Citation
Effects of ethylene and its action inhibitor (1-Methylcyclopropene) for regulating ripening and extending the postharvest life of avocado

Material Information

Title:
Effects of ethylene and its action inhibitor (1-Methylcyclopropene) for regulating ripening and extending the postharvest life of avocado
Creator:
Jeong, Jiwon
Publication Date:
Language:
English
Physical Description:
xiii, 171 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Avocados ( jstor )
Cell walls ( jstor )
Ethylene production ( jstor )
Figs ( jstor )
Fruits ( jstor )
Ripening ( jstor )
Sugars ( jstor )
Tomatoes ( jstor )
Waxes ( jstor )
Weight loss ( jstor )
Avocado -- Postharvest technology ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF ( lcsh )
Ethylene -- Physiological effect ( lcsh )
Horticultural Science thesis, Ph. D ( lcsh )
Plant growth inhibiting substances -- Physiological effect ( lcsh )
Plant regulators -- Physiological effect ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

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

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
028216836 ( ALEPH )
49320795 ( OCLC )

Downloads

This item has the following downloads:


Full Text









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




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E8U3R0ADV_18MC58 INGEST_TIME 2013-03-05T20:37:10Z PACKAGE AA00013530_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

EFFECTS OF ETHYLENE AND ITS ACTION INHIBITOR (1-METHYLCYCLOPROPENE) FOR REGULATING RIPENING AND EXTE DING 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

PAGE 2

ACKNOWLEDGMENTS I ould 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. Ste en Sargent Dr. Charles Sims Dr. Jonathan Crane and Dr. Rebecca Darnell who have all g1 en 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. 11

PAGE 3

T ABL OF CONT NTS CKNOWLEDGMENTS . ... .. ............ ..... .. .. .......... .. ............ .. .. ... ... .. .......... ....... .. ......... ii LI T OF TABLES .... ............ .. .. .. ...... ..... .. ... ........... ... .. ................... ....... ... .................... . v LIST OF FIGURES................................. .. . ............. .... .. .. ... .... .. .. ..... ... ... ......... .. .. .. ..... iii LIST OF ABBREVIATIONS ............................ .. ... .. .. .. ............ ... .... ................ .. ....... .. . xi ABSTRACT ...... .. ... ...... ..... .. ........................... .. ...... .. ... ................. ... .... .. ..... .. ........ .. .. .. .. xii CHAPTERS 1 INTRODUCTION .............. ...... .. .. ...... .. .. .. .. .. .. .. ....... ............... .. .. ................................ 1 2 LITERATURE REVIEW ....... .......... .. ... .. .. .......... .. ... ......... .... ... .... ............................. 3 Introduction ... ... ............... ....... .. .. ....... .. ..... ... .. ............................ ...... ..... .. ... .. .......... 3 Avocado ......... .. .. .. .. .. .. ...... .. .. .. ..... .. .. .. .. .. ..................................... .. ..... .. .... ...... .. ........ 3 Fruit Development .. ... .. .. ...... ... . .. .......... ...... .. .. ....... .. .. .. .................. .. .... ... .. ... .. .. ...... 4 Avocado Fruit Ripening .. ... .. .. .. .. .. .. .. ... ..... .. .. .. .. .. ... .. ... .. ... .... ............ ..... .... .... .. .. .... 4 Climacteric Characteristics ........ ..................... ....... .. .. ......... .. ..... .. ..... .... ... .... .. .. ....... 5 Fruit Softening .. .............................................. .. ... ... ... .. ... ... .. .................................... 5 Cell wall changes ............ .. .. .. .. ... .. .. .. .. .. .. ....... ............ ... ..... ... .... ... ..... .. ............... 6 Cell wall hydro lases .......................................... ... ............ ...... ............. ..... .. .......... 6 Ripening-induced compositional changes ... ...... .. ......... .. .. .. ....... .. ........................ 9 Modification of Ripening ........................................ .... ... .. .... ... .... ... ........ ... ... ... .. ... 10 E th y l e n e ... .... .. .. ... .. .......... .. .. .................. ....... .. ............ .. ......... ............... .. ................. 10 Gas ous Plant Growth Regulator.. .. .. .. .... .. .. ... .. .... .. .... ... .. ......................... .. ........ 11 th y l e ne Biosynthetic Pathway ............................................. .. ... ..... .. .. ... ............. 11 thyl n Perception and Signal Transduction ...... .................. ................ .. ................ 11 Practical Use of E thylene ......... .. .. .................. .. .. ........ .. ......... ................................. 12 Et h y l ene Antagonists .................. .. ... .. .. ....... .. ... .... .. . .... . ... .... .... ......................... 13 1-M thylcyclopropene (1-MCP) ... .. .. ......... ......................... .. .... ..... ...... . ................... 15 Inhibitor of Ethyl ne Responses .. ... ...... ....... .. .. .. .. .... .. ... .. .... .. ...... .... .......... ....... .. 15 Tr atment of Plants .......... .. .. .......... .. .. .................................. ...... ........ .. ... ... ... .. .. .. . 17 Effi ct of concentrations exposure times and temperatur of 1-MCP ... ............ 17 Effi ct on respiration and ethylen production of 1-MCP .. ... . .. .... ..... ... .. .... ......... 19 0th r r sponses of 1-MCP ... .. ...... ... ................... ... .. . . ...... ................................. 19 Ill

PAGE 4

3 POSTHARVE T ETHYLENE TREATMENT FOR UNIFORM RIPENING OF WE T INDIAN-TYPE AVOCADO FRUIT. ... . .. .. . .. .... .. .. .. . ... .. ................................... 21 Introduction ........... ............... .. .. ............. . .. .. ........ .. . .................... .. .. .. .... ........ ......... 21 Materials and Methods .. .. .. .. .. ........................ .. .. ..... .. .......... .. ... .. ... ..................... 22 Results .. ................ .. ..... .. .. ..... .. .. .. .. . .. ................ .. ..... ...................... .... .. ............. 27 Discussion .... .. .. ...... .. ..... .................... .. .. .... .. .... ........................................................ 44 Summary ....... .. .. .. .......... .... .. .. ..... .. ..... ....... ................................ .. ...... .... .......... ...... 46 4 INFLUENCE OF 1-METHYLCYCLOPROPENE (1 -MCP ) ON RIPENING AND CELL WALL MA TRIX POLYSACCHARIDES OF A YOCADO FRUIT ........... . ....... .48 Introduction .. ...... .... .. ......... ..... .. . ............... .... ..... . .. .. .. .. .. .............. ........ .. .. .. ....... .. 48 Materials and Methods .. .. .. .. .. ........ ............... .............. .................. .. ... .. .. ... .. .. .. .... 50 R es ults ... .. .. .... . ............................................... . ........... .. .. .. .. .. .. .... ............................... 57 Di s cussion .. ....... .. ... .. .. .. ......... .. .... ... ..... .. .. ..... .. ............... ....... .... .... ................ .... 65 Summary ... ......... .. . .. ...... .. .. .. .. .. .. ... . .. .. ........... .. ..... ....... .. .. ... .. ... .. .. .. .......... ...... .. 71 5 THE EFFECTS OF 1-METHYLCYCLOPROPENE (1-MCP) AND WAXING FOR REGULATING THE RIPENING AND EXTENDING THE STORAGE LIFE OF AVOCADO FRUIT .. .. .. ...... .. ......................... .. .. .. .. ...... .............................................. 82 Introduction ...... .. ................ .. ......................................................................................... 82 Materials and Methods .. ... ....... .. .. .. ........................ .......... ..... .......................... .. ........... 83 Results .... .. ........ .. ... .. ............ .. .. ......... ...... .. ......... .. .. .. .. ............... ......... ... .. .............. 87 Discussion .. .. ... .... .......... .. ...... ...... . ...................... .. .. .. ..... .. .. ............. .. ...................... 93 Summary ............................... .. ............................................... .. ......... ...... .... . ...... .. ..... 97 6 INFLU NCE OF ETHYLENE AND ITS ACTION INHIBITOR (1-MCP) ON RIP ENING AN D CELL WALL MATRIX POLYSACCHARIDES OF AVOCADO F R U IT ....................................................... .. .. ................... .. ........... .. ........ .. ... .. .............. 103 Introduction .. ... .. ........... ..... .. ...................... ...... ..... .. ...... .......... ................................. 103 Materials and Methods .. .......... .................. ... ..... .. . ... .. ...... ....... .. .. .. .... ... .... .. .. .. .. .. .. 104 Results .... .. .. .. .... ... .. .. ... .. .. .. .. ...... ................... .. .. .. .. ... ..... .... .. .............. ... .. ............ 113 Discu s ion . .................... ....... ..... .. .. .. .. ........ ... . .. .. .. ... ............................................ 13 2 umm a r y ............................................. ..... . ................. ... ..... ... .... .... .. ... ......... .. .. ... 138 7 UMMARY AND CONCLUSIONS .. .. ..... .. .. .. .. ......... ... .. .. ...................................... 153 R ~ R N ................................................................................................................ 15 MP I II A K T H ............................................... . . .... .. ... .. .... ... ....... .. .. .. .... 1 71 lV

PAGE 5

LIST OF TABLES Tabl 3-1. Fruit quality evaluation at the full-ripe stage for Simmonds a v ocados from early -h arvest (06 / 29 / 98) gassed immediately for 48 hat 20 C and transferred to 10 C or stored at 10 C for 7 d then gassed for 48 hat 20 C and then transferred to 10 C .. ...... ................... .. . . .. . ........... ....... .. .... . . ........ 28 3-2. Fruit quality evaluation at the full-ripe stage for Simmonds avocados from mid-harvest (07 / 13 / 98) gassed immediately for 48 hat 20 C and transferred to 12 C or stored at 12 C for 7 d then gassed for 48 hat 20 C and then transferred to 12 C . . ............. .............. .............. .. . . .. .... .. . .. . .. .. . .. .. .. 29 3-3. Fruit quality evaluation at the full-ripe stage for Simmonds avocados from lat e-harvest (07 /27 / 98) gassed immediately for 48 hat 20 C and transferred to 13 C or stored at 13 C for 7 d then gassed for 48 hat 20 C and then transferred to 13 C ........................................................................ ..... .. 3 0 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 treatments ............................... .. .. .. .. .. .. .... .... ... ................. .. .. .. .. .. 33 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 .. ... ... .. ..... .. . . . ........... 34 3-6. Fruit quality evaluation for Booth 7' avocados from late-harvest (10/17 / 98) tored for 7 d at l 2 C and transferred to 20 C 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 12 C with different ethylene treatments .... .... .......... ... ........ 36 3-8 Fruit quality evaluation for Monroe avocados from ear l y-harvest (11 / 20 / 98) stored for 7 d at 13 C and transferred to 20 C with different ethylene tr atments ..... . .. ... ... ............. ....................... . ....... . . ..... ........... ... .. 3 3-9. Fruit quality valuation for Monroe avocados from early-har t ( 1 l / 20 / 98 tor d for 14 d at 13 C with different ethylene tr atm nt ... . .. ... . ...... .. ... .......

PAGE 6

3-10. Fruit quality evaluation for Monroe avocados from mid-harvest (12/04/98) s tor d for 7 d at 13 C and transferred to 20C with different ethylene treatments ......... .. ... ........... ............ ... ... .. .... ... ............... ... .. .. ... . ... 40 3 -11. Fruit quality e aluation for Monroe avocados from mid-harvest ( 12 / 04 /9 8) stored for 14 d at 13 C with different ethylene treatments .............................. 41 3 -1 2 Fruit quality evaluation for Monroe avocados from late-har ves t (12/18/98) stored for 7 d at 13 C and transferred to 20C with different ethylene tr ea tm ents ........... .. ... ...................... .. .. .. .. ... .......... ........ .. .. .. ............ 42 3-13. F ruit 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 Da ys to peak and maximum amount CO 2 and C 2 H 4 production for Simmonds avocados stored at 20C with 1-MCP treatments Fruit were treated with two 1-MCP concentrations (0.45 and 0.09 L L1 ) and three exposure periods (6 12 and 24 h ) ........ .. ........... .. .. .. .. ... .... . ... .. .... . .. .. .. . .. .. ...... .. . ... .. . .... 59 4 2. P ee l color of Simmonds' avocados stored at 20 C with 1-MCP treatments ........... 60 5-1. Da ys to peak and maximum amount CO 2 and C 2 H 4 production for Tower II avocados treated with 1-MCP (0.9 L L1 for 12 hat 20C) and / or wax and store d at 20C .... .. .. .. .... . ....................................... . .. ... .. ................. 89 5-2. Da ys to peak and maximum amount CO 2 and C 2 H 4 production for Booth 7 avocados stored at 13C after wax treatment with or without 1-MCP ( 0 .9 L L1 for 12 hat 20C) .......... ............................................................ 91 5-3. Peel color of 'T ower II avocados treated with 1-MCP (0.9 L L1 for 12 h at 20C) and/or wax and stored at 20C ........................................................ 92 6-1. P e l color of 'Boot h 7' avocados stored at 13 C after 1-MCP tr ea tment (0.9 L L1 for 1 2 hat 20C) and then transferred into 20C (85% RH) om fru it were tr ea t e d with C 2 H 4 (100 L L1 for 12 hat 20C) before transfi r to 20C ........ . ...................................................................... 119 6-2. ugar compos ition of water-soluble UA released from E IS prepared from av cad to red at 13 C for 12 d and then transferred to 2 0 C ................ .. ........ .. 1 25 6-3. u gar c mposition of water-so lubl e UA from E IS prepar e d from avocado tr at d with 1-M P or 1-M P & C 2 H 4 .............. .. .................................... 1 26 6-4. ugar mpo iti n of DT A-soluble UA from E IS prepared from avocado tor dat 1 3 for 1 2dan dth ntransferr dto20 C .. .... .. .. .. .... ....................... 1 2 7 V I

PAGE 7

6-5 ugar composition of CDT A-soluble UA from I prepared from avocado treat d with 1-MCP or 1-MCP & C 2 H 4 . .. .. .. ...... .. ............... ................... . 129 6-6. Sugar composition of 4 M alkali-soluble hemicellulose released from EI prepared from avocado stored at 13 C for 12 d and then transferred to 20C ................................ .............. ... .. .... .. .... .. ........... ...... ........ . .. 130 6-7. Sugar composition of 4 M alkali-soluble hemicellulose from EIS prepared from avocado treated with 1-MCP or 1-MCP & C 2 H 4 .... ..... ... ..... ...... ............ 131 Vll

PAGE 8

LIST OF FIGURES Figure 4-1. Fruit firmness (Newton) and weight loss (%) of 'Simmonds avocados stored at 20 C with different 1-MCP treatments .. ... .................... .................... ... . ... 73 4-2. Effect of 1-MCP (0.45 L L1 for 24 h) on PG and PME activities of avocados stored at 20 C . .. .... .... ...... .. ............ .. .... ...... .... ........................... ...... 74 4-3 Effect of 1-MCP (0.45 L L1 for 24 h) on Cx-cellulase and aand ~-galactosidase activities of avocados stored at 20C .................................................. .. ....... 75 4-4. Effect of 1-MCP (0.45 L L1 for 24 h) on the amount of EIS in the mesocarp tissue and on the changes in water-, CDT A-soluble UA, and total UA in EIS from avocados stored at 20C ......................................... . .... ....... .... ......... 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 CDT A-soluble polyuronides from EIS prepared from avocado treated with 1-MCP ( o ) and without 1-MCP ( ) .. .. . ... .. . . .. ... .. .... 78 4-7 E f ct of 1-MCP (0.45 L L1 for 24 h) on the changes in the extractable amount of 4 M alkali-soluble h micellulose in EIS from avocados stored at 20 c .. ............ .. ....... .. ......... .............. ........... ...... .. .. ............... .. 79 4-8. Mo! cular mass profiles of 4 M alkali-so luble hemicellulose from EIS prepared from avocado treated with 1-MCP ( o ) and without 1-MCP ( ) .... ... . .. ........ . ... 80 4-9 Mol cul a r mass profiles of xyloglucan in 4 M alkali-soluble hemicellulose from I prepared from avocado treated with 1-MCP ( o ) and without 1-M P ( ) .... .. ... ... .. . ................... .......................... ... ........ ... .. ... . 81 5-1. Fruit firmn es s (N) and weight loss(%) of 'Tower II avocado treated with 1-M P (0.9 L L 1 for 12 hat 20 C) and/or wax and tored at 20 .. . ... ............. 99 5-2. F ruit firmn e (N) and w ight lo (%) of Booth 7 avocado tor d at 13C a ft r ax tr a tm nt with ( o ) or without( ) 1-M P (0.9 L L1 for 12 hat 2 0 ) ................... .. .. ..... . .. . ... ...... ........ ..... . . .... .............. ..... .. 100 Vlll

