

Consistent with previous reports, the ripening of avocado fruit was accompanied

by increases in Cx-cellulase and PG activities (Awad and Young, 1979; Christofferson et

al., 1984; Pesis et al., 1978) and a decline in PME activity (Awad and Young, 1979;

Awad and Young, 1980; Zauberman and Schiffmann-Nadel, 1972). Of the cell wall

enzymes measured in 1-MCP-treated fruit, PG exhibited the strongest response, showing

little or no recovery over the 12-day post-treatment storage period. Feng et al. (2000)

found that 30, 50, or 70 nL L' 1-MCP suppressed PG activity about 10 to 30% and

delayed the increase in cellulase activity by 4 days in 'Hass' avocado.

Although PG activity did not recover in 1-MCP-treated avocado, the firmness

ultimately reached values comparable to those of control fruit, indicating that PG is not

required for the major component of avocado fruit softening. This conclusion is

consistent with reports for tomato fruit, wherein antisense suppression of PG activity had

minimal influence on fruit softening until the very late stages of ripening (Carrington et

al., 1993; Kramer et al., 1992). Although not statistically significant, 0.45 pL L'' 1-

MCP-treated avocados at the full-ripe stage remained slightly firmer than control fruit.

This may indicate that PG in avocado fruit, as for tomato fruit, is more important in the

late stages of ripening.







Consistent with the marked suppression of PG levels in 1-MCP-treated

'Simmonds' avocado fruit, the solubilization and degradation of polyuronides was

significantly delayed and reduced in 1-MCP-treated fruit. At the pre-ripe stage (day 0),

water- and CDTA-soluble UA constituted 22 % and 7.9% of the total EIS UA content,

respectively. At the full-ripe stage (10-20 N), water- and CDTA-soluble polyuronides of

control fruit comprised approximately 65% and 7.5%, respectively, of the total UA

content, whereas those of 1-MCP treated fruit comprised approximately 38% and 9.7%,

respectively, of the total UA content. The large difference in the levels of water-soluble

polyuronides expressed as a percentage of total EIS UA was in large part due to the

persistence of the UA levels in EIS of 1-MCP-treated fruit and a 31% decrease in UA

levels of control fruit. The 31% decrease in total EIS UA is consistent with the 28%

decline in total UA reported for 'Lula' avocado (West Indian-Guatemalan hybrid)

(Wakabayashi et al. 2000). Owing to the extensive depolymerization of polyuronides

characteristic of ripening avocado fruit (Huber and O'Donoghue 1993; Sakurai and

Nevins 1997; Wakabayashi et al. 2000), the persistence in total UA in 1-MCP-treated

fruit is likely explained on the basis of the more limited molecular mass downshifts in the

polyuronides of these fruit. In the present study, both water and CDTA-soluble

polyuronides of control fruit exhibited the characteristic molecular mass downshifts

reported in earlier studies, with polymers in ripe fruit eluting near the total column

volume. Through 8 d of storage (Fig. 4-5C, 4-6C) 1-MCP treated fruit showed

considerably less extensive breakdown of both water- and CDTA-soluble polyuronides.

Further downshifts in polyuronide molecular mass were evident in 1-MCP-treated fruit

after 12 d of storage. These data suggest that the low activity of PG in 1-MCP-treated







fruit is sufficient to depolymerize avocado polyuronides; however, we note that the

quantities of polyuronides extracted from 1-MCP treated fruit at 12 d are proportionally

much lower than those from control fruit. Additionally, Wakabayashyi et al. (2000) have

shown that limited molecular mass downshifts in avocado polyuronides, as evident from

gel filtration analyses, can be brought about by deesterification, independently of PG

action. Consequently, the relatively normal levels of PME in these fruit might have

influenced the gel filtration behavior of polyuronides from 1-MCP-treated fruit.

Total extractable a- and P-galactosidase activities decreased during avocado

ripening; however, the use of total protein extracts in our assays would have masked

differential responses of specific isozymes of these proteins. For example, Pressey

(1983) and Carey et al. (1995) reported that total P-galactosidase activity in tomato

remained relatively constant throughout ripening, whereas the levels of one isozyme (P-

Gal II) increased about 4-fold. Smith and Gross (2000) found that transcript

accumulation for tomato p-gal II, one of 7 P-gal transcripts detected, was significantly

impaired in rin, nor, and Nr fruit relative to wild-type accumulation, indicating that P-gal

II may be upregulated by ethylene.