PAGE 9

5-3. PG activity of Tower II avocados treated with 1-M P (0.9 L L 1 fo r 1 2 h a t 20 ) a nd / or ax and s tor e d at 20C . . ... .. ... .... .. .. . . ........ .. .. .. ..... . .. . 101 5-4. ffi ct of 1-M P on PG activity of Booth 7 avocados sto r d at 13 after wax tr a tment with ( o ) or without( ) 1-MCP ( 0.9 L L1 for 1 2 hat 20C) ..... ... 102 6-1. Fruit firmness (N) and weight lo ss (%) of Monroe avocados gassed immediately with C 2 H 4 (100 L L 1 ) for 12 hat 20C, then either s tor e d at 13 C or continuously treated with 1-MCP (4.5 L L1 ) for 24 hat 20C and then transferred to 13 C ...................... .................................. . . .. .. 141 6-2 Fruit firmness (N) and weight loss(%) of Monroe avocados treat e d with 1-MCP (4 .5 L L1 for 24 hat 20 C) and stored at 13 C .. .... . ...... . . . . ....... .. .. 14 2 6 3. Fruit firmness (N) of Booth 7' avocados treated with 1-MCP ( 0 .9 L L1 for 1 2 hat 20C) stored at l 3 C for 19 d and then transferred in 20C . . ........ ...... .. 143 6 4. Weight loss( %) of Booth 7' avocados treated with 1-MCP (0.9 L L 1 for 12 h at 20C), stored at 13 C for 19 d and then transferred in 20C ........ ............ 144 6-5 Carbon dioxide (mgkg1 h1 ) and ethylene (Lkg1 h1 ) production of Monroe avocados gassed immediately with C 2 H 4 (100 L L1 ) for 12 hat 20C, then either stored at 13 C or continuously treated with 1-MCP (4.5 L L1 ) for 24 h at 20C and then transferred to 13 C ......................................................... 145 6-6 Carbon dioxide (mgkg1 h1 ) and ethylene (Lkg1 h1 ) production of Monroe avocados stored at 13C after 1-MCP treatment (4.5 L L1 for 24 hat 20C) . .. ..... 146 6-7. Ethylene production 1 h1 ) of Booth 7' avocados treated with 1-MCP ( 0.9 L L1 for 12 hat 20 C) stored at 13C 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 L L1 for 1 2 h at 20C) stored at 13 C 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 L L1 for 12 hat 20C), stored at l 3C for 19 d and then transf e rr e d in 20C ...... ....... ... ........ .... ....... .... ......... ..... .. .... .. ..... . .... .... . 14 9 6-10. The changes on the amount of EIS in the mesocarp tissue and on th e change in water-, CDT A-soluble UA, and total UA in EIS from Booth 7' avocados treated with 1-MCP (0.9 L L1 for 12 hat 20C), stored at 13 C for 19 d and th en transferred in 20C .. ... ........ ........... .................. .... .... .. .. .. ... 1 0 lX

PAGE 10

6-11 Mol cular ma s profiles of water-soluble polyuronides from EI prepared from Booth 7 a ocado .................................................................. .. 151 6 -1 2. Molecular ma profiles of CDT A-soluble polyuronides from EIS prepared from Booth 7 a ocado . ... ... ........ ... ... ....... ... .... ............................... 15 2 X

PAGE 11

a-Gal ~-Gal CDTA CMC DMC DP EIS 1-MCP PG PME RH UA XG LIST OF ABBREVIATIONS a-galactosidase ~-galactosidase trans-1 2-cyclohexylenediamine-N N N' N' -tetraac etic acid carboxymethylcellulose dry matter content degree of polymerization ethanol insoluble solids 1-methylcyclopropene polygalacturonase pectinmethy lesterase relative humidity uronic acid xy loglucan Xl

PAGE 12

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-M THYLCYCLOPROPENE) 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 rip ning Avocado fruits from immediate ethylene treatment had more uniform ripening and b tt r 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. Th 1-Methylcyclopropene (1 -MCP) treatment delayed ripening of avocado fruit charact riz d by a significant reduction in the rate of fruit softening and in th timing and int n ity f th thylene and respiratory climacterics. Avocado treat d with 1-M P al h w d ignificantly 1 ss w ight loss and r tained more green color than control fruit (n t xp d t 1-M P). Inhibiti n f p lygalactur na (P ) activity a th trong tr pon t 1-M P nd d littl r n ry o r th torag p riod. on i t nt with th m rk d uppre 1 n in 1-M P-tr at d a ocado fruit th olubilization and d gradati n f polyuronid s wa significantly d la d and r due d in 1-M P-tr at d Xll

PAGE 13

fruit. 1-M P tr atm nt did not significantly affect quantity or comp sition of then utral ugar of 4 M alkaliolubl h micellulose during ripening. The 1-M P tr atment ho r ignificantly reduced molecular mass downshifts in 4 M alkali-soluble hemic llulo sand xyloglucan. In addition to its effect on PG 1-MCP treatment significantly delayed the acti iti f -c llulase pectinmethylesterase and total extractable aand ~-galacto ida e during avocado fruit ripening. Selected cell wall enzymes (PG ~-galactosidase and C x -cellulase) were upregulated by ethylene. Exogenous ethylene treatment before or after 1 MCP treatment did not influence fruit firmness, weight loss respiration or C 2 H 4 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 C 2 ~ action and delays C 2 H 4 -dependent ripening of avocado fruit. Xlll

PAGE 14

CHAPTER 1 INTRODUCTION Th onset of ripening in a ocado 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 pectinmethyl t rase Cx-cellulase, and aand ~-galactosidases. Th importance of ethylene in regulating fruit ripening has been clearly demon trated from analyses of fruits exhibiting suppressed ethylene biosynthesi or action. The e thyl ne action inhibitor 1-methylcyclopropene (1-MCP) has b en hown to block ethyl n r c ptors preventing ethylene effects in plant tissu s fore t nd d period ( i s l r a nd r k, 1997 isl er et al. 1996a; Sisler et al. 1996b) and has provid d a facil approach fi r am inin g r lation hip between ethylen and fruit rip ning in a ran g of lim act ric fruit (Abd i t al. 1998 Fan and Matthei 1999 F ng et al. 2000 Golding tal. 1998 t I ., 2000) ldin g t a l. 1999 lievre et al. l 997b Nakat uka t al. 1997 Watkins

PAGE 15

2 Th objecti e of the res arch described herein were the follow in g: 1 to xamm th ffi cts of po tharvest application of ethyl neon avocado ripening uniformit y a nd fruit qualit y 2) to characterize physiological and biochemical r es pon ses of avocado fruit to 1-MCP tre a tment 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 wax ing on ripening characteristics in avocado fruit ; and 4) to investigate the influenc e 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.

PAGE 16

CHAPTER2 LITERATURE REVIEW Introduction Avocado The a v ocado (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 s ubtropical and semitropical fruits on the basis of increasing cold hardiness and general climactic adaptation (Bergh, 1976 Knight 1980 Seymour and Tucker 1993). Fruit charact ristic including size and skin texture vary considerably among the strains (Kn i g ht 1980 ). r 40 c ul ti va r of avocado are grown commercially in Florida. They compri e thr g n ra l g roup : cul ti var of the West Indian race cul ti var of th Guatemalan race and h ybrid c ulti va r which ar mo tly crosses of W st Indian and Guat malan race (Hatton and amp b 11 1960 Hatton t al. 1964). W st Indian avocado are grown comm rcially in th continental Unit d tat sonly in south rn Florida wh r they matur during th umm rand arly fall. Th Guatemalan vari ti in contra t mature 3

PAGE 17

4 during th fall and inter. Hybrid varieties constitute nearly 90 p rcent of the Florida avocado and th y mature during the fall and winter (Hatton and ampbell 1960). Fruit Development The avocado fruit is classified botanically as a berry comprising s d and pericarp which is separated into rind ( exocarp ), flesh (mesocarp) and the thin papery layer next to the seed coat ( endocarp) (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 20C depending on physiological maturity. The best ripening temperatures are between 15 5 and 24C 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 19 5 3 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 substanc transmitt d from the tree to the fruit (Seymour and Tucker 1993).

PAGE 18

5 Climacteric Characteristics The a ocado 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 re s pirator 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 chan g es in the structural properties of the cell walls. The cell wall structure of fruit ti ss u h a long been a subject of interest because the changes in cell wall com p o ition and ri g idity greatly affect the firmness of the whole fruit. Such o r ga ni za ti n a l chang es ar an integral part of the endogenously controlled fruit-ripening

PAGE 19

6 Cell wall change Fruit t xtural changes are due to the modification of variou c 11 wall component s including crystalline cellulose microfibrils hemicelluloses pectin s and structural proteins (McNeil et al. 1984). The primary cell wall of fruit s 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 ith 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 synthesi z ed some of which are cell wall-directed hydrolytic enzymes. Hydro lases 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 (C E.C. 3.2.1.4) and aand ~-galactosidases In avocado fruit both polygalacturonase and C x -cellulase activities have been reported to increase during ripening whereas pectinmethylesterase activity declines during the same period (Awad and Young 1979 ; Pesis et al. l 978a Raymond and Phaff 1965).

PAGE 20

7 Pol galacturonase. The polygalacturonase (PG) enzymes (particularly the endo form) ha 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 ofendo-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 po tclimacteric 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 C -cellulase activity (Awad and Young 1979) These observations suggest that PG is a dominant factor in th extensi d g radati n f polyuronid s during avocado fruit rip ning (Hob on 1962). Pcctinmethyle tera e. P ctinmethyle t rase (PME) catalyz the removal of m th xy l g r up f e t rifi d p ctin and ha b en implicated in cell wall softening even

PAGE 21

8 though its contribution to the process is not entirely clear (Hub r 198 3 b ). Pectinmethyl sterase is responsible for the deesterification of pectin r e quir e d b e for e polygalacturonase-mediated depolymerization of pectins during ripening (Awad and Young 1980 ; Wakabayashi et al. 2000). The presence of PME in avocado fruit s and it s rapid d clin 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 ~-1 4-linked glucans (Hatfield and Nevins 1986). C x -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). Alphaand 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 ; Burns, 1990; Gross et al. 1986). Beta-galactosidase appears early in fruit ripening and before the appearance of PG (Pres y 1983 Watkin et al. 1988). Studies on tomato (Watkins et al. 1988) mango and papa a (Lazan and Ii

PAGE 22

9 1993 appl (Bartle 1974) kiwifruit(WegrzynandMacRae 1992) andhotpepper Gross et al. 1986) have reported dramatic increases in the activity of ~-galactosidase during ripening One tomato ~-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 ~-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 ~-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 (McColl um et al. 1989). Typically the molecular weight of the chelator s oluble polyuronides decreases during ripening. Exceptions have been noted with apple (Knee 1978) and strawberry (Huber 1984). Neither of these fruits contains active ndo-PG In avocado fruit the solubility of polyuronides increases substantially during rip ning oncomitant with marked downshifts in molecular mass (O'Donoghue and Hub r 1992). Alth ugh change in the total amount of extractable hemicellulos s are not u u a ll y b r v d rip ning is oft n characteriz d by a low ring of th molecular weight of th p l y m r ( Hub r 1984 Mc ollum t al. 1989 Tong and Gro 1988). During

PAGE 23

10 ripening of avocado hemicelluloses exhibit a reduction in large mol c ular ma s p o l y m e r and a proportional increase in lower molecular mass polymer s (O'Dono g hue and Hub r 1992) Another facet of cell wall degradation during fruit ripening is the loss of non cellulo ic neutral sugars presumably solublilized by the action of cell wall glycosidas es 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 0 2 (2 to 5%) and high CO 2 (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 consid red to be the initiator of

PAGE 24

11 chang s in color texture aroma and flavor and other biochemical and physiological attributes ( Brady 1987 Oetiker and Yang 1995). Ethylene Bio ynthetic Pathway The ethylene biosynthetic pathway is established as S-adenosylmethionine 1-aminocyclopropane-1-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 i p e rceived by a family of histidine kinase-like receptor homodimers: ETRl E R 1 ETR2 E IN4 and ERS2 in Arabidopsis. The ETRI (ethylene-resistant) gene th first e th y l e n e receptor gene cloned (Chang et al. 1993) encodes a membrane-spanning prot in that consists of an amino terminal domain a putative histidine kinase domain and a r ceiv r domain (Chang et al. 1993; Chang and Meyerowitz 1995) The ERS (ethylen n r ) which lack s the rec iver domain was lat r i olat d by cross h ybr idi za tion with TRI (Hua et al. 1995). The membrane-locali z d thylene-binding it r quir a co pp r cofactor and prop r r ceptor function r li s on a copper transport r

PAGE 25

12 (Rodriguez t al. 1999). A tomato homologue ofETRl called NR ha s b e en clon e d fro m the ripening-impaired tomato mutant Never rip e (Nr) (Wilkinson et al. 1995 ) eve r r ipe mutants are affected in ethylene perception and show no accumulation of N r mRN A (Payton et al. 1996) Never ripe mutants synthesize reduced quantities of ethylen e 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 CTRl 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 CTRl (Clark et al. 1998; Hua and Meyerowitz 1998). Binding of ethylene on the other hand inhibits receptor activation of CTRl (Hua and Meyerowitz 1998) Downstream EIN2 is a structurally novel protein containing an integral membrane domain (Alon s o et al. 1999 ; Chang and Shockey, 1999) The EIN3 family ofDNA binding proteins regulates transcription in response to ethylene and an immediate target of EIN3 is a DNA-binding protein of the EREBP (ethylene-responsiv element binding protein) family which relates to the AP2 family of transcription factor (Chang and hock 1999) Practical U e of Ethy l ene thyl n affects the physiology of various fruits in storage. Classic examples of th use of thyl near its commercial application to ripen bananas and tomatoes and to

PAGE 26

13 degr n citru fruits. It has been established that ethylene treatment of detached mature a ocado fruit promotes the onset of ripening (Biale 1960 Eaks 1966 Eaks 1978 Eaks 1980 Er ick son and Yamashita 1964 Gazit and Blumenfeld 1970 ; Zauberman and Fuch 1973 ). Treatment of mature Hass avocados with 100 L L" 1 ethylene for 24 h after har st 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 E th y l e ne responses can be controlled by regulating ethylene 's production or action. The u se 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 exog nous sources of ethylene. Silver thiosulfate (STS) is a known inhibitor of ethylene action (Veen 1983) il ver thiosulfate probably acts through the irreversible interaction of silver ions with ethy l n binding sites ( isler et al. 1986; Veen 1983) and its ef ct is considered to b non-c mp titi v (Bey r 1976) ilver thiosulfate has be nus d with much uccess on cut flow r and p tt d plants (Ve n 1983) Although TS is widely us d to reduce thyl ne acti n nvir nm ntal cone ms have re tricted its application in some countries (Serek and R id. 1 993). The earch for inhibitors of thyl ne action has concentrat d on thyl n ana l gu In tudi xam ining th nature of thyl n binding and th natur of the

PAGE 27

14 binding it a numb r of organic molecules that appear to block th e thylene r e ceptor for e xt nd d p ri d have been identified (Sisler and Blankenship l 993a i s ler et al. 1986 Sisl r t al. 1993) The 2 5-Norbornadiene (NBD) a competitive inhibitor of ethylene binding ( isl er 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 offensi v e 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 delicio a) persimmon (Diospyros kaki) tomato (Sisler and Blankenship 1993b ; Sisler and Lallu 1994) and several ornamentals including carnation geranium (Pelargonium zo nal e ) 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 -M CP) and 3.3-dimethylcyclopropene (3.3-DMCP) bind to the thyl ne receptor and prevent the physiological action of ethylene for extended periods in a number of plant (Sisler and Serek 1997; Sisler et al. 1996a ; Sisler et al. 1996b) All ofth ar g a ou

PAGE 28

15 at room temp e rature and have no obvious odor at the concentrations needed to protect against the e ffects 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 24 C remained insensitive to ethylene for 12 days and then ripened normall y 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 conc e ntrations as low as 0.5 nl r 1 (Sisler and Serek 1997). The 1-MCP is a very effective alternative to STS as a pretreatment for ethylene s nsitiv flow rs (Serek et al. 1994a ; Serek et al. 1995a). At very lo concentration 1-M P i a ffi ctiv a T not only in preventing the effects of xog nous ethyl n but a l in d l ay in g n c nc of flow rs whose natmal enesc nee i mediated by a rise in nd th y l n production ( rek t al. 1995a) The 1-MCP has substantially

PAGE 29

16 improved prop rties over DACP (Serek et al. 1994a) Th effi ctiv concentration o f 1MCP is more than 10-fold lo wer than that for DACP (Sis! r et al. 1996b ). Th 1-M P competes with ethy l ene for the ethylene receptor. Yueming et al. (1999) report d 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 L 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 r ceptor 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).