The mol mass distribution of hemicelluloses from 'Simmonds' was generally

similar to that of hemicelluloses from 'Lula' avocado fruit (O'Donoghue and Huber,

1992). During ripening of 'Simmonds' avocado at 200C, hemicelluloses at each

developmental stage exhibited a gradual but limited molecular mass downshift. 1-MCP

treatment did not significantly affect the quantities of 4 M alkali-soluble hemicellulose

during ripening (Fig. 4-7). 1-MCP treatment, however, significantly reduced molecular

mass downshifts in 4 M alkali-soluble hemicelluloses and xyloglucan (Fig. 4-8, 4-9). The







changes in polymer distribution were more obvious as a gradual peak sharpening over

molecular mass range.

Therefore, treatment of avocado fruit with 1-MCP reduced the rate of ripening

and extended the postharvest life of avocado while ensuring high quality.


Summary

West Indian-type avocado (Persea americana Mill. cv. Simmonds) fruit were

treated with two different concentrations (0.09 and 0.45 pL L') of 1-methylcyclopropene

(1-MCP) for three exposure times (6, 12, and 24 h) at 200C. The fruit were then stored at

20 C in ethylene-free air for ripening assessment. Firmness, weight loss, respiration and

C2H4 production, peel color, selected cell wall enzymes (polygalacturonase,

pectinmethylesterase, a-, P3-galactosidase, and Cx-cellulase) and cell wall matrix

polysaccharides (polyuronides and hemicellulose) were monitored during storage.

1-MCP treatment at 0.45 uL L'1 for 24 h at 200C delayed ripening of avocado fruit

by 4 d at 200C. This delay was characterized by a significant reduction in the rate of fruit

softening and in the timing and intensity of the ethylene and respiratory climacterics.

Avocado treated with 1-MCP (0.45 pL L') for 24 h at 200C also showed significantly

less weight loss and retained more green color than control fruit at the full-ripe stage (10

to 20 N). The delay in avocado ripening was influenced by 1-MCP concentration,

exposure duration, and exposure temperature.

1-MCP treatment affected the activity trends of all cell wall enzymes measured

and completely suppressed the appearance of polygalacturonase activity for up to 12 d.

At the full-ripe stage, water- and CDTA-soluble polyuronides of control fruit comprised

approximately 65% and 7.5% of the total polyuronide content, respectively, whereas







those of 1-MCP-treated fruit comprised approximately 38% and 9.7% of the total

polyuronide content. When fully ripe, polyuronides from 1-MCP treated fruit exhibited

reduced molecular mass downshifts compared with control fruit. 1-MCP treatment also

delayed and slightly reduced the depolymerization of 4 M alkali-soluble hemicelluloses,

including xyloglucan.

Inhibition of ethylene action with 1-MCP during the early stages of climacteric

produces changes in subsequent ripening behavior. 1-MCP profoundly blocked C2H4

action and delayed C2H4 dependent ripening of avocado fruit. This study has shown that

1-MCP has the potential to control the ripening of avocado fruit. Further work on the

effects of 1-MCP will allow its future commercial use and, in conjunction with

appropriate harvesting and environmental conditions, will extend the storage period of

avocado and other fruit. he storage period of avocado and other fruit.











100
--e 1-MCP (0.45 pL L'1 for 24 h)
-o- 1-MCP (0.45 pL L'1 for 6 h)
80 -- 1-MCP (0.45 pL L"' for 12 h)
1 -MCP (0.09 pL L' for 12 h)
"- --- 1-MCP (0.09 pL L' for 6 h)
) 60 -0- Control (no 1-MCP)


40
U.

20
A
0


8 B


6

0
4


2


0

0 2 4 6 8 10 12 14
Storage period (days)


Figure 4-1. Fruit firmness (N) and weight loss (%) of 'Simmonds' avocados stored at
200C with 1-MCP treatments. Fruit were treated with two 1-MCP concentrations (0.45
and 0.09 plL L') and three exposure periods (6, 12, and 24 h). Vertical bars represent
standard deviation of 6 independent samples.










35

30 A
30

-| 20
E
15

E 10

5

0

4 B -- 1-MCP (0.45 pL L-1 for 24 h)
Control (no 1-MCP)

3

CUE
2

1 "




0
0 2 4 6 8 10 12 14
Storage period (days)


Figure 4-2. Effect of 1-MCP (0.45 piL L'' for 24 h) on PG and PME activities of avocados
stored at 200C. Fruit were treated with (o) or without (o) 1-MCP. Vertical bars represent
standard deviation of 3 independent samples.












4

>-- 3

E
c-: 22
7_ E


00
8 18




S1-MCP (0.45 pL L"1 for 24 h) 8 o
S-Control (no 1-MCP) 7 0o x
6 **
5 -E
S4 o
3 Mi
B o2
2 ? 6 E
1




2

0 CD

E C


0 2 4 6 8 10 12 14

Storage period (days)




Figure 4-3. Effect of 1-MCP (0.45 iL L'1 for 24 h) on Cx-cellulase and a- and P-
galactosidase activities of avocados stored at 200C. Fruit were treated with (o) or
without (*) 1-MCP. Vertical bars represent standard deviation of 3 independent samples.











240
220
200
180
160
140
120


A


1-MCP (0.45 pL L-1 for 24 h)
-- Control (no 1-MCP)












D


0
120
100
80
60 0
o
40 ?
20
20


0)
W

E
o


1050
1000
950
900
850
800
75;n


0 2 4 6 8 10 12 14


Storage period (days)



Figure 4-4. Effect of 1-MCP (0.45 pL L'' for 24 h) on the amount of EIS in the mesocarp
tissue and on the changes in water-, CDTA-soluble UA, and total UA in EIS from
avocados stored at 200C. EIS were incubated sequentially in distilled water and 50 mM
CDTA in 50 mM Na-acetate, pH 6.5, each for 4 h at 340C. Suspensions were filtered and
UA content determined. Fruit were treated with (o) or without (*) 1-MCP. Data are
means standard deviation of 3 independent samples.











18
15 Vo Vt A
12
9
6
3
0
15 o 1-MCP (0.45 pL L for 24 h) g
0 12 -- Control (no 1-MCP)
0
U 9



o 0
15
m 12
S 9
6
S 3
0' ^r a.- .--
0
15 D
12
9
6
3
0
10 20 30 40 50 60 70 80
Elution volume (mL)



Figure 4-5. Molecular mass profiles of water-soluble polyuronides from EIS prepared
from avocado treated with (o) and without (e) 1-MCP. Polyuonides (= 0.5 mg
galacturonic acids equivalents) were applied to Sepharose CL-2B-300 and individual
fractions were measured for UA content. Data for each fraction expressed as a percentage
of the total eluted UA. Day 0 (A); Day 4 (B); Day 8 (C); Day 12 (D). Vo, Void volume;
Vt, total volume.











15
12 Vo Vt A
9
6
3
0 0 -- -
12 -- Control (no 1-MCP) B
_-o 1-MCP (0.45 pL L' for 24 h,
0 9
6

o 3


4-9
o 0
S12 C







VD
9
6
3
0

10 20 30 40 50 60 70 80

Elution volume (mL)


Figure 4-6. Molecular mass profiles of CDTA-soluble polyuronides from EIS prepared
from avocado treated with (o) and without (*) 1-MCP. Polyuonides (= 0.5 mg
galacturonic acids equivalents) were applied to Sepharose CL-2B-300 and individual
fractions were measured for UA content. Data for each fraction expressed as a percentage
of the total eluted UA. Day 0 (A); Day 4 (B); Day 8 (C); Day 12 (D). Vo, Void volume;
Vt, total volume.

















20

--- Control (no 1-MCP)
18 1-MCP (0.45 pL L'for 24 h)
0


uW 16






10 o




0 2 4 6 8 10 12 14






Figure 4-7. Effect of 1-MCP (0.45 pL L'' for 24 h) on the changes in the extractable
amount of 4 M alkali-soluble hemicellulose in EIS from avocados stored at 200C. Fruit
were treated with (o) or without (e) 1-MCP. Data are means SD of 3 independent
samples.