PAGE 30

17 Treatment of Plant ith 1-MCP Effect of concentration, exposure time, and temperature Th 1-MCP is effective in inhibiting ethylene action in various plant tissues at nL L1 le els. There are large differences in the 1-MCP concentrations required to inacti ate eth lene responses in different plant species and tissues and the 1-MCP concentration required to protect plants against ethylene depend on the time of exposure. With long r xposure periods lower 1-MCP concentrations are effective (Sis ler et al. 1996b ). The 1MCP concentrations required to protect plants against ethylene also depend on the temp rature 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(Poratetal. 1995 ; Sereketal. 1994a ; Sereketal. 1994b) The 1-MCP cone ntrations as lo w as 0.5 nL L1 were sufficient to protect carnation (Dianthus caryop h y llu ) flowers for several days against ethylene (Sisler and erek 1997) r atm nt with 40 nL L1 of 1-MCP for 6 h was required for complete inhibition of thylene effi cts in p a seedlings (Sis ler and Serek 1997) Pr tr atm nt with low cone ntrations (1 to 20 nL L1 range) of 1-MCP for 6 h eliminated th th lene-induced abscis ion or wilting of alstromeria b gonia carnation matthiola phalaenopsis and ro e (Porat t al. 1995 r k t al. l 994a erek t al. 1995a). Th 1-M Pat 20 nL L1 pr v nt d th y n -induced bud flow r and l eaf ab ci sion and flo r nescenc mpt m ( r k t al. 1994a Ornam ntal tr at d ith 0.5 nL I

PAGE 31

18 1-MCP did not r spond to ethylene even at concentrations as high as 1000 nL 11 ( r k et al. l 994a isler et al. 1995) With fruits 1-MCP tends to behave differentl y dependin g on th fruit type. 1MCP dela ys ripening and improves storage quality of climacteric fruits s uch as pears ( P yrus communi L. cv. Passe-Crassane) (1elievre et al. 1997b) bananas (Musa s p .) (Go ldin g 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 11 for 24 h was needed for complete protection against ethylene (Sisler and Serek 1997 ). 1-MCP treatment at 0.5 nL 11 for 24 h protected banana against exogenous ethylene (Sisler and Serek, 1997). 1-MCP treatment of apricot fruit at 1 1 for 4 hat 20 C 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 11 extended the storage life by ;::: 35% at 20C and 150% at 5C. At higher 1-MCP concentrations (500 nL 11 ) however there was an accelerated softening due mostl y to the onset of rotting with a 30% to 60% decrease in storage life at both 20 and 5 C (Ku et al. 1999) The storage life of broccoli increased with increasing 1-MCP concentration and exposure time at both 5 and 20C (Ku and Wills 1999). Broccoli tr at d with 1-M P

PAGE 32

19 1 L L1 ) at 2 0 C exhibited a significantly longer storage life than that treated ith 1MCP at 5 C ( Ku and Wills 1999) Effect on re piration 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 L1 for 4 hat 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 L L1 for 1 h) 6 or 12 h after propylene treatment (500 L L1 ) (Golding et al. 1998) Other responses of 1-MCP The 1-MCP delays symptoms of senescence (such as electrolyte leakage and lipid fluidity) in P e tunia (Serek et al. 1995b ). The 1-MCP retards storage-induced leaf yellowing but reduces rooting ability of stored cuttings (Muller et al. 1997). Th e 1-M P treatment enhanced xylanase-induced ethylene production in tomato ( nd e r n t al. 1996) and inhibited ethylene epinasty in tomato plants (Cardinale et al. 1995 ) In P a ra an pears 1-MCP treatment resulted in reduced accumulation of A o x id as transcript and thylene production during chilling (Lelievre et al. 1997b ). Apric t fruit tr at e d with 1 L L 1 1-M Pat for 4 hat 20 C showed a reduction in titr a t a bl e a cidit y lo during torage at 0 or 20 C and delayed production of volatile a lcoh I a nd t e r during rip ning at 20 (Fang et al. 2000). Th 1-MCP (0.45 mmol m

PAGE 33

20 3 for 12 h) inhibited color changes in apple fruit (Fan and Mattheis 1999). While 1-M P (45 LL_, for 1 h) delay d ripening of bananas it also caused uneven peel degreening and suppressed volatile aroma production (Golding et al. 1998). The 1-MCP (50 100 nL L1 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).

PAGE 34

CHAPTER3 POSTHARVEST ETHYLENE TREATMENT FOR UNIFORM RIPENING OF WEST INDIAN AND WEST INDIAN-GUATEMALAN HYBRID AVOCADO FRUIT Introduction E th y lene plays a vital role in the ripening of climacteric fruits and whether applied exoge nously or produced naturally initiates ripening and softening. The avocado ( P er sea americana Mill.) is one of the most rapidly ripening of fruits often completing ripenin g within 5 to 7 d following harvest (Seymour and Tucker 1993) Ethylene treatment of detached mature avocado fruit promotes the onset of ripenin g (Eaks 1966 1980 ; Gazit and Blumenfeld 1970 ; Zauberman and Fuchs 1973 Zauberman et al. 1988). Treatment of mature 'Hass' avocados with 100 L L1 ethylene for 24 h after harvest hastens the onset of ripening making them ready to eat in 3 or 4 d ( ak 1966 ). A number of fruits notably banana (Inaba and Nakamura 1986) tomato (Jahn 1975 ) pear (C hen et al., 1996) and mango (Barmore, 1975) are commercially har v sted prior to the onset of ripening and treated with ethylene gas to compress the ripenin g p riod and allow uniform ripening among fruits. Avocados, especially West Indian cultivars or their hybrids have potential to b market d a a premium-quality product. As with all avocado types they do not ripen until harv st d How v r thi s conv nience creates difficulty in mark ting avocados with uniform rip n s ine th timing of ripening initiation can ary id l within fruit lots d p ndin g up n the t ag of maturation at harve t and th variety. Howe r th influ n e f th y I n n th rip ning of these cul ti var has not pr viou ly been studied 21

PAGE 35

22 unlike Guatemalan typ s Mexican types or their hybrids Programmed ripening would give shipp rs th ability to ship high quality uniformly ripe avocados and lead to th implem ntation of a premium-quality avocado program. The obj ctive 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 lifi of earl y -s ason ( 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 tran port d to the Po tharv st 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 dat 422.4 g) and shape (average diameter at equatorial r gion early-harv t dat

PAGE 36

23 9.4 cm mid-har est date 9.1 cm and late-harvest date 9.1 cm) and then were surface t riliz d in a 15% (90 mM NaOCl) commercial bleach solution rinsed and dried. Midea on ('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). Lateeason ('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 r gion early-harvest date 10.7 cm, mid-harvest date 10.4 cm, and late-har e t
PAGE 37

24 and mid(13 July 1998) harvest dates respectively, and stor d for 5 or 12 days. For late (27 July 1998) harvest dates, ethylene-treated fruit were transferred to air at 13C (85 % RH) and stored for 5 or 9 d. After 5 d storage fruits were transferred to 20C (85% RH and evaluat d on a daily basis for fruit quality until fully ripe. Full-ripe stage was defin d as the point at which fruits softened to 10 to 15 N of firmness values considered too oft for commercial handling by author. For the delayed ethylene treatment ethylene was applied to fruits at 20C 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 l 2C (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 l3C (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 l r I ethylene at a flow rate of 4000 mL min1 CO 2 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 20C) and 85% relative humidity (RH). Following the ethylene treatments fruit were transferred to air at l 2C (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 20 (85% RH) and evaluated on a daily basis for fruit quality until fully rip

PAGE 38

25 Late-sea on ('Monroe') cultivar Fort fi e fruit were placed in a chamber (174 L) and immediately treated with flo -thr o u g h ai r or 100 L L1 ethylene at a flow rate of 4000 mL min1 Ethylene treatment as p e rformed for two exposure periods (12 or 24 h) at two temperatures (13 or 20C) and 85% relative humidity (RH). Following the ethylene treatments all fruits were transferred to storage rooms at 13C (85% RH). Control fruit (not exposed to ethylene) were maintained under identical storage conditions. After 7 d fruits from each treatment were transferred to 20C (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 percent age 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 In s trument (Model 4411 Canton, MA USA) fitted with a flat-plate probe (5 cm in diam ete r ) and 50 kg load cell. After establishing zero force contact between the probe and th e eq u a t o rial region of the fruit the probe was driven with a crosshead speed of 10 mm min1 T h force was recorded at 2.5 mm deformation and was d t rmined at two qui di tant p int n th e e quatorial region of each fruit. Peel Color ach fruit was m a rk d at th e equatorial region (2 regions p r fruit) and color at th ame l ca ti n was r cord d v ry other day as L hu angle and chroma value with a Min ta hr ma Met r ( R-2000 Minolta Cam ra o Ltd. Japan Th chroma met r

PAGE 39

26 wa calibrat d with a white standard tile. The color was reported a hu angl e H 0 w ith a value of 90 representing a totally yellow color and 180 a totally green color. Th results are presented as lightness (L *) chroma (C*) and hue angle (H 0 ). The chroma and hue angl were calculated from the measured a* and b* values using the formulas C* = (a* 2 +b* 2 ) 1 12 and H 0 = arctangent (b *la*) (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 60C in an oven for 48 h and then r -weighed Dry matter content was determined by the following equation: Pulp dry matter content (%) = (M/MJ X 100 Where M; is the initial weight of the fresh sample and Mis the final weight of dried sample. Oil Content Oil content was determined for the midand 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, Lunzem, 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 H 2 0 dried with anhydrous sodium sulfate for 12 h and filtered into tar d t t tub Th oluti n

PAGE 40

27 as e aporat d ith an Evapomix (Buchler Instruments Inc.) maintained below 50C for 12 h Th e tub e s were again weighed for estimation of total oil content. Stati tical Analysis The experiments were laid out in a completely randomized design Statistical procedures w ere performed using the PC-SAS software package (SAS-Insititute 1985) Data were subjected to ANOV A 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 earlymid, 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 e thyl e ne-treated fruit exhibited no significant differences in average firmness v alu es aft e r 5 d at l 0 C followed by 3 d at 20 C or 12 d at l 0 C (Table 3-1 ). After 12 d at 10 a v ocado fruit from the immediate thylene treatment showed less variability in firmn ess than those from control and delayed ethylene treatment. In mid-harvest fruit firmn e s d e clin e d dramatically during storage for 5 and 12 d at 12 C and significant (p < 0 05 ) di ffi r nc s b e twe n control and ethylene-tr ated fruits ere vid nt (Tabl 32) o ntr 1 fr uit t o r e d for 5 d at 12 were s ignificantly firm r (31.4 5 5 N data not h w n ) th a n th 1 n -tr a t d fruit (14 9 2 6 N) and r quir d 2 d at 20 C for all fruits to r ac h th full rip e t ag (10 15 N). Aft r 12 d at 12 fruits from both immediate and

PAGE 41

28 delayed thyl ne treatments w re softer and showed more uniformity in firmne s v alu s than control fruit tared under similar condition. Tabl 3-1. Fruit quality evaluation at the full-ripe stage for Simmonds avocados from early-har e t (06 / 29 / 98) gassed immediately for 48 hat 20 C and transferred to 1 o c or tared at 10 C for 7 d then gassed for 48 hat 20 C and then transferred to 10 C Value s follow d by the ame letter do not differ significantly according to Duncan s Multiple Rang Test (p < 0 05) Data are means standard deviation of 6 independent samples Treatment At harvest Control (no C 2 H4 gassing) Immediate C 2 H 4 gassing Control (no C 2 H4 gassing) Immediate C2~ gassmg Delayed C 2 H 4 gassing Days to Fruit fully ripe firmness 5 d at 10 c & 3 d at 2o c 5 d at 1o c & 3 d at 2o c 12 d at 1o c 12 d at 1o c 12 d at 1 o c (N) 107.4 9.5 14.0 1.7 14.3 2.3 13.5 2.8 13.3 1.3 11.5 2 3 Decay incidence (%) 0 0 0 0 0 Dry L Chroma matter value value (%) 12.9 40 9 1.0 1.8 11.8 a 11.8 a 12.9 a 11.6 b 11.8 b 43.5 a 44.2 a 49.5 a 46.2 b 46.3 b 24 5 2 2 24.2 a 26.6 a 25.8 b 33.4 a 23 9 b Hue angle (Ho) 125 7 1.8 125.2 a 123.9 a 117 8 a 119.6 a 119.1 a In late-harvest fruit, firmness declined during storage for 5 and 9 d at l 3C (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 13 C As with control fruit from the mid-harvest date control fruit stored for 5 d at 13 C re significant! firm r (19 0

PAGE 42

29 3 .4 data not hown) than ethylene-treated fruit (10.8 2.0 N) and required 2 d 20C for all fruit tor ach the full-ripe stage (10 to 15 N). After 9 d at 13C fruit from imm diate 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 hat 20 C and transferred to 12 C or stored at 12 C for 7 d then gassed for 48 hat 20 C and then transferred to 12 C 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 At harvest Control (no C2H4 gassing) Immediate 2 H 4 ga mg ontrol ( no 2 H4 gas ing) Imm diat e 2 H4 gassing Days to fully ripe 5 d at 12 c & 2 d at 2o c 5 d at 12 c 12 d at 12 c 12 d at 12 12 d at 12 Fruit firmness (N) 103.1 15.5 11.9 1.8 14.9 2.6 13.4 2.7 10 1 1.8 10.0 1.4 Decay incidence (%) 0 0 16.7 16.7 16 7 Dry matter (%) 13.2 L* value 40.9 .0 .4 12.3 a 12.1 a 12.2 a 10.9 b 11.8 a 49.3 a 47.6 a 50.6 C 71.5 a 53.1 b Chroma value 26 1 2.9 24.4 a 25.1 a 28.9 b 46 8 a 31.7 b Hue angle (Ho) 125.0 1.9 119.4 a 119.7 a 116.1 a 91.7 C 112.0 b

PAGE 43

30 Peel color and decay incidence At harvest the avocado peel was moderately green (hue angl e= 125 7 125.0 and 122.0 in early mid, and late-harvest fruit respectively where pure yellow = 90 and pur 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 gr en 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 hat 20 C and transferred to 13 C or stored at 13 C for 7 d then gassed for 48 hat 20 C and then transferred to 13 C. 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 (no C 2 H 4 13 C & 2 12.9 16.7 12.5 49.5 38.0 118.2 gassing) d at 20 C 2.5 a a a a Immediate C 2 H 4 5 d at 10 8 0 12.3 48.3 34.9 119.5 gassmg 13 C 2.0 a a a a Control (no C2H 4 9 d at 10.2 25.0 12.2 49.3 36.6 118 5 gassing) 13 C 2.6 a b b a Immediate C 2 H4 9 d at 8.7 16.7 12.3 50.9 41.7 114.7 gassmg 13 C 1.2 a ab a b Delayed C 2 H 4 9 d at 10.5 16.7 12.7 52.9 42.4 113.2 gassmg l 3 C 3.1 a a a b

PAGE 44

31 In earl y -h ar est fruit no significant differences in the hue angle of peel color among treatments were observed due to ethylene treatment after 5 d at 10 C followed b 3 d at 20 C or 12 d at 10 C (Table 3 -1 ). There were however significant differences in the L alue ( L *) and chroma value (C) of the peel color among fruit from all treatments after 12 d at 10 C In mid-harvest fruit, no significant differences in peel color were observed between control and ethylene-treated fruits after 5 d at l 2C (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 l 2C. 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 13 C (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 l 2C. Control fruit had the highest hue angle (118.5) (Table 3-2). Fruit stored continuously for 5 d at 10 12, or 13C exhibited no decay symptoms except for control fruit (16 7%) from late-harvest (Tables 3-1 3-2 and 3-3). Early har v est 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%) aft r 1 2 d s tora g a t l 2 C (Table 3-2) In late-harvest fruit control fruit exhibited decay (16 7 %) a ft r 5 d storag at 13 C followed by 2 d at 20C and showed the highest decay (2 5 %) aft r 9 d s torage at 13 C (Table 3-3). Pulp dry matter content Initi a l pulp dry matt r content (DMC) of arlymidand lat -harv st fruit av rag d 1 2.9 1 3 .2 and 12.3% r spectively (Tables 3-1 3-2 and 3-3) In early-harvest

PAGE 45

32 fruit th r w r no ignificant differences in DMC among treatment for avocado s aft r 5 d at 10 follow d by 3 d at 20 C ( able 3-1). During storage DMC of fruit from all treatm nts d er as d compared with DMC of freshly harvested fruit. Ethylene-treated fruit lost significant l y more dry matter (lower DMC) than control fruit after 12 d storage at 10C. There were no significant differences in DMC among treatments for midand 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 midand 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 l 2 C, and fruit from all treatments exhibited significant (P < 0.05) differences in average firmness values after 7 d at 12C (Table 3-4). Fruit treated with ethylene for 24 hat 20 C were softer (20.9 .8 N) than those from control and other ethylene treatments and had less variability in firmness. Fruit from all treatments after 7 d at l 2 C were somewhat firm (over 21 N) and required 3 d at 20C to reach the full-ripe stage (10 to 15 N). Fruit from all treatments reached full-ripe firmness after 14 d at l 2C (Table 35). In late-harvest fruit fruit from all treatments exhibited significant (P < 0.05) differences in average firmness values after 7 d at 12C (Table 3-6). Late-harvest fruit treated with ethylene for 24 hat 20C were softer (18.6 3.6 N) than those from control and other ethylene treatments. Fruit from all treatments after 7 d at l 2C were somewhat firm ( ov r 19 N) and required 3 d at 20C to reach the full-ripe stage. Fruit from all treatments reached full-ripe firmness after 14 d at l 2C (Table 3-7). Fruits treated with ethylene for 24 hat 12 C and for 12 hat 20C showed less variability in firmness although the differences wer not statistically significant (Tabl 37).

PAGE 46

Tabl -4. Fruit qualit e aluation for Booth 7 avocados from mid-harvest (10 / 03 / 98) stored for 7 d at 12 C and transferr e d to 2 0 ith di f r nt eth lene treatments Data are means standard deviation of 6 independent samp les. Storage time and temperature Initial eth lene 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) (%) (%) (Ho) (N) (%) (%) (%) (Ho) w 12 0 46.6.5 0 14.6 122.3 10.7.6 0 14.4 3.8 120.1 w 12 12 54.2 5 0 14.4 123 7 10 .3. 0 0 14.5 4.8 120.2 12 24 60.6.6 0 14.2 124.8 12.7.5 0 12 .6 4.5 121.7 20 12 75.9.8 0 14.5 124.4 12 .5.3 0 13.7 4 3 122 5 20 24 20 9.8 0 14 6 121.4 10.7.8 16.7 13.0 3 6 117 8

PAGE 47

34 Tabl 3-5. Fruit quality evaluation for Booth 7 avocados from mid-harvest 10 /03 / 98 s tored for 14 d at 12 with diffi rent ethylene treatment s. Yalu s fo ll owed by th am l tt r do not diffi r significantly according to Duncan s Multiple Rang est (p < 0.05 Data ar mean standard d viation of 6 indep e ndent samp l es. Storage time and temperature tr atm nt 14 d at 12 C Temp Time Fruit Decay Dry Oil Hue angle firmness incidence matter content (C) ( h ) (N) (%) (%) (%) (Ho) 12 0 11.6.7 0 13.8 4.5 120.7ab 12 12 10.8.2 0 14.7 4.6 122.3a 12 24 11.4.0 0 13.8 3.5 120.5ab 20 12 13.0.9 0 14 1 3.3 120.8ab 20 24 11.1 .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 midand late-harvest fruit, respectively). In mid-harvest fruit fruit treated with ethylene for 24 hat l2 C and for 12 hat 20 C retained more green color after 7 d storage at 12 C or 7 d at 12 C followed by 3 d at 20C, although the differences were not statistically significant (Ta ble 3-4). After 7 d at l2C followed by 3 d at 20C or 14 d storage at 12C the peel of fruit treated with ethylene for 24 h at 20C showed more loss in greenness (Ta bl es 3 -4 a nd 3-5) In lat e -harvest fruit, there were no significant differ e nce s in peel color among tr ea tments after 7 or 14 d storage at l 2C (Tables 3-6 and 37 ). Fruit tr eated with ethylene for 24 hat 20C showed significant loss in greenness after 7 d at l 2C followed b y 3 d at 20C (Table 3-6).

PAGE 48

T a bl 3-6. Fruit qualit e aluation for Booth 7' avocados from late-harvest (10/17 / 98) stored for 7 d at 12 C and transferred to 20 ith di ffi r nt th lene treatments Values followed by the same letter do not differ significantly according to Duncan s Multiple Ran ge T s t (p < 0 05). Data are means standard deviation of 6 independent samples. Storage time and temperature Initial eth lene 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) (%) (%) (Ho) (N) (%) (%) (%) (Ho) L,..) Vl 12 0 29 7 2 0 15 8 123.1 11.7.3 0 15.3 5 3 118 0a 12 12 29.4 7 0 15.1 124.0 11.2.4 0 14 7 5 3 120.9a 12 24 22.1 3.0 0 14.4 123.9 11.0.1 0 15.4 4.9 120.3a 20 12 23.8 7.0 0 15.7 122.7 10.2.5 0 15.0 5.4 118.1 a 20 24 18.6.6 0 15 2 121.0 10.2.8 0 14.7 5.0 114.2b

PAGE 49

36 Table 3-7. Fruit quality valuation for Booth 7' avocados from l at -harve s t (10/17 /9 8 stored for 14 d at 12 C with different thylene treatment s. Data ar mean standard deviation of 6 independ e nt samples. Initial ethylene Storage time and t e mp rature treatm nt 14 d at 12 C Temp Time Fruit Decay Dry Oil Hu e angle firmness incidence matter content ) ( h ) (N) (%) (%) (%) (Ho) 1 2 0 11.0.9 0 14 7 5.0 121.3 1 2 12 10.5.0 0 15 0 5.6 120.3 12 24 10.3.3 0 15.4 6.1 120 .3 20 12 10.9.2 0 15.2 6.4 119 6 20 24 9.5 2.0 0 14.8 4.9 119 .2 Mid-harvest avocado fruit from all treatments stored continuously for 7 d at l 2C exhibited no decay symptoms (Table 3-4). However following an additional 3 d at 20 C over 16 7 % of fruits from the 24 -h ethylene treatment at 20 C showed decay. Surface decay of control and ethylene -treated fruits stored for 14 d at l2 C was not observed (Ta ble 3 -5 ). Late-harvest fruits from all treatments exhibited no decay symptoms after 7 or 14 d storage at 12 C and 7 d at l2C followed by 3 d at 20C (Tables 3-6 and 3 -7 ). Pulp dry matter and oil contents Initial pulp dry matter content (DMC) of midand late-harvest fruit averaged 15 .3 and 17.4% respectively. In midand late-harvest fruit there were no significant differences in DMC among treatments after 7 or 14 d storage at l 2 C and 7 d at l 2C followed b y 3 d a t 20 C (Tables 3-4 3-5 3-6 and 3-7). Initial oil content(%) of mid and late-har ves t fruit averaged 5 1 and 4.9% respectively and show d slight change during storage (Tables 3-4, 3-5, 3-6 and 3-7). However control and thyl ne -tr ea t e d

PAGE 50

37 fruit exhibited no significant differences in average oil content at the full-ripe stage (10 to 15 N). Late-Sea on ('Monro ') Cultivar Fruit firmne s Initial firmnes o f earlymidand 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 13C or after 7 d at 13C followed by 4 d at 20 C (T able 3-8). After 14 d at 13 C avocados from all ethylene treatments were softer (around 13 N) than control (not exposed to ethylene) fruit (33 N) however ethylene tr ea t e d fruit exhibited significantly less variability in firmness (Table 3-9). Although control fruit r eac hed full-ripe firmness within 2 d of transfer to 20 C after 14 d at 13 C over 50 % of the fruit s howed decay prior to reaching the full-ripe stage (data not shown). In mid-har ves t fruit firmness declined dramatically during storage for 7 or 14 d at 13 C but di ffere nces 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 l 3C followed b y 3 d at 20C (Table 3-10). In late-harvest fruit fruit from all treatments exhibited significant (P < 0.05 ) diff e r e nce s in average firmness values after storage for 7 d at 13 C (Table 31 2 A wit h co nt ro l fr uit from the earlyand mid-harvest date fruit held continuou ly in air fir 1 4 d at 1 3 ex hibit e d hi g h variability in firmness (Table 3-13). E thylene-treat e d fr uit t r d for 7 d at 13 C followed by 3 d at 20C or 14 d at 13 C were softer and how d i gn ifi an tl y mor e uniformity in firmness than control fruit stor d under similar condition (Tab l s 3 -1 2 and 3-13).

PAGE 51

Table 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 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 + 4 d at 20 C Temp Time Fruit Decay Dry Hue angle Fruit Decay Dry Oil Hue angle firmness incidence matter firmness incidence matter content CC) (h) (N) (%) (%) (Ho) (N) (%) (%) (%) (Ho) w 00 13 0 41.8.5 0 15.4 123.7 10.9.0 50 14 6 6.3 119 .3 13 12 46.1.0 0 17.0 124.1 13.1.3 50 15.1 5.9 119.8 13 24 44.5.8 0 16.8 125.4 15.2.1 0 14.1 5.3 1 22.0 20 12 39.4.6 0 15.2 124.1 14 .3 .5 16 7 14 .2 5.4 120.3 20 24 26 .9 7.1 0 15.6 124.5 11.0 1.8 50 14 .6 5.6 1 21.1

PAGE 52

39 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 de iation of 6 independent samples. Initial ethylene Storage time and temperature treatment 14 d at 13 C Temp Time Fruit Decay Dry Oil firmness incidence matter content (C) ( h) (N) (%) (%) (%) 13 0 32.9.9 16.7 15.6 13 12 12.8.5 16.7 13 9 5 0 13 24 13 .3.9 16.7 14.5 6.0 20 12 12.8 .5 16.7 15.4 6.8 20 24 13.5.9 16.7 13.9 4.7 Oil content was not measured because fruits were not fully ripe. Peel color and decay incidence Hue angle (Ho) 123 2 121.4 121.0 122.5 120 .2 At har ves t the avocado peel was moderately green (h ue angle = 126.4 124.1 and 126 .5 for earlymid, and late-harvest fruit respectively where pure ye llow = 90 and pure gr e n = 180) and showed little change during storage. At the full-ripe stage there were no s i g nificant differences in peel color among fruit from all treatments (Tables 3-8 to 3 -13) In ea rl y -h arvest, avocado fruits stored continuously for 7 d at 13 C exhibited no y mptom s of d ecay (Table 3-8). Following an additional 4 d at 20C to reach the full-rip stage howev r fruit from the 24-h ethylene treatment at l 3C had no incidenc of d ca ur face d ca in control and ethyl ne-treated fruit stored for 14 d at 13 C as obs r ed but wa n t r e Tabl 3-9). In mid-harvest fruit from th 24-h thyl n treatment at 13 had n in cid nc f d cay after 7 d at 13 followed by 3 d at 20 and showed th tin id nee fdecayofa lltr atm nts 14dat 13 C(Ta bl 3-10and3-11).

PAGE 53

Table 3 -10. Fruit qu a lit y eva lu at i on fo r Monroe avocados from mid-harvest (12 / 04 /9 8) stored for 7 d at 1 3 C and transferred to 20 C with di ffere nt ethy l ene treatments Data are means standard deviation of 6 independent samples. Storage time a nd temperature Initial ethylene treatment 7 d at 13 C 7 d at 13 C + 3 d at 20 C Temp Time Fruit Decay Dry Hue angle Fruit Deca y Dr y Oil Hue angle firmness incidence matter firmness incid e nce matter content (C) (h) (N) (%) (%) (Ho) (N) (%) (%) (%) (Ho) 13 0 47.3 6 0 16.3 125 7 19 .3 1 3 .3 50 16.4 6.7 122 7 0 13 12 36.1.7 0 17.7 125.9 15.9 .3 33.3 15. 2 5.3 122.3 13 24 29 .3 .5 0 16.2 1 22. 7 14.9 .2 0 16.1 5 5 120 .3 20 12 29.6.8 0 16.5 1 24. 0 1 2.6.6 16 .7 17.4 5.9 119 2 20 24 27.6.9 0 17.5 1 24.7 15. 9.6 16.7 16.1 5.9 120.0

PAGE 54

41 Table 3-11 Fruit qualit y evaluation for Monroe avocados from mid -h arvest (12 / 04 / 98) store d for 14 d at 13 C with different ethylene treatments. Values followed by the same 1 tter do not differ significantly according to Duncan s Mu l tip l e Range Test (p < 0.05) Dat a are means standard de v iation of 6 indepe n dent samples Initial ethylene Storage time and temperature treatment 14 d at 13 C Temp Time Fruit Decay Dry Oi l Hue angle firmness i n cidence matter content (C) ( h) (N) (%) (%) (%) (Ho) 13 0 13 8 3 33.3 16.0b 5.6 122 5 13 12 11.7.3 33.3 18 6a 7 0 121.0 13 24 14.6.9 16.7 16.0b 6.0 121.9 20 12 12.8 7 33 3 1 8.2a 6.6 120.5 20 24 14 3.0 33.3 17.0ab 6.0 118.7 Control fruit stored for 7 d at 13 C followed by 3 d at 20C exhibited the highest deca y incidence (50%) of all treatments (Table 3 10). In late harvest fruit from the 24 h ethylene treatment at 13C also had no incidence of decay after 7 d at 13C and 3 d at 20C or after 14 d at 13 C (Tab l es 3 12 and 3 13). Pulp dry matter conte n t a n d oil co n tent Initi a l pulp dr y matter content (DMC) of early midand late harvest fruit averaged 16 7 17 .9 and 16.7% respectively. In early-harvest fruit there were no s i gn ificant di ffere nces in DMC among treatments after 7 or 14 d storage at 13 C or 7 d at 1 3 fo ll ow d b y 3 d at 20C (Tables 3-8 and 3-9). In mid-harvest fruit there were no i gn ific a nt di ffi r nc in DMC among treatments for avocados after 7 d at 13 (Table wer significant (P < 0.05) differences in DM among treatments afte r 14 d at 1 3 (Tab l 3 -11).

PAGE 55

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 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 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) (%) (%) (Ho) (N) (%) (%) (%) (Ho) 13 0 95.8.8 0 15.8 125.0 20.8.6 33.3 16.9 6.1 122.1 N 13 12 61.7.9 0 16.3 124.8 12.3.6 0 16.4 6.2 121.8 13 24 51.3.5 0 16.2 125.2 15.3.0 0 16.6 6.3 122.5 20 12 50.8.5 0 16.8 125.7 13.9.0 16.7 15.8 6.6 123.5 20 24 28.3.0 0 18.4 124.0 14.7.3 16.7 16.5 6.8 122.1

PAGE 56

43 Tabl 3-13. Fruit quality eva l uation for Monroe avocados from late-harvest (12/18 / 98) tor d for 14 d at 13 C ith different ethylene treatments. Data are means standard d iation of 6 independent samp l es. Storage time and temperature treatm nt 1 4 d at 13 C Temp Time Fruit Decay Dry matter Oil Hue angle firmness incidence content (C) (h) (N) (%) (%) (%) (Ho) 13 0 42.2.0 1 6.7 1 4.7 5.6 121.7 13 12 14.5.4 33.3 16.5 5 0 120.0 13 24 14 0.6 0 15 6 5 5 118.0 20 12 12 0.6 16.7 15 8 5 3 1 1 8.4 20 24 12.0.9 33.3 16.4 5 7 119 5 Fruit from the air treatment and the 24-h et h y l ene treatment at 13C followed by 14 d of storage at 13 C 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 13 compar d with DMC of freshly harvested fruit (Tables 3-12 and 3-13). Initial oil cont nt (%) f arlymid, and late-harvest fruit averaged 5.4 5.4 and 5.3 % r pectiv l y a nd did not change significantly during storage (Tables 3 8 to 3-13). There w r no ignificant diff e r nces in oil content among tr atments aft r 7 d at l 3 C follow d by 3 d at 20 or 14 d at 13 C (Tables 3-8 to 3-13).

PAGE 57

44 Discussion In th present study sev ral parameters (firmness peel color dry matter content oil content and decay incidence) were examined to determin the effects of postharv t 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 imn1ediate ethylene treatment ripened more quickly and uniforml y 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 midand late-harvest required 3 d at 20C following 7 d storage at 12C to reach full-ripe stage, whereas fruit were already soft after 14 d storage at 12C. Comparing 'Monroe' fruit from the three harvest dates, after 7 d storage at 13 C, ethylene-treated avocados from the early harvest required 4 d at 20 C to reach the full-ripe stage (10 to 15 N), whereas those from the middleand late-harvests required 3 d at 20C (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 13 C, ethylene-treated fruit from the three harvest dates were already soft Control fruit (not exposed to ethylene) from the earlyand late harvests stor d for 14 d at 13C were firmer (32.9.9 and 42.2 0 respectively) than ethylene-treated fruit and required 2 to 3 d at 20C 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 i not metabolized

PAGE 58

45 during ripening (Dolendo et al. 1966). Unlike the Mexican and Guatemalan type a ocados hich ha e higher oil content (about 15 to 30%) estimating pulp oil using dr matter content was not feasible with the West Indian-type due to the wide variabilit m oil cont nt b e tween 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 20C the pulp showed gray-black discoloration and blackening of the vascular bundle. Most fruits stored at 10C became unacceptable during storage at 20C. The incidence and severity of chilling injury increased with storage time at 20C. No specific internal symptoms of chilling injury such as discoloration of the flesh, were observed in any fruit during storage at 13 C. However following storage at 20C 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 13C followed by 2 d ethylene gassing at 20C) showed more mesocarp discoloration and vascular browning than control fruit (7 d storage at 13 C followed by 2 d at 20C). Simmonds avocados gassed immediately at 20C for 2 d and stored at 13 for 7 d showed less mesocarp discoloration and va cular browning than control fruit and d layed ethylene-treated fruit. Stem-end rot was observed in fully rip fruit aft r 12 d torage at 10 and 12C or after 9 d storage at 13 In Booth 7 a c a do n ith r p cific internal symptoms of chilling injury nor discoloration of th m c a rp w r b rv d during the storage at 12 How v r following rip ning at 2 0 mmor m ocarp di c !oration and localiz d va cular bro ning wa observ d. t me nd d e e y wa al o obs rv don som full rip fruit but th incidenc was not

PAGE 59

46 severe. In Monro avocados no specific internal symptom of chilling injury s uch a di coloration ofth flesh (mesocarp) were observed in any fruit during storage at 13 However following ripening at 20C minor mesocarp discoloration and localized ascular 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 thylene treatment. It was not possible to estimate pulp oil content of W t Indian typ avocado fruits using dry matter content due to wide variability of oil content b twe n individual fruits. From the tests with Simmonds' avocados, it was determined that fruits from immediate thylene treatment had more uniform ripening and better pulp quality than fruit from delayed ethylene treatment. Tests with 'Booth 7 avocados revealed that b t fruit quality was obtained with immediate ethylene treatments at 20 for 12 h or at 1 2

PAGE 60

47 for 24 h. fter ubsequent storage at 12C in air for 5 or 12 d fruit from this treatment de lop d no chilling injury remained acceptably firm and ripened normally. Monroe a ocados exposed to ethylene (100 L L1 ) at 13C for 24 h ripened more uniformly than fruit treated at 20C for 12 h or control fruit. During 14 d of storage at 13 C fruit from this treatment ripened normally while maintaining marketable fruit firmness had good qualit and also had lower incidence of decay. Ethylene treatment did not affect fruit qualit y as det er mined by peel color dry matter content and oil content.