80





15 -I III II
12
12 Vo Vt
9
6
3
0





o Q9
6





i 9
e 6
E 3

0
4 12 D








Elution volume (mL)



Figure 4-8. Molecular mass profiles of 4 M alkali-soluble hemicellulose from EIS
prepared from avocado treated with (o) and without (*) 1-MCP. Two ml of
hermicellulose was applied to the Sepharose CL-6B-100 and individual fractions were
measured for total sugar. Data for each fraction expressed as a percentage of the total
eluted sugar. Day 0 (A); Day 4 (B); Day 8 (C); Day 12 (D). Tick marks at the top of the
figure indicate void volume (Vo), 70, 40, 10 kDa (middle), and glucose (right).
Figre4-8 Mleulrmsprflso4 alaislbehmclus rmEI

prepared 0rmaoaotetdwt o ndwtot()1MP w lo
hemielllos wa0ple oteSpaoeC-B-0 n niiulfatoswr
measured 9o oa ua.Dt o ahfato epesda ecnaeo h oa











12

9 Vo Vt

6

3


| ~- MCP (0.45 pL L-1 for 24 h) B
o 9


4- 6
o
3
0
c -- Control (no MCP)
9 D

o 6

3

0

9

6

3

0
10 20 30 40 50 60 70 80

Elution volume (mL)


Figure 4-9. Molecular mass profiles of xyloglucan in 4 M alkali-soluble hemicellulose
from EIS prepared from avocado treated with (o) and without (*) 1-MCP. Two ml of
hemicellulose was applied to the Sepharose CL-6B-100 and individual fractions were
measured for total sugar. Data for each fraction expressed as a percentage of the total
eluted sugar. Day 0 (A); Day 4 (B); Day 8 (C); Day 12 (D). Tick marks at the top of the
figure indicate void volume (Vo), 70, 40, 10 kDa (middle), and glucose (right).












CHAPTER 5
THE EFFECTS OF 1-METHYLCYCLOPROPENE (1-MCP) AND WAX COATING
FOR REGULATING THE RIPENING AND EXTENDING THE STORAGE LIFE OF
AVOCADO FRUIT


Introduction

The avocado (Persea americana Mill.) is a climacteric fruit that is characterized

by a surge in ethylene production at the onset of ripening. This climacteric increase in

ethylene production is associated with hastened ripening of fruits. Avocado is one of the

most rapidly ripening of fruits, often completing ripening within 5 to 7 d following

harvest (Seymour and Tucker, 1993).

As ethylene plays as important role in regulating fruit ripening, inhibiting

ethylene biosynthesis or action should slow the ripening process and extend the fruit's

postharvest storage life. 1-methylcyclopropene (1-MCP), one of the synthetic

cyclopropenes, blocks ethylene receptors and prevents ethylene effects in plant tissues for

extended periods (Sisler and Serek, 1997; Sisler et al., 1996a; Sisler et al., 1996b). This

material is nontoxic, odorless, and effective when plants are treated at concentrations as

low as 0.5 nL L'1 (Sisler and Serek, 1997). Although 1-MCP binding to the ethylene

receptor sites is irreversible, it appears that new receptors can be formed during

climacteric (Sisler et al., 1996a).

1-MCP has been shown to delay fruit ripening and improve storage quality of

climacteric fruits such as pears (Pyrus communis L. cv. Passe-Crassane) (Lelievre et al.,

1997b), banana (Musa sp.) (Golding et al., 1998; Golding et al., 1999; Sisler and Serek,







1997), plums (Prunus salicina Lindl) (Abdi et al., 1998), tomato (Lycopersicon

esculentum Mill) (Nakatsuka et al., 1997; Sisler and Serek, 1997), apple (Malus sylvestris

L.) (Fan and Mattheis, 1999; Watkins et al., 2000), and avocado (Persea Americana Mill)

(Feng et al., 2000). 1-MCP, therefore, has provided a valuable tool to investigate ethylene

metabolism in ripening climacteric fruit (Nakatsuka et al., 1997) and has the potential to

extend the storage life of horticultural products.