PAGE 61

CHAPTER4 INFLUENCE OF 1-METHYLCYCLOPROPENE (1-MCP) ON RIPENING AND C LL WALL MA TRJX POLYSACCHARIDES OF AVOCADO FRUIT Introduction The a ocado (Persea americana Mill.) is a climacteric fruit that is characterized b y 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 t al. 1996) and impaired in ethylene response (Lanahan t al. 1994). In addition to the use of fruit lines with suppressed ethylene synthesis or perception the application of compounds that block ethylene action ( isler and erek 48

PAGE 62

49 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-Norbornadiene (NBD) was perhaps the first widely employed cyclic olefin utilized for its ability to compete with ethylene at the receptor le vel (Sisler et al. 1986 ho ev r the effectiveness of NBD a toxic and offensive smelling compound (S isler 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 L1 (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 r cov ry of the ripening process (Sisler et al. 1996a). 1-M P has been shown to delay fruit ripening and improve storage quality of climact ric fruit including pears (Pyrus communis L. cv. Passe-Crassane) (Lelievre et a l. 1997b ) bananas (Mu asp.) (Golding et al. 1998 ; Golding et al. 1999; Sisler and r k 1997) plum (Prunus alicina Lindi) (Abdi et al. 1998) tomatoes (Lycoper icon e s culen tum Mill) (Nakatsuka et al. 1997 ; Sisl rand erek 1997) appl s (Malus

PAGE 63

50 tri L. (Fan and Matth is 1999 Watkins et al. 2000) and avocados (Per ea Am ricana Mill) ( eng t al. 2000). 1-MCP therefore has provid d a valuable tool to inv tigat thylen 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 postharve t 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 l 3C 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 select d for uniformity of weight (757 31 g) and shape ( diameter at equatorial region 10.5 0.7) and th n were surface sterilized in a 15% (90 mM NaOCl) commercial bleach solution rinsed and dried.

PAGE 64

51 1-MCP Treatment T elve 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 L L1 were achieved through addition of lor 5 mg of the po w der respectively to 100 mL of FloraLife buffer following manufacturer s instructions (Floralife, Ethyblock product specification sheet) Following a ddition of the buffer to the 1-MCP the beakers were tranferred to the 18-L containers w hich were sealed immediately. 1-MCP treatment at each concentration was performed for three exposure periods (6, 12 and 24 h) at 20C and 85% relative humidity (RH). Immediately following 1-MCP treatments the fruit were removed from the chambers and transferred to 20C storage facilities (85% RH). Control fruit (not exposed to 1-MCP) we re 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 co n idered too soft for commercial handling by the author). Fruit quality was assessed on th e basis of fruit firmness weight loss CO 2 and C 2 t1.1 production and peel color. Mesocarp tissue derived from the equatorial region of selected fruit was stored at -30 C and u se d for analysis of cell wall enzymes and structural polysaccharides. Fruit Firmness Firmness was determined on whole, unpeeled fruit using an Instron Universial Testing In s trum e nt (Model 4411 Canton MA USA) fitted with a flat-plate probe (5 cm in diam t r) and 50 -k g load c 11. After establishing ze ro force contact b tween th prob nd th quat r i a l r gion of the fruit th e probe was driv e n with a cro head speed of 10 mm min1 Th fi rce was record d at 2.5 mm d efo rmation and was d termined at two

PAGE 65

52 equidistant points on the equatorial region of each fruit. The sa m e four fruit of each tr atm nt r mea ured repeatedly every other day until they reach e d the full-ripe stag Re piration and Ethylene Evolution R spiration and ethylene production were measured every other day using the ame four fruit of each treatment. Fruit were individually sealed for 30 min in 2-L pla s tic container 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 0 ) with a value of 90 representing a totally yellow color and 180 a totally green color. The results are presented as lightness (L *) chroma (C*), and hue angle (H 0 ). The chroma and hue angle were calculated from the measured a* and b* values using the formulas C* = (a* 2 +b* 2 ) 1 1 2 and H 0 =arctangent (b *la*) (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 4 C. The supernatant was discard d and the p 11 t

PAGE 66

53 r r u pend din 50 mL of ice-cold 80% EtOH for 1 min and again centrifuged at 7840 g fo r 1 O min at 4C 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 (5C), 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 NaCl for 30 min in ice-cold (5C) 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 L aliquot of the cell-free protein extract with 500 L (2 mg) of polygalacturonic acid (from orange peel Sigma Chemical Co St. Louis MO U A) dissolved in 30 mM KOAc pH 5.5 containing 100 mM KCl. After 30 min at 34C uronic a cid r e ducing groups were measured using the method of Milner and Avigad ( 1967 ) PG activity was expressed as mol D-galacturonic acid equival nts produced per kg prot in p r minute. Pectinmethylesterase (PME E.C. 3.1 1.11) was measured using modification s of th method of Hagerman and Austin (1986) A 0.5% (w / v) solution of citru p ctin ( i g ma Chemical Co St. Louis MO USA) was pr par d in 0.1 M Na 1 and a dju t d to pH 7 5 A 0.01 % (w / v) solution of bromothymol blu a pr pared in 0.003 M pota s ium pho phate pH 7 5 In a cuvette 2.0 mL of the 0.5% citrus pectin w r mi x d ith 0 1 5 mL f brom thymol blu and 0.83 mL of wat r pH 7 5. The reaction

PAGE 67

54 as initiat d by adding 20 L of the cell-free protein extract adjust d to pH 7 .5 a nd th decreas m 620 was recorded. PME activity was expressed as M620 per mg prot ei n p er minute. C llulase (endo-1 4-~-glucanase ; E C. 3 2.1.4) activity wa measured viscometrica ll y. A 100 L aliquot of the cell-free protein extract was added to 1.5 mL of a 2.5% o luti on of carboxymethylcellulose (CMC.7HSP Fisher Scientific Co, Fair Lawn J 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. aand ~-galactosidase activities were measured using modifications of the method of Pharr et al. (1976). p-N0 2 -phenyl aand ~-D-galactop yranosides (Sigma Chemical Co St. Louis MO USA) were used as substrates. Substrates were prepared at 2 mg mL1 in 0.1 M NaOAc pH 5.2. A 200 L aliquot of the cell-free protein extract adjusted to pH 5.2 was added to 200 L of substrate, and the reaction mixture incubated at 37C for 15 min. The release of p -N0 2 -phenol was measured spectrometrically at 400 nm Activity was expressed as moles of N0 2 -phenol equivalents released per kg protein per minute. N02 -phenol concentration was determined using free N0 2 -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 witzerland) 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

PAGE 68

55 Whatman) and ashed with 100 mL of 95% ethanol. The EIS ere transferred to 100 ml of chloroform : methanol (1: 1 v / ) and stirred for 30 min at room temperature. The EI ere filter d under vacuum through glass fiber filters (GF/C Whatman) and washed with 100 mL of ac tone. EIS samples were dried in an oven at 40C for 5 h and stored in a desiccator at room temperature. Pectin Extraction and Analysis Water and CDT A (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 hand quantified in terms of extractable uronic acid (UA) content. UA were determined by the hydroxydiphenyl assay (Blumenkrantz and Asboe-Hansen 1973) and expressed as g mt 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 CDT A-soluble uronic acids were concentrated using rotary evaporation to a final UA concentration of approximately 500 g mL1 Two-ml aliquots (1 mg UA equivalents) were passed through a Sepharose CL-2B-300 (Sigma Chemical o t. Loui MO USA) column (1.5 cm wide and 30 cm high) op rated with 200 rnM amm nium ac tate pH 5.0 (Mort et al. 1991). Two-ml fraction w r coll cted at a flow rat f 40 mL h1 and aliquots (0 5 mL) were used for the determination of UA content. Th c lumn oid (V ) and total (VT) volumes were id ntified by th lution positions of Blu D xtran (2000 kDa ) and glucose respectiv ly. Th UA content in each column fracti n wa xpr s d a a p re ntag of th total U r co er d.

PAGE 69

56 Hemicellulo e Extraction H mic lluloses were isolated using the method of Huber and Nevins (1981) as modifi d by de V tten and Huber (1990). To remove the major portion of pectin prior t o hemicellulos 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 40C for 5 h For hemicellulose extraction 50 mg of EIS were incubated in 5 mL of 4 M KOH including 26 mM NaBH 4 for 12 hat 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 g glucose equivalents in 200 mg EIS. Gel Permeation Chromatography of Hemicelluloses Two ml of the hemicellulose extracts (at approximately 1 mg mL1 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 assay d for total sugars (Dubois et al. 1956) and xyloglucan (Kooiman 1960 ). The column V 0 and Vt were identified by the elution positions of Blue Dextran 2000 and gluco e

PAGE 70

57 resp cti The column was calibrated with dextran standards of 70 40 and 10 kDa (Sigma t. Louis MO). Statistical Analysis The experiments were laid out in a completely randomized design. Statistical procedure were performed using the PC-SAS software package ( A -Insititute 1985). Data were subjected to ANOV A 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 4-1 A. Control fruit softened rapidly and completed ripening (softened to 10 to 20 N) within 8 d of storage 1-MCP at 0.09 L L1 for 6 or 12 h had no significant effect on fruit firmness relative to control fruit. Firmness decrease of fruit treated with 1-M Pat 0.45 L L1 was significantly delayed and varied with time of exposure to the gas. After 8 d of storage at 20C, fruit treated with 1-MCP at 0.45 L L1 for 6 12 or 24 h xhibit d firmness values of 20.1, 28.5 and 41.6 N respectively. Firmness of fruit treat d with 1-MCP at 0.45 L L1 for 6 and 12 h reached a full-ripe firmness value (10 to 20 N) aft r about 10 days. Fruit treated with 0.45 L L1 for 24 hr quired an additional 2 d tor ach the full-ripe stage. Weight lo s trends for immonds avocado fruit during storag at 20C are h wn in Figur 4-lB. ntrol fruit and fruit treated at 0.09 L L1 for 6 or 12 h how d n diffi r n in th magnitud and rat of weight loss ft r 8 d at which time fruit r fu ll ripe cumulativ w ight loss ranged between 6.5 and 7.0 %. The rate of

PAGE 71

58 eight los in fruit treat d with 1-MCP at 0.45 L L1 paralleled the effe ct s of the gas o n firmne Aft r 8 d of storage fruit treated with 1-MCP at 0.45 L L 1 for 6 1 2 or 24 h show d w ight loss values of 5 0 4.5 and 3.9 % respectively. Final weight loss valu es of fruit treated with the higher 1-MCP concentration (0.45 L L1 ) were not significantly different from control fruit and fruit treated with 1-MCP at 0.09 L L1 for 6 12 and 24 h Regardless of treatment, fruit when ripe showed overall weight loss values ranging from 6 to 7 % (Fig. 4-lB). Respiration and Ethylene Evolution Respiration trends in fruit treated with 1-MCP at 0.45 L L1 for 12 or 24 h were atypical. Respiration in control fruit and fruit treated with 1-MCP at 0.09 L L1 for 6 or 12 h began to increase after 2 d storage at 20C (not shown) and CO 2 production reached maxima of 51.7 55.4 and 47.5 mg kg1 h1 respectively after 6 to 7 d storage at 20C (Table 4-1). CO 2 production of fruit treated with 1-MCP at 0.45 L L1 for 6 h increased initially after 4 d storage at 20C (not shown), reaching a maximum of 43.6 mg kt 1 h1 after 9 3 d storage at 20C (Table 4-1 ). There were no significant differences in the maximum CO 2 production rate between control fruit and fruit treated with 1-MCP at 0.09 L L1 for 6 h but the application of 1-MCP at 0.09 L L1 for 12 hand at 0.45 L L1 for 6 h slightly suppressed the magnitude of the respiratory peak (Table 4-1 ). The CO 2 production of fruit treated with 1-MCP at 0.45 L L1 for 12 and 24 h showed a d e layed and attenuated climacteric pattern. Increased respiration for fruit treated with 1-M CP at 0 .4 5 L L1 for 12 and 24 h was first evident at day 6 and day 10 respectively (not shown). A distinct peak of CO 2 production did not occur during the storage period and maximum CO 2 production rates were reduced nearly 40 % compar d with all other treatments when experiments were terminated (Table 4-1 )

PAGE 72

59 Tabl 4-1 Da s to peak and maximum amount CO2 and C21Li production for 'Simmonds a ocado tor d at 20C with 1-MCP treatments. Fruit were treated with two 1-MCP concentration (0.45 and 0.09 L L1 ) and three exposure periods (6 12 and 24 h) Initial rates of C 2 H 4 and CO 2 production were 0.5 L kgh1 and 61.3 mg kt 1 h1 respectively Data are m ans standard deviation of 4 independent samples. CO2 C21Li Treatments Days to Maximum Days to Maximum peak (mg kg1 h1 ) peak (L kt 1 h1 ) Control (no 1 MCP) 6 51.7.9 6 124.2 39 0 1-MCP (0.09 L L1 for 6 h) 7.3 55.4 9.8 6 130.3 69 0 1-M P (0.09 L L1 for 12 h) 6.7 47.5 1.6 6 103.5 37 8 1-MCP (0.45 L L1 for 6 h) 9.3 43.6 8.3 9 117.0 40.0 1-MCP (0.45 L L1 for 12 h) lOX 25.8 17.5x lQX 45.9 64 6x 1-MCP (0.45 L L1 for 24 h) 12 y 30.3 13.5y 12 y 50.5 57.0 Ethylene and respiratory climacteric peak did not occur during storage at 20C 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 tr ated with 1-MCP at 0.09 L L1 for 6 or 12 h showed charact ristic climacteric pattern durin g storage at 20C. Ethylene production began to incr a e after 2 d in torag (not shown) and reached maximum values of 124.2 130.3 and 103.5 l kg1 h1 r sp ctiv ly after 6 d storage at 20C (Table 4-1 ). Ethylen production of fruit tr at d with 1-M Pat 0.45 L L1 for 6 h b gan to rise after 4 d torage at 20 r aching a ma x imum f 117 l kg1 h 1 aft r 9 d torage at 20 Th r w r n ignificant diffi r nc in the maximum amount of thyl ne production b tw n control fruit and fruit tr a t d ith 1-M P at 0.09 L L 1 fi r 6 and 12 hand 0.45 L L 1 fi r 6 h (Tabl 4-1 ).

PAGE 73

60 thylene production in fruit tr ated with 1-MCP at 0.45 L L 1 for 12 or 24 h began to increase after 6 and IO d of storage, respectively ( data not s hown). As for CO2 production a distinct peak of ethylene production was not observed during storage at 20C 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 20C 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 L 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 Ran ge Test (p < 0.05). Initial L chroma and hue angle were 46 5 24.2 and 123.6 respectively. Days to fully L* Chroma Hue angle Treatments ripe at 20C (Ho) Contro l (no 1-MCP) 8 46.3 be 33.2 ab 122.3 ab 1-MCP (0.09 L L1 for 6 h) 8 47.5 ab 33.9 ab 120 8 b 1-MCP (0.09 L L1 for 12 h) 8 49.7 a 37.4 a 120.0 b 1-MCP (0.45 L L1 for 6 h) 10 46.1 be 32.4 ab 121.0 ab 1-MCP (0.45 L L1 for 12 h) 10 45.3 be 31.6 b 123 5a 1-MCP ( 0.45 L L1 for 24 h) 12 44 1 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 increa e in

PAGE 74

61 chroma alue reflected increasing intensity of yellow color. At the full-ripe stage fruit treated ith 1-MCP at 0.45 L L1 for 12 and 24 h retained more green color than fruit tr ated ith 1-MCP at 0 09 L L1 for 6 and 12 h. Fruit treated with 1-MCP at 0.45 LL1 for 24 h had the lowest L* value (44.1) and chroma value (26.0) with the highest hue angle (123 .5) (Ta ble 4-2). These data indicate that the peel of avocado fruit treated with 1-MCP at 0.45 L L1 for 24 h retained moderate green color with low color intensity ( light) during 12 days storage at 20 C. 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 L L1 for 24 h). The activities of cell wall enzymes in cell-free protein extracts of avocado fruit treated with 1-MCP at 0.45 L L1 for 24 hare 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 increa s during the postclimacteric phase (Fig. 4-2A). The pattern of PG accumulation in 'S immonds avocado fruit is consistent with activity levels reported for Fuerte avocado (A ad and Y ung 1979 Zauberman and Schiffmann-Nadel 1972). PG acti ity in immond fruit treated with 0.45 L L1 1-MCP for 24 h remain d at levels comparable t or li ght l y b low thos e detected at harvest (Fig. 4-2A). PM activ ity in control fruit declined from a maximum valu at harve t to a minimum I v I at th full-ripe (day 8) stage (Fig. 4-2B) The tr nd for 1-MCP treated fruit parall 1 d that for control fruit although th d cline in acti it wa lightly delay d.

PAGE 75

62 Th le I of PME in 1-M P tr ated fruit after 12 d were similar t th control fruit at 8 d. not e d fo r C -cellulase ( ndo-1 4-P-glucanase) activity was not detected in fruit m e a s ured within 24 h of harvest (Fig 4-3A). In control fruit C x -cellulase l e v ls 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 L L1 1-MCP for 24 h was not detectable until day 4. C x -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 L L1 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 L L1 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 20C. 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 waterand CDT A-soluble UA content total UA content and EIS levels for both control fruit and fruit treated with 0.45 L L1 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 U A 1 v ls in 1-MCP-treated fruit remained constant throughout the 12-day to ra g

PAGE 76

63 p riod Water-soluble UA in control fruit increased significantly after 4 d at 20C and an additional 2.1-fold when fruit were fully ripe (Fig. 4-4B). The levels of CDT A-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 aft r 8 days at 20 C and reached levels 1.8-fold higher than those at day 0 (Fig. 4-4B). In contra t to control fruit the levels of CDT A-soluble UA in 1-MCP-treated fruit remained constant through 12 d of storage at 20C (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 20C. Gel permeation profiles of water -soluble polyuronides from control and 1-MCP treated avocado on days O 4 8, and 12 are shown in Figure 4-5. At the pre-ripe stage (Day 0 Fig. 4-SA) water-soluble polyuronides, representing approximately 20% of total EI UA eluted as a polydisperse population. As ripening proceeded (Day 4 through 8 Fig. 4-SB 4-SC) 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-M P-treated fruit were considerably more limited throughout 8 d of storage. At the full-rip stage (Day 12) the water-soluble polyuronides of 1-MCP-tr at d fruit showed vid nc of furth r mol mas downshifts (Fig. 4-5D) howev r as e id nt from Figur 44 (A B) the I vels of wat r-soluble polyuronides in 1-MCP-treat d fruit represented a pr p rti nally much l w r p rcentage (40%) of total EIS UA compar d with the 70% for c ntr I fruit.

PAGE 77

64 Th mol cular mas distributions and downshifts of CDT A-soluble pol y uronid e parall led th e vid nt for wat roluble polymers (Fig. 4-6) Mol m a down hi ft for rip control fruit (Day 8 Fig. 4-6C) and consid rably les s pronoun c d for rip 1-M P-treated fruit (12 d Fig. 4-6D). CDTA-soluble polyuronides were a relativ ly 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 20C 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 L L1 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, w ith peak elution volumes noted at 40 42 and 44 ml for hemicelluloses from da y 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).

PAGE 78

65 s illustrated in Figure 4-9 changes in molecular mass of xyloglucan (XG) parallel d those observed for total hemicelluloses though XO-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 (e lution 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 C 2 H 4 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 treatm nt consistent with the fact that softening is one of the most ethy l ene -sensiti ve rip nin g proc ss s (Lelievre et al. 1997a). Significantly delayed soft ning by 1-MCP sub tantiat that ethylene is involved in augmenting the activity of softening-related m tabolism. imilar effects of 1-MCP in attenuating fruit softening have been obser ed. Feng t al. (2000) report d that treatments for 24 h with 30 to 70 nL L1 1-MCP prior to ex po ur to thylene delayed ethy l ne-induced softening of Ha s' avocado a Mexicanu at malan h ybr id by 10 to 12 d. In apricot fruit tr ated with 1 L L1 1-M P for 4 hat 20 had ignificantly high r firmn ss compared to untr at d fruit aft r 10 d torage at 20 (Fan t a l. 2000). Rupa ingh et al. (2000) found that Mcinto h and Delicious

PAGE 79

66 apples tr at d with I L L1 1-MCP for 18 hat 20 C showed a significant delay in fruit softening. The concentration of and length of exposure to 1-MCP significantly influenc e d the effect of the gas in delaying avocado fruit ripening. Avocado fruit treated with 0.45 L L1 1-MCP for 24 hat 20C required 2 to 4 more days at 20C to reach the full-ripe stage compared with fruit treated with 0.09 or 0.45 L L1 1-MCP for 6 or 12 hat 20 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 L1 caused a progressive delay in fruit softening of 'Hass' avocado. 1-MCP significantly de l ayed 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 CO 2 production did not fully recover in avocado fruit treated with 0.45 L L1 1 MCP for 12 or 24 hat 20C with maximum production rates remaining 50 % and 70 % lower, respectively, than those for all other treatments. De l ayed 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 thylene effects also depends on the exposure temperature Ku and Wills (1999) found that the to rage life of broccoli increased with an increase in the cone ntration and

PAGE 80

67 xposure p riod of 1-MCP treatment at 5 and 20C and also reported that broccoli treated ith 1 L L1 1-MCP at 20 exhibited a significantly longer storage life than that treated at 5 ha e observed that 1-MCP treatment at 20C had more effect on delaying a ocado fruit ripening than at 12 C (Jeong, unpublished). In carnation the effectiveness of 1-MCP is 4-fold higher at room temperature (24C) than at 4 C (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 L L1 for 24 h) did not result in a further delay in ripening compared with treatment at 0.45 L L1 1MCP for 24 h (data not shown). This indicates that treatment with 0.45 1 1-MCP for 24 his sufficient to exert maximal delay of avocado ripening. The concentration of 1MCP 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 L1 Sisler 1996a ; Sisler 1997) ethylene-induced ab cission and flower senescence for potted flowering plants (20 nL L1 I Serek et al. 1994 b) and ripening of banana fruit (0. 7 nL L1 Sisler and Serek 1997) the levels necessary to delay avocado rip ning (0.45 L L1 ) are comparable to those found effective for delaying rip ning of apricot ( 1 r 1 Fan t al. 2000), and McIntosh and Delicious apples (I L L1 Rupa ingh t al. 200 0) In an analysis of the absorption capacity of tomato and avocado fruit for 1-M P an e quivalent mas of avocado tissue absorbed 2 to 3-fold higher le of 1-M P ga (J ong, unpublished) The gr ater amount of 1-MCP r quired to block 2 H 4 acti n in avocado could be due to the high oil cont nt in these fruit which could act

PAGE 81

68 a a comp titive r rvoir for 1-MCP. The gradual (but incompl e te) recovery of C2!1i production in 0.45 L L1 1-MCP-treated avocados during storage at 20C suggests e ith r the synth i of new r ceptor proteins metabolism of the 1-MCP receptor-protein complex or dissociation of 1-MCP from the receptor sites (Sisler and erek, 1999 ; isler et al. 1996a). Consistent with previous reports the ripening of avocado fruit was accompanied by increases in C x -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 L1 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 L L1 1MCP-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 mor important in the late stages of ripening

PAGE 82

69 Consistent ith the marked suppression of PG levels in 1-MCP-treated immonds a ocado fruit the solubilization and degradation of polyuronides was significantly delayed and reduced in 1-MCP-treated fruit. At the pre-ripe stage (day 0) aterand DTA-soluble UA constituted 22 % and 7.9% of the total EIS UA content respectiv l y At the full-ripe stage (10-20 N) waterand 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 3 8% 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 xplained on the basis of the more limited molecular mass downshifts in the polyuronides of these fruit. In the present study, both water and CDT A-soluble polyuronid s f control fruit exhibited the characteristic molecular mas downshift r p rt d in arlier studi s with polymers in ripe fruit eluting n ar th total column olum e Thr u g h 8 d of torag (Fig. 4-SC 4-6C) 1-M P treated fruit show d c n id e rabl y l e e x t n iv breakdown of both waterand CDT Aolubl polyuronide urth rd w n hift in p lyuronid molecular mass w r vid nt in 1-M P-tr at d fruit aft r 1 2 d f t rag Th e data uggest that the low activity of PG in 1-M P-treat d

PAGE 83

70 fruit i u f fici nt to d p lym ri z a ocado polyuronid s hl"\u , oup not that th quantiti of polyur nid xtract d from 1-MCP tr at d fruit at 1 2 d ar proporti o n a ll y much lo r than tho from control fruit. Additionally Wakaba yas h y i et al. (2000) have hown th a t limit d mol cular mass downshifts in avocado polyuronid es as evident from gel filtration analyses can be brought about by deesterification ind e p e ndently of PG action Cons quently the relatively normal levels of PME in these fruit might ha ve influenced the gel filtration behavior of polyuronides from 1-MCP-treated fruit. Total extractable aand ~-galactosidase activities decreased during avocado ripening how ver the use of total protein extracts in our assays would have masked diffi rential re ponses of specific isozymes of these proteins. For example Pressey ( 1983 ) and Carey et al. (1995) reported that total ~-galactosidase activity in tomato remained relatively constant throughout ripening whereas the levels of one isozyme Gal II) increased about 4-fold. Smith and Gross (2000) found that transcript accumulation for tomato ~-gal II one of 7 ~-gal transcripts detected was significantly impaired in rin, nor and Nr fruit relative to wild-type accumulation indicating that ~-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 ). Durin g ripening of Simmonds' avocado at 20C hemicelluloses at each dev e lopmental 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 (F i g 4-8 4-9) T h

PAGE 84

71 hanges in pol mer 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 L L1 ) of 1-methylcyclopropene (1-MCP) for three exposure times (6 12, and 24 h) at 20C. The fruit were then stored at 20 C in ethylene-free air for ripening assessment. Firmness, weight loss, respiration and C 2 H 4 production peel color, selected cell wall enzymes (polygalacturonase, pectinmethy lesterase a, ~-galactosidase, and Cx-cell ulase) and cell wall matrix polysaccharides (polyuronides and hemicellulose) were monitored during storage. 1-MCP treatment at 0.45 L L1 for 24 hat 20C delayed ripening of avocado fruit by 4 d at 20 C. This delay was characterized by a significant reduction in the rate of fruit oftening and in the timing and intensity of the ethylene and respiratory climacterics. Avocado treated with 1-MCP (0.45 L L1 ) for 24 hat 20C also showed significantly less weight loss and retained more green color than control fruit at the full-ripe stag (10 to 20 N). Th delay in avocado ripening was influenced by 1-MCP concentration xpo ure duration and exposure temperature. 1-M P tr atment affected the activity trends of all cell wall nzyme measur d and c mp! t ly uppress d the appearance of polygalacturona acti ity for up to 12 d. t th full-rip tag at rand DT Aolubl polyuronid of control fruit compri d a ppr x im a tel 65% and 7.5% of the total polyuronide content r p ctively whereas

PAGE 85

72 those of 1-M P-treated fruit comprised approximately 38% and 9 7 % ofth total polyuronide content. Wh n fully ripe polyuronides from 1-MCP tr at d fruit exhibit d reduced molecular mass downshifts compared with control fruit. 1-M P treatment al s o delayed and slightly reduced the depolymerization of 4 M alkali-soluble hemicellulo s s 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 C 2 H 4 action and delayed C 2 H 4 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.

PAGE 86

73 100 -+1-MCP (0.45 L L 1 for 24 h) 80 -o1-MCP (0.45 L L 1 for 6 h) -T1 MCP ( 0.45 L L 1 for 12 h) z -----'1-1-MCP (0 09 L L 1 for 12 h) ----1-MCP ( 0.09 L L 1 for 6 h) ti) ti) 60 Control (no 1-MCP) (1) C: E .... t;::: 40 ::::s .... u. 20 A 0 8 8 6 0 ti) ti) 0 ... 4 .r:. C) (1) 2 0 0 2 4 6 8 10 12 14 Storage period (days) igur 4-1. Fruit firmne s (N) and weight loss(%) of immond a ocados stored at 20 with 1-M P tr atm nts. Fruit were treated with two 1-M P cone ntrations (0.45 and 0.09 L I ) and thr x posure p riods (6 12 and 24 h). V rtical bar repr sent tandard d viation of 6 ind p nd nt ampl

PAGE 87

74 35 30 "' C 25 >< C 20 > E ns "7 (!) Cl Q. Q) 15 0 10 E 5 0 4 8 1-MCP (0.45 L L1 for 24 h ) --Control (no 1-MCP) "7 3 ~ = E > -..... U Cl ns E 2 w 0 N ID Q.
PAGE 88

75 4 >. A :~ 3 .... (.) c: ro (1) E en .... 2 ro 0) ::J E Q) (.) 1 I >< u 0 1-MCP (0.45 L L1 for 24 h) 8 ~,qs;: 0 :.:; "l""" -+----Control (no 1-MCP) 7 (.) >< ro 6 (1) .... en C: 5 ro E "'O en .... 4 0 0) 3 (.) (1) ro 8 ro 0 2 0) E I 1 t$ ~,qs;: 0 6 :.:; "l""" (.) >< ro (1) .... 5 en C: ro E "'O en '7 4 0 0) (.) (1) ro ro 0 3 C 0) E I C1 2 0 2 4 6 8 10 12 14 Storage period (days) igur 4-3 f[i ct of 1-M P (0.45 L L. 1 for 24 h) on Cx-c llulase and aand~ galact idas activiti of avocados stor d at 20 Fru i t wer tr at d w i th ( o ) or ithout ) 1-M P. V rtical bars r pr s nt standard deviation of 3 ind p n d ent amp

PAGE 89

..... C (f) C W o""'" (.) 'c, < E :::> ti) ro c, ..... 1 0 t..... C Q,) ..... C 0 (.) < (f) :::> w Q,) C) -g E 76 240 --------------~ 220 200 180 160 140 A 120 ~--------------------------~ 120 B 100 80 60 40 ---------------20 30 ---------------o1-MCP (0.45 L L1 for 24 h) 25 --Control (no 1-MCP) 20 15 0 C) <{> 2: 10 < C to u 5---------------------------1050 D 1000 950 900 850 800 ---------------750 0 2 4 6 8 10 12 14 Storage period (days) ..... C Q,) ..... C 0 (.) < (f) :::> w Q,) C) -g E 0 C) ti) 1 I ... Q,) ..... ro 3: ti) :E Q. ... o ro ti) (.) Q,) 0 ti) .c Q,) ::, E 0 C) ti) co N 0 C C: ro c, .c E wFigure 4-4 Effect of 1 MCP ( 0 .45 L L1 for 24 h ) o n t h e amount of EIS in the mesocarp tissue and on the c h anges i n water C O TA so lu b l e UA and total UA in EIS from avocados s t ored at 20C EIS were incu b ated seq u entia ll y in distilled water and 50 mM COTA in 50 mM Na acetate pH 6.5 eac h for 4 h at 34C. Suspensions were filtered and UA content determ in ed F ru it were treated wit h ( o ) or without( ) 1-MCP Data are means sta n dard deviation of 3 independent samples.

PAGE 90

"'O (1.) '(1.) > 0 (.) (1.) '.... 0 .... 0 ti) "'O (.) (.) C: 0 '::::> 77 18 --------------15 12 9 6 3 A 0 .,__._ ________ _;:;;:;;a.&;;Mliaif 15 ---o--1 MCP ( 0.45 L L" 1 for 24 h) 8 12 ---Control (no 1-MCP) 9 6 3 0 ~:i..:::i,,o ,a... _______ ...c.. ~~a.o.:~ 15 12 9 6 3 0 ....... ~=::;., ___ _;~~ ....... 15 12 9 6 3 D 0 e..a.o"'""' ~-------~ ~~~ 10 20 30 40 50 60 70 80 Elution volume (ml) igure 4-5 M 1 cular mass profiles of water soluble polyuronid s from EIS prepared from av cad tr at d with ( o ) and without ( ) 1-MCP. Polyuonid ( ~ 0 5 mg g alacturonic a cid quival nts) w r appli d to Sepharos L-2B-300 and individual fr a ction r m asured fi r A cont nt. Data for each fraction xpr ed as a percentag o f th e t tal lut d UA Day 0 (A) Day 4 (B) Day 8 ( Day 12 (D). Vo Void vol um Vt t tal !um

PAGE 91

"'C a, a, > 0 0 a, co ..... 0 ..... .... 0 78 15 -----------------. 12 9 6 3 A 0 ... iAA.--------~li&iA ....... 12 -Control ( no 1 -MCP ) B 9 ---o-1 MCP ( 0.45 L L 1 for 24 h 6 3 o ~~ ------....: ~~~ 12 9 6 3 0 ~~~;;._ _____ ___,; ~ ~~ :AQ 12 9 6 3 D 0 o.:::i.::i..::..oiiil--------....;,;i; ~;Q,Q,i~ 10 20 30 40 50 60 70 80 Elution volume (ml ) Figure 4-6. Molecular mass profiles of CDT A-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 expre sed as a percentage of the total eluted UA. Day 0 (A) Day 4 (B); Day 8 (C) Day 12 (D). Vo Void olume Vt total volume.