Waxing is also known to extend the storage life of avocado by reducing water loss

and modifying the fruit internal atmosphere (Joyce et al., 1995). The postharvest life of

avocado fruit is longer when water loss is reduced (Adato and Gazit, 1974; Joyce et al.,

1995). Similarly, reduced water loss has been associated with shelf life extension in

ripening pear and banana (Littmann, 1972). In addition, waxing delays softening and

improves appearance of avocado fruit (Durand et al., 1984; Lunt et al., 1981) and

depressed respiration and ethylene production of avocado fruit (Durand et al., 1984).

The objectives of the present research were to characterize physiological and

biochemical responses of avocado fruit to 1-MCP and/or wax treatment during avocado

fruit ripening and to evaluate its ability as a postharvest tool for regulating the ripening of

avocado fruit. Hypothesis: Application of wax coatings to 1-MCP-treated avocado will

reduce the rate of ripening to a greater extent than treatment with 1-MCP alone.


Materials and Methods

Plant Material

'Tower II' and 'Booth 7', mid-season cultivars, were selected for these

experiments. They are "hybrid" cultivars, which are crosses of West Indian and

Guatemalan races (Hatton and Campbell, 1960; Hatton et al., 1964). Recommended







storage temperature is 130C to avoid chilling injury (Seymour and Tucker, 1993).

Mature avocado fruit were obtained from a commercial grower in Homestead, Florida,

packed in fiberboard cartons, and transported to the Postharvest Horticulture Laboratory

in Gainesville within 24 h after harvest. Fruit were selected for uniformity of size

(weight, 'Tower II' 575 48 g and 'Booth 7' 526 40 g) and shape (diameter at

equatorial region, 'Tower II' 9.0 0.3 cm and 'Booth 7' 9.5 0.2 cm), and then were

surface sterilized in a 15% (90 mM NaOCI) commercial bleach solution, rinsed, and

dried.

1-MCP and Wax Treatment

Twelve fruit were placed in 18-L containers and exposed to 1-MCP by releasing

the gas from a commercial powdered formulation (Ethyblock Floralife, Burr Ridge, IL).

The concentration selected, 0.9 [pL L', was achieved through addition of 10 mg of the

powder to 100 mL of FloraLife buffer following manufacturer's instructions (Floralife,

Ethyblock product specification sheet). Following addition of the buffer to the 1-MCP,

the beakers were tranSferred to the 18-L containers, which were sealed immediately.

1-MCP treatment was performed for 12 h at 200C and 85% relative humidity (RH). In the

first experiment with 'Tower II', immediately following 1-MCP treatment, the fruit were

removed from the treatment chambers and then half of the fruit were waxed (Sta-Fresh

819F, FMC co.). Fruit were dipped in the wax concentrate for 1 min and allowed to

drain and air-dried with a fan. The fruit were subsequently stored at 200C in ethylene-free

air at 85% relative humidity. Control fruit (not exposed to 1-MCP and not waxed) were

maintained under identical storage conditions. In the second experiment with 'Booth 7',

immediately following 1-MCP treatment, the fruit were removed from the treatment







chambers and then waxed. The fruit were subsequently stored at 130C in ethylene-free air

at 85% relative humidity. Control fruit (not exposed to 1-MCP but waxed) were

maintained under identical storage conditions. Samples of fruit from each treatment were

evaluated for fruit quality on a daily basis until they reached the full ripe stage (10 to 20

N, too soft for commercial handling). Fruit quality was assessed on the basis of fruit

firmness, weight loss, CO2, and C2H4 production, as well as peel color changes.

Mesocarp tissue derived from the equatorial region of selected fruit was stored at -300C

and later used for analysis of polygalacturonase activity.

Fruit Firmness

Firmness was determined on whole, unpeeled fruit using an Instron Universial

Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat-plate probe (5 cm

in diameter) and 50-kg load cell. After establishing zero force contact between the probe

and the equatorial region of the fruit, the probe was driven with a crosshead speed of 10

mm min-'. The force was recorded at 2.5 mm deformation and was determined at two

equidistant points on the equatorial region of each fruit. The same four fruit of each

treatment were measured repeatedly every other day until they reached the full-ripe stage.