PAGE 92

79 20 ----------------------CL> ti) 0 :::s 18 "a;ci) -~w E c, 1 6 1E CL> 0 -o .CN ..2 c: 14 O t{> C, :.: E ns ---Control ( no 1-MCP) -o1-MCP (0.45 L L 1 for 24 h) 1 0 __, _________________________ __,. 0 2 4 6 8 10 12 14 igure 4-7. ffect of 1-M P (0.45 L L1 for 24 h) on the changes in th xtractable amount of 4 M alkali-soluble hemicellulose in EIS from avocados stored at 20C. Fruit were tr at d ith ( o ) or wi thout ( ) 1-MCP Data are m ans D of 3 independ nt amp

PAGE 93

80 15 I I I A 12 V o V t 9 6 3 0 "'C 12 ---o1-MCP (0.45 L L 1 for 24 h ) B (l) a.. (l) > 9 0 0 (l) 6 a.. "' ..... 3 0 ..... ...... 0 0 0 12 C (l) 1/) 0 9 ::J (l) 6 0 E 3 (l) .r:. 0 .0 12 D ::J 0 1/) 9 I "' 6 .!c:: "' 3 -.::t' 0 10 20 30 40 50 60 70 80 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 hemicellulose was applied to the Sepharose CL 6B-100 and individual fractions were measured for tota l sugar. Data for each fraction expressed as a percentag of the total eluted s u gar. Day 0 (A); Day 4 (B); Day 8 (C); Day 12 (D). Tick marks at the top of th figure indicate void volume (V 0 ) 70 40 10 kDa (middle) and gluco (right).

PAGE 94

81 12 I I I 9 Vo V t A 6 3 "C 0 a, '-----0MCP (0.45 L L1 for 24 h) a, B > 9 0 (.) a, '6 (0 0 3 \t0 0 0 C: ----Control (no MCP) (0 (.) 9 ::::J C') 0 6 >< 3 0 9 D 6 3 0 10 20 30 40 50 60 70 80 Elution volume (ml) 1gur 4-9. Mol cular mass profil s of xy loglucan in 4 M alkali-soluble hemicellulos from I pr par d from avocado treated with ( o ) and without( ) 1-M P. Two ml of hemic llul was app li d to th pharos CL-6B100 and individual fractions were rn a ured for total sugar. Data for each fraction expressed as a p rcentage of the total Jut d ugar. Day 0 (A) Day 4 (B) Day 8 ( ) Day 12 (D). Tick mark at the top of th fi g ur indi at void olurn (V 0 ) 70 40 l 0 kDa (rniddl ) and gluco (right).

PAGE 95

HAPTER 5 TH EFFE T OF 1-METHYLCYCLOPROPENE (1-MCP) AND WAX COATING FOR REGUL TING THE RIPENING AND EXTENDING THE STORAGE LIFE OF AVOCADO FRUIT Introduction The a ocado (Persea americana Mill.) is a climacteric fruit that is characteriz e d b 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 th e most rapidly ripening of fruits often completing ripening within 5 to 7 d following harvest (Sey mour and Tucker 1993). As th y lene plays as important role in regulating fruit ripening inhibiting ethyl n bios y nthesis or action should slow the ripening process and extend the fruit s po tharvest storage life. 1 methylcyclopropene (1-MCP) one of the synthetic c clopropenes 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 L1 (Sisler and Serek 1997). Although 1-MCP binding to the ethylene rec ptor sites is irreversible it appears that new receptors can be formed during climacteric ( isler 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) (Lelie r t al. 1997b) banana (Musa sp.) (Golding et al. 1998 ; Golding et al. 1999 isler and r k 82

PAGE 96

83 1997 ), plum ( Prunu alicina Lindl) (Abdi et al. 1998) tomato (Lycopersicon e ul e ntum Mill) (Nakatsuka et al. 1997 Sisler and Serek 1997) apple (Malus sylvestris L.) ( Fan and Mattheis 1999; Watkins et al. 2000), and avocado (Per ea 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 a ocado 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 ripenin g 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 reduc th rate of ripening to a greater extent than treatment with 1-MCP alone. Materials and Methods Plant Material T w r II and Booth 7' mid-season cultivars wer select d for these ex p rim nt Th y ar hybrid cul ti var which are cros s of W t Indian and u a t m a l a n rac (Hatton and ampbell 1960 Hatton t al. 1964). Recommended

PAGE 97

84 torag t mp rature is 13 to avoid chilling injury ( eymour and Tuck r 1 993 Mature avocado fruit were obtained from a commercial grower in Homestead lorida packed in fib rboard cartons and transported to the Postharvest Horticulture Laboratory in Gaine vill within 24 h after harvest. Fruit were selected for uniformity of s i ze (w i g ht Tow r 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 NaOCl) 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 L L1 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 hat 20C 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 8 l 9F 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 20C in ethylene-free air at 85 % r l a tive humidity. Control fruit (not exposed to 1-MCP and not waxed) were maintained und e r identical storage conditions. In the second experiment with Booth 7 immediately following 1-MCP treatment the fruit were removed from the treatm nt

PAGE 98

85 chamb r and then axed. The fruit were subsequently stored at l3C in ethylene-free air at 85 % r lati humidity Control fruit (not exposed to 1-MCP but waxed) were maintain d und r identical storage conditions. Samples of fruit from each treatment were e aluated 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 CO 2 and C 2 H 4 production, as well as peel color changes. Mesocarp tissue derived from the equatorial region of selected fruit was stored at -30C 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 min1 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 w re measured repeatedly every other day until they reached the full-ripe stage Re piration and Ethylene Evolution R e piration and ethylene production were measured every other day using th am four fruit of each treatm nt. Fruit were individually sealed for 30 min in 2-L pla tic nt a in e r pri rt sampling A 0.5 mL gas sample was withdrawn by syringe through a rubb r ptum and carbon dioxide d termined using a Gow-Mac gas chromatograph ( e ri s 580 Bridge water NJ U A) equipped with a thermal conductivity detector (T D ). thyl n wa m a ur d by inj cting a 1.0 mL ga amp! into a HP 5890 ga

PAGE 99

86 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 0 ) with a value of 90 representing a totally yellow color and 180 a totally green color. The results are presented as lightness (L *), chroma (C*) and hue angle (H 0 ) The chroma and hue angle were calculated from the measured a* and b* values using the formulas C* = (a* 2 +b* 2 ) 1 1 2 and H 0 =arctangent (b *la*) (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 4 C. 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 4C. The pellets were transferred to 50 mL of ice-cold acetone for 10 min followed by centrifugation (7840 g, 10 min, 4 C). 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 (5C), 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 NaCl for 30 min in ice-cold (5C) wat r bath and c ntrifuged. The supernatant was analyz d for enzyme activities as described below. Prot in cont nt was

PAGE 100

87 measured using the bicinchoninic method (Smith et al. 1985) with bovine serum albumin as a standard. Polygalacturonase Assay Pol ga lacturonase (PG E C. 3 2 1.15) activity was assayed reductometrically by incubatin g a 100 L aliquot of the cell-free protein extract with 500 L (2 mg) of pol y galacturonic acid (from orange peel Sigma Chemical Co St. Louis MO USA) dissolved in 30 mM KOAc pH 5 5 containing 100 mMKCl. After 30 min at 34C uronic acid reducing groups were measured using the method of Milner and A vi gad (1967). PG activity was expressed as mol D-galacturonic acid equivalents produced per kg protein per minute. Statistical Analysis The e x periments were conducted in a completely randomized design. Statistical procedures were performed using the PC-SAS software package (SAS-Insititute 1985) Data were subjected to ANOV A using the General Linear Model (Minitab, State College PA) Differences between means were determined using Duncan s multiple range test. Results Fruit Firmne sand Weight Loss han g es in Tower II avocado fruit firmness following 1-MCP treatment (0 9 L L 1 for 1 2 hat 20 ) and / or wax treatments are shown in Figure 5-1 A Control fruit so ft n d r a pidl y and completed ripening (10 to 20 N) within 7 days of torage at 20C. A ft r 7 d f tora g at 20 fruit treated with wax 1-MCP or both 1-MCP and wax x hibit d firmn valu of 31.2 36 6 and 51 5 N respectively Firmn ss decrease of fruit tr a t d w ith 1-M P and / or wax was significantly d layed and vari d with different

PAGE 101

88 treatments. Firmness of waxed fruit without 1-MCP reached the full-ripe stage (10 to 20 N) aft r about 9 d while that of fruit treated with 1-MCP or both 1-M P and wax reached th e full-ripe stage after about 11 d. Weight loss trends of Tower II avocado fruit during storage at 20C are shown in Figure 5-IB. After 7 d of storage at 20C at which time control fruit were fully ripe weight loss was 7.3 %, whereas fruit treated with 1-MCP wax, or both 1-MCP and wax showed weight loss values of 5.4 2.0 and 1.9 %, respectively. At the full-ripe stage (10 to 20 N) weight loss of non-waxed fruit treated with 1-MCP was significantly higher (9. 3 %) than that of control fruit (7.3%) and waxed fruit with and without 1-MCP treatment (2.7 and 3.2% respectively). The rate of weight loss in control fruit and non waxed fruit treated with 1-MCP paralleled the effects of the gas on firmness. Waxed fruit treated with and without 1-MCP showed no differences in the magnitude and rate of weight loss Regardless of 1-MCP treatment waxed fruit at the full-ripe stage showed overall weight loss values ranging from 2.5 to 3.5 % (Fig 5-lB). Changes in Booth 7' avocado fruit firmness following 1-MCP (0.9 L L1 for 1 2 hat 20C) and / or wax treatments are shown in Figure 5-2A. Waxed fruit without 1-M C P treatm e nt softened and completed ripening (10 to 20 N) within 19 d of storage at l 3C. After 19 d of storage at l 3C fruit treated with both 1-MCP and wax exhibited firmness va lu e of 76.8 N. Firmness decrease of waxed fruit treated with 1-MCP was significantly delayed and r eac hed firmness value of 26.5 Nat day 37 (Fig. 5-2A). Although waxed fruit treated with 1-MCP could not reach the full-ripe stage due to decay incidence after about 37 d fruit were edible.

PAGE 102

89 eight loss trends of Booth 7 avocado fruit during storage at l 3C are shown in Figur 5-2B. Weight loss of waxed fruit without 1-MCP treatment was 4.5 % at the full ripe stage After 19 d of storage at 13C, waxed fruit treated with 1-MCP showed weight loss alues of 3.8 %. After about 37 d, weight loss of waxed fruit treated with 1-MCP (7.5%) was significantly higher than that of waxed fruit without 1-MCP treatment (4.5%) at the full -rip e stage after 20 d (Fig. 5-2B). Table 5-1. Days to peak and maximum amount CO 2 and C 2 ~ production for 'Towe r II avocados treated with 1-MCP (0.9 L L1 for 12 hat 20C) and/or wax and stored at 20C. Initial rates of C 2 H 4 and CO 2 production were 1.2 L kg1 h1 and 38.3 mg kg1 h1 respectively 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 4 independent samples. CO2 C2~ Treatments Days to peak Maximum Days to peak Maximum (mg kg1 h1 ) (L kg-I h1 ) Control 6b 144.9 15.1 4c 114 7 10 1 (no 1-MCP & no wax) Wax only 5b 156.8 22.7 5 be 89.4 13.9 1-MCP only 8a 145.5.8 8a 330.0 66.5 1-MCP & wax 9a 150.9 7.6 7 ab 130.5 35.3 Re piration and Ethylene Evolution Tow r II avocado fruit from all treatments showed characteristic respiratory climact ric patterns during storage at 20C (Table 5-1 ). Respiration in control fruit and waxed fruit without 1-M P treatment began to increase after 1 day torag at 20 (data not h wn) and 0 2 production r ach d maximum values of 144.9 and 156.8 mg kg1 h1 r s p cti ly aft r 5 to 6 d torage at 20C (Tabl 5-1). CO 2 production of fruit treated ith 1-M P r b th 1-M P and wax increa ed initially aft r 3 d storage at 20 (data not

PAGE 103

90 shown) r aching a maximum of 145.5 and 150.9 mg kg1 h1 respectively after 8 to 9 d storage at 20C (Table 5-1). 1-MCP treatment delayed the respiratory climacteric pattern of waxed and non-waxed fruit by 2 to 4 d respectively. The time to attain the maximum ethylene production closely paralleled that for respiratory values in all treatments (Table 5-1 ). Ethyl ne production in control fruit and waxed fruit without 1-MCP treatment began to increase after 1 din storage (not shown) and reached maximum ethylene production values of 114.7 and 89.4 l kt 1 h1 respectively, after 4 to 5 d storage at 20C (Table 5-1 ). Ethylene production of fruit treated with 1 MCP or both 1-MCP and wax began to rise after 3 d storage at 20C, reaching a maximum of 330.0 and 130.5 l kg1 h1 respectively, after 7 to 8 d storage at 20C. 1-MCP treatment significantly delayed the ethylene climacteric pattern of waxed and non-waxed fruit by 2 to 4 d respectively. The application of 1 MCP also increased the maximum ethylene production of waxed and non waxed fruit about 1.5 and 2.9 fold, respectively, compared with waxed and non waxed fruit without 1 MCP treatment. There were no significant differences in days to peak ethylene production between waxed and non waxed fruit, while there were significant (P < 0.05) differences in the maximum amount of ethylene production between waxed fruit and non-waxed fruit (Table 5-1 ). Maximum ethylene production of waxed fruit with and without 1-MCP treatment was reduced about 60% and 22% respectively compared with non-waxed fruit. Booth 7' avocado fruit without 1-MCP showed characteristic respiratory climacteric patterns during storage at l 3C (Table 5-2). Respiration in waxed fruit without 1-MCP began to increase after 7 d storage at l 3C (not shown) and CO 2 production reached a maximum of 65.7 mg kg1 h1 after 17 d storag at 13 CO 2

PAGE 104

91 production of a ed fruit treated with 1-MCP increased initially after 22 d storage at 13 C ( data not shown). A distinct respiratory peak of waxed fruit treated with 1-MCP was not obs r ed during storage at 13C, but the maximum CO 2 production rates (60.7 mg ki 1 h1 ) ere similar to those of waxed fruit without 1-MCP when experiments were terminated (Table 5-2). 1-MCP treatment delayed the respiratory climacteric pattern of waxed fruit by over 16 d (Table 5-2). Table 5-2 Days to peak and maximum amount CO2 and C2H4 production for 'Booth 7 avocados stored at 13C after wax treatment with or without 1-MCP (0 9 L L1 for 12 h at 20 C). Initial rates of C 2 H 4 and CO 2 production were 1.6 L kg1 h1 and 70.1 mg kg1 h1 respectively. 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 4 independent samples. CO2 C2 H4 Treatments Days to peak Maximum Days to peak Maximum (L kg-I h1 ) (mg kg1 h1 ) Control (wax only) 17 b 65.7 0.7 16 b 32.5 10.9 Wax & 1-MCP 33xa 60.7 6.9 X 25 a 40.6 15.7 Respiratory climacteric peak did not occur during storage at 13 C and data were measured before experiments were terminated due to decay incidence. Booth 7 avocado fruit from both treatments showed characteristic ethylene climacteric patterns during storage at l 3C (Table 5-2). Ethylene production in waxed fruit without 1-MCP treatment began to increase after 7 d in storage at l 3C (not shown) and r ach d maximum ethylene production values of 32.5 l kg1 h1 after 16 d storage at 13 wh r as thyl ne production of waxed fruit treated with 1-MCP began to rise after 20 d tora g at l 3 C r aching a maximum of 40.6 l kg1 h1 aft r 25 d storage at l 3C ( abl 5-2) 1-M P tr atm nt significantly delayed the ethylen climacteric pattern of waxe d fruit b y ab ut 9 d Th maximum thyl ne production of wax d fruit with 1-M P