Respiration and Ethylene Evolution

Respiration and ethylene production were measured every other day using the

same four fruit of each treatment. Fruit were individually sealed for 30 min in 2-L plastic

containers prior to sampling. A 0.5 mL gas sample was withdrawn by syringe through a

rubber septum, and carbon dioxide determined using a Gow-Mac gas chromatograph

(Series 580, Bridge water, NJ, USA) equipped with a thermal conductivity detector

(TCD). Ethylene was measured by injecting a 1.0 mL gas sample into a HP 5890 gas







chromatograph (Hewlett Packard, Avondale, PA, USA) equipped with a flame ionization

detector.

Peel Color

Individual fruit were marked at the equatorial region (2 regions per fruit), and

color at the same location was recorded every other day as L*, hue angle, and chroma

value with a Minolta Chroma Meter (CR-2000, Minolta Camera Co Ltd., Japan). The

chroma meter was calibrated with a white standard tile. The color was reported as hue

angle (H'), with a value of 90 representing a totally yellow color and 1800 a totally

green color. The results are presented as lightness (L*), chroma (C*), and hue angle (H).

The chroma and hue angle were calculated from the measured a* and b* values using the

formulas C* = (a*2+b*2) 1/2 and H = arc tangent (b*/a*) (McGuire, 1992).

Preparation of Cell-Free Protein Extract

Partially thawed mesocarp tissue (10 g) was homogenized with 40 mL of ice-cold

95% EtOH for 1 min in an Omnimixer (Model 17150, Newtown, CT, USA) and

centrifuged at 7840 g for 10 min at 40C. The supernatant was discarded and the pellets

were resuspended in 50 mL of ice-cold 80% EtOH for 1 min and again centrifuged at

7840 g for 10 min at 40C. The pellets were transferred to 50 mL of ice-cold acetone for

10 min followed by centrifugation (7840 g, 10 min, 4C). After 2 additional acetone

washings, the pellets were suspended in 50 mL of ice-cold 80% EtOH, stirred with a

spatula, kept for 10 min in an ice-cold water bath (50C), and then centrifuged (7840 g, 10

min, 4C). The pellets were transferred to 30 ml of 10 mM Na-acetate, pH 6.0,

containing 1.8 M NaCI, for 30 min in ice-cold (50C) water bath and centrifuged. The

supernatant was analyzed for enzyme activities as described below. Protein content was







measured using the bicinchoninic method (Smith et al., 1985) with bovine serum albumin

as a standard.

Polygalacturonase Assay

Polygalacturonase (PG, E.C. 3.2.1.15) activity was assayed reductometrically by

incubating a 100 pL aliquot of the cell-free protein extract with 500 1IL (2 mg) of

polygalacturonic acid (from orange peel, Sigma Chemical Co, St. Louis, MO, USA)

dissolved in 30 mM KOAc, pH 5.5, containing 100 mMKC1. After 30 min at 340C,

uronic acid reducing groups were measured using the method of Milner and Avigad

(1967). PG activity was expressed as mol D-galacturonic acid equivalents produced per

kg protein per minute.

Statistical Analysis

The experiments were conducted in a completely randomized design. Statistical

procedures were performed using the PC-SAS software package (SAS-Insititute, 1985).

Data were subjected to ANOVA using the General Linear Model (Minitab, State College,

PA). Differences between means were determined using Duncan's multiple range test.


Results

Fruit Firmness and Weight Loss

Changes in 'Tower II' avocado fruit firmness following 1-MCP treatment (0.9 pL

L'' for 12 h at 200C) and/or wax treatments are shown in Figure 5-1A. Control fruit

softened rapidly and completed ripening (10 to 20 N) within 7 days of storage at 200C.

After 7 d of storage at 200C, fruit treated with wax, 1-MCP, or both 1-MCP and wax

exhibited firmness values of 31.2, 36.6, and 51.5 N, respectively. Firmness decrease of

fruit treated with 1-MCP and/or wax was significantly delayed and varied with different




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