PAGE 105

92 was slightly higher than that of waxed fruit without 1-MCP (Tabl 5-2 ) consi s t e nt with r suits obtained for Tower II fruit (Table 5-1). Peel Color The peel of Tower II' avocado fruit prior to storage had a moderate green color (hue angle = 125.3 where pure yellow = 90 and pure green = 180). At the full-ripe stage (10-20 N) ther were significant (P < 0.05) differences in the L value (L *) chroma value (C) and hue angle of the peel color among fruit from all treatments (Table 5-3). Table 5-3. Peel color of Tower II' avocados treated with 1-MCP (0.9 L L1 for 12 hat 20C) and / or wax and stored at 20C. Peel color was measured at the full-ripe stage. 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 43.5 30.1 and 125.3 respectively. Treatments Control (no 1-MCP & no wax) \1/ax only 1-MCP only 1-MCP & wax Days to fully ripe at 20C 7 9 11 11 L* 49.5 a 42.8 b 46.2 a 40.7 b Chroma 39.3 a 27.8 b 36.6 a 26.0 b Hue angle (Ho) 117 6 b 121.9a 120.7 ab 124.1 a Changes in hue angle constituted the major alteration of fruit color coordinates 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 and / or wax had more green color than control fruit. Waxed fruit with or without 1-M P treatment had significantly lower L value and chroma value than non-wax d fruit. Fruit treated with both wax and 1-MCP had the lowest L* value (40.7) and chroma alue (26 0) with the highest hue angle (124.1) (Table 5-3)

PAGE 106

93 Pol galacturonase Acti ity T h act i iti es of pol yg alacturonase ( PG) in Tower II and Booth 7 a ocado fr ui t t r a t e d ith 1-M C P ( 0.9 L L1 for 12 hat 20 C) and/or wax are shown in Figures 5 3 and 5-4. The activity of polygalacturonase (PG) in control fruit was very low in fr eshl y harv es ted fruit increased during the climacteric period and continued to increase durin g the po s tclimacteric phase. This pattern of PG accumulation in Tower II and Booth 7' a v ocado fruit is consistent with that shown for Fuerte avocado (Awad and Young 1979 Zauberman and Schiffmann-Nadel 1972). In Tower II avocado PG levels of waxed and non-waxed fruit without 1-MCP treatment increased significantly and r e ached l ev els at the full-ripe stage 8 3and 7 8-fold higher respectively than le v els a t har ves t (F i g. 5-3 ) PG levels of waxed fruit treated with 1-MCP increased and reached 5 0fo ld hi g h e r than levels at harvest when fruit were fully ripe whereas PG levels of non w a xe d fruit tr e ated w ith 1-MCP were completely suppressed through 9 d of storage and then incr e as e d reaching values 4.6-fold higher than levels at harvest when fully ripe (Fig. 5-3) In Booth 7' avocado PG levels of waxed fruit without 1-MCP increased significantl y during the climacteric period continued to increase during the po s tclim a ct ric pha se and reached 8.5-fold higher than levels at harv s t (Fig. 5-4 ). PG l eve l of wax d fr uit tr e at e d with 1-MCP however remained at lev l comparable to or li g htl y b I w th ose d t ct d at harvest through 37 d of storage at 13 C (Fig. 5-4). Di cus ion In th p r ese nt tud y 1-M P and wax tr e atm nt significantly d layed th np nm g of Tow r II avo cado s tor d at 20 and Booth 7' avocado s tor d at 13C as evaluated b y firm n w i g ht I s r pirati n and C 2 H 4 production p l color and PG activity

PAGE 107

94 T w r II avo cado fruit treat e d with 1-MCP required 2 to 4 m o r e d ay a t 2 0 C t o reac h th full-rip sta g e (10 to 20 N) than fruit without 1-MCP tr e atment (F i g 5-l A). In B oot h 7 avocado wax and 1-MCP treatments significantly delayed fruit s oft e nin g and firmness values decreased from > 170 N to about 25 N over a 5-week period at 13 C (F i g. 5-2A) F irmness of both cul ti vars was significantly retained in respons e to 1-M C P treatment consistent with the fact that softening is one of the most s e nsiti ve rip e nin g process es to eth y lene (Lelievre et al. 1997a). Significantly delayed softening b y 1MC P in the pres e nt study substantiates that ethylene is involved in augmenting the activit y o f softening-related metabolism. Similar effects of 1-MCP in attenuating fruit softening ha ve been obs e rved for Hass avocado (Feng et al. 2000) apricot (Fan et al. 2000 ) and McIntosh and Delicious apple (Rupasinghe et al. 2000) fruits. Waxed Tower II avocado fruit treated without or with 1-MCP required 2 or 4 more days respectively at 20C to reach the full-ripe stage than control fruit ( Fig 5-lA ). This observation indicates that wax treatment also helped retain fruit firmness and e x tended the storage life of avocado by reducing weight loss as has been shown pre v iousl y for Hass avocado (Adato and Gazit 1974 ; Joyce et al. 1995 ). The present r e sults indic a te that there was a positive relationship between firmness and weight loss but the y w e re not correlated with each other. Although non-waxed fruit treated with 1-MCP had significantly more weight loss (7.2%) than waxed fruit without 1-MCP treatment ( 2 7) at day 9 they had higher firmness values (26.1 N compared to 20.4 N for wa xe d fruit w ithout 1-MCP) (Fig 5-2B). Combined 1-MCP and wa x treatment e xe rts ma x imal d e la y of avocado fruit ripening by reducing the rate of fruit s oft e nin g and w e i g ht lo s

PAGE 108

95 1-MCP treatment significantly delayed the onset of climacteric ethylene and respiratory patterns of both Tower II and Booth 7 cul ti vars (Tables 5-1 and 5-2) Similar effects of 1-MCP on climacteric behavior have 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 application of 1-MCP slightly increased the magnitude of the ethylene peak in both Tower II and Booth 7 cultivars although the differences were not statistically significant (Tables 5-1 and 5-2) as has been reported with banana (Golding, 1998). Mullins (2000) found the application of 1-MCP causes an immediate increase in ethylene production of grapefruit (Mullins 2000). However the gradual recovery of C 2 H 4 production in 1-MCP-treated avocados during storage at 13 or 20 C suggests either the synthesis of new receptor proteins metabolism of the 1-MCP receptor protein complex or release of the bound 1-MCP from the receptor sites thus regaining sensitivity (Sisler and Serek 1999; Sisler et al. 1996a). Both Tower II and 'Booth 7' treated with 1-MCP showed suppressed ethylene production and respiration for a significantly longer time than those treated without 1-MCP (Ta bles 5-1 and 5-2). The application of wax did not significantly affect climacteric ethylene and respiratory patterns but reduced the maximum of ethylene production In Tower II avocado wax s ignificantly suppressed the magnitude of ethylene peak but did not influ nc th magnitude of th e respiratory peak. In 'Booth 7 fruit tr ated with wax and 1-M P did not r cover normal r spiratory behavior during storag at l 3C. Their ma x imum 2 production rate m asured imm diately prior to terminating the x p rim nt wa I w r than that of wax d fruit without 1-MCP. Durand et al. (1984)

PAGE 109

96 r port d that axing cau ed an incr a in internal 0 2 lev I and a r duction in int rnal 0 2 one ntrati n of Fuerte avocado during storage Although th e pre se nt s tud y s h w d normal thy 1 n and r spiratory patterns elevated CO2 and reduced 0 2 concentrations in th internal atmosphere may delay fruit ripening. This assumption is further supported b y th vid nee showing that controlled and modified atmosphere (CA and MA) storage delays rip ning and improves the keeping quality of Hass avocado (Meir et al. 1994 Meir et al. 1997). Thus extension of shelf life by wax treatment can be attributed to a combination of reduced water loss and modified internal atmosphere. The peel of Tower II avocado fruit treated with both wax and 1-MCP had better r tention of green color with low color intensity (light) during 11 d storage at 20C. E ither 1 MCP or wax treatment significantly helped to retain moderate green color. In the previous 1-MCP experiments for Simmonds' avocado 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 (data not shown). Thus PG was measured in the present study. The rapid ripening of control fruit was accompanied by marked increases in PG activities as reported for avocado fruit (A ad and Young 1979 ; Christofferson et al. 1984; Pesis et al. 1978). 1-MCP treatment of both Tower II and Booth 7 significantly suppressed PG accumulation whereas wax treatment had no evident effect on the enzyme. In 'Tower II fruit PG levels of waxed and non-waxed fruit treated with 1 MCP at the full-ripe stage (10 to 20 N) were reduced about 40 and 42% respectively compared with those without 1-MCP treatment (Fig. 5-3). There were however no significant differences in PG levels between waxed and non-waxed fruits at the full-ripe stage. PG levels in Booth 7' avocado fruit treated with

PAGE 110

97 a and 1P remained at levels comparable to or slightly below those detected at harvest through storage at l 3C even during the climacteric period and the postclimacteric phase (Fig. 5-4). Softening of 1-MCP treated fruit progressed slowly but normall caused by significant suppressed PG activity. Feng et al. (2000) found that 30 50 and 70 nL L1 1-MCP suppressed PG activity about 10 to 30% in Hass avocado although fruit ripened and softened normally. Therefore application of wax coatings to 1-MCP-treated avocado reduced the rate of ripening to a greater extent than treatment with 1-MCP alone. Summary 1-methylcyclopropene (1-MCP) an inhibitor of ethylene action has been shown to extend the storage period of avocado fruit. Waxing is also known to extend the storage life of avocado by reducing water loss and modifying the fruit internal atmosphere. In this study 1-MCP and waxing were used to investigate their effects on ripening characteri tics in avocado fruit. Preclimacteric avocado (Per ea americana Mill. cv Tower II and Booth 7') fruit were treated with 1-MCP (0.9 L L1 ) for 12 hat 20C Half of th fruit were waxed (Sta-Fresh 819F, FMC co.) after 1-MCP treatment. The fruit w r sub s equently stored at 13C or 20C in ethylene-free air at 85% relati e humidity. A valuated by fruit firmness ethylene evolution and respiration rate 1-M Pat 0 9 L 1 for 12 hat 20 C and wax treatment delayed ripening f both Tower II and B th 7 a cad Fruit tr at d with both 1-MCP and wa had b tt r r t ntion of gr n p ee l c I r a nd fruit firmn and d lay d climacteric ethyl n oluti n and respiration rat 1-M P tr a tm nt ignificantly uppressed polygalacturona accumulation.

PAGE 111

98 Waxing r duced weight loss and retarded softening but did not delay climacteric ethylene evolution and respiration rates. Whereas firmness of control fruit decreased from > 100 N to 20 N in as few as 7 d at 20C fruit treated with both 1-MCP and wax requir d over 11 days at 20C to reach firmness values of 20 N. The firmness of Booth 7' avocados treated with both 1-MCP and wax decreased from> 170 N to about 25 N over a 5-week period at l 3C. Inhibition of ethylene action with 1-MCP treatment and reducing water loss and modifying internal atmosphere with waxing during the early stages of climacteric produces changed in subsequent ripening behavior of avocado fruit.

PAGE 112

99 140 -------------------120 100 z :::::s "80 60 LL 40 20 A 0---------------------. 12 --------------------____._ Control (no 1-MCP & no wax) 10 _._ Wax only ---01-MCP only 8 0 V, V, 0 6 .... .c C') Q.) 4 3: 2 0 -61-MCP & wax 0 2 4 6 8 Storage period (days) B 10 12 Figur 5-1. Fruit firmness (N) and w ight loss(%) of 'T ow r II avocados treat d with 1-M P (0.9 L L1 for 12 hat 20C) and / or wax and stored at 20 Fruit wer tr at d w ith wax( _. ) 1-M P ( o ) both 1 -MCP and wax ( /1 ) or witho u t both 1-MCP and wax tr atm nt ( ). V rtical bar r pr nt standard de iation of 6 ind p nd nt samp l H ri z ntal bar r pr nt 1-M P tr atm nt.

PAGE 113

100 200 ---------------------,i 180 160 140 120 100 80 60 40 20 o~------------------... 10 ----------------------. 0 "' "' 0 8 6 4 2 0 0 ------Control (wax only) -o--1-MCP & wax 4 B 8 12 16 20 24 2 8 32 36 4 0 Storage period (days) Figure 5-2 Fruit firmness (N) and weight loss(%) of 'Booth 7 avocados stored at 13 after wax tr atment with ( o ) or without ( ) 1 MCP (0.9 L L1 for 12 h at 20 C). Vertica l bars represent standard deviation of 6 independent samples. Horizontal bar represents 1-MCP treatment.

PAGE 114

101 70 -eControl (no 1-MCP & no wax) 60 Waxonly -< >,, .... ..... 40 > C +i E 0 cu ..... 30 C) C) a. Q,) 0 H E 20 10 0 0 2 4 6 8 10 12 Storage period (days) igure 5-3. P activity of Tower II avocados treated with 1-M P (0.9 L L1 for 12 hat 20 and / r wax and stored at 20C. Fruit were treated with wax ( ) 1-MCP ( o ) both 1-M P and wax ( fl) or without both 1-MCP and wax treatment ( ). Vertical bars r pr nt tandard d v iation of 3 ind pendent samples. Horizontal bar repr ents 1-M P tr atm nl.

PAGE 115

102 30 --Control (wax only) 25 --0-1-MCP & wax &t) 0 T'9 20 >< >, .... I > C +:i E 15 CJ cu .... (!) C, a.. Cl) 0 10 E 5 0 4 8 12 16 20 24 28 32 36 40 Storage period (days) Figure 5-4. Effect of 1-MCP on PG activity of 'Booth 7 avocados stored at 13 C after wax treatment with ( o ) or without( ) 1 MCP (0.9 L L. 1 for 12 hat 20C). Vertical bar represent standard deviation of 3 independent samples. Horizontal bar represents 1-MCP treatment.

PAGE 116

CHAPTER 6 LUENCE OF ETHYLENE AND ITS ACTION INHIBITOR (1M T HYL YCLOPROPENE) ON SOFTENING RIPENING AND CELL WALL MA TRIX POLYSACCHARIDES OF AVOCADO FRUIT 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 of int re t since changes in cell wall composition and rigidity greatly affects fruit firmn ess uch organizational changes are an integral part of the endogenously controll e d fruit ripening process. th y l e ne plays a vital role in the ripening of climacteric fruits and whether appli e d e x o ge nously or produced naturally initiates ripening and softening. The importanc of thylene in regulating fruit ripening has been clearly demonstrated from s tudi es in v ol v ing suppressed thylene biosynthesis or action Th application of eth l n action inhibit r which bind r ceptors so that ethylene cannot licit ignal tran duction ( i 1 r a nd r k 1997 ; Yu ming and Jiarui 2000) has nabl d am r in-depth analy i f th r l a ti n hip b e twe n thyl n and fruit ripening 1 m th y lc y cloprop n e (1-M P) a synthetic cycloprop n has been shown to bl oc k th y n r ce ptor pr v nting thylene effects in plant ti u for xtended period 103

PAGE 117

104 (Si !er t al. 1996a ; i l r et al. 1996b; isler and erek, 1997). Although 1-M P binding t th thyl n r ceptor sites is irreversible it appears that new receptors can allo ntual r cov ry of the ripening process (Sisler et al. 1996a). 1-MCP 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 1-MCP and ethylene treatment during avocado fruit ripening and 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 neutral sugar composition. Hypothesis: Treatment of avocado fruit with 1-MCP will effectively protect fruit against exposure to exogenous ethylene. Materials and Methods Plant material 'Booth 7' a mid-season cultivar, and 'Monroe', a late-season cultivar were selected for this experiment. Both cultivars are "hybrid" crosses of West Indian and Guatemalan races (Hatton and Campbell 1960 ; Hatton et al., 1964). 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 ( Booth 7 446.8 36.8 g and Monroe' 575 48 g) and shape (diameter at equatorial region Booth 7 9.1 0.2 cm and 'Monroe' 9.0 0.3 cm), and then were surface sterilized in a 15% (90 mM NaOCl) commercial bleach solution, rinsed and dried.

PAGE 118

105 1-MCP treatment In the first experiment Monroe avocados ( 45 fruit) were placed in a chamber (174 L) and treated with flow-through air or 100 L L1 ethylene at a flow rate of 4000 mL min1 for 12 hat 20C (85% RH). CO 2 levels in the treatment chambers did not exceed 0.05%. Following ethylene treatment, half of the fruits were treated with 1-M P before transferring to 13C (85% RH). Twelve fruit were placed in 18-L containers and exposed to 1-MCP by releasing the gas from a commercial powdered formulation (Ethy block Floralife Burr Ridge IL). The concentration selected 4.5 L L1 was achieved through addition of 50 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. Total 1-MCP treatments were performed for 24 hat 20 and 85% relative humidity (RH). Immediately following treatment the fruit we re remov d from the treatment chambers and transferred to 13 C storage facilities. Control fruit (not exposed to C2H 4 and 1-MCP) were maintained under identical storage condition amples of fruit from each treatment were evaluated for fruit quality on a dail y ba i until they reached the full-ripe stage (firmness 10 to 20 N). Fruit quality wa assessed on th basis of fruit firmness weight loss peel color a w 11 as CO 2 and 2 H 4 production In th s cond experim nt Monroe avocados (12 fruit) w re placed in 18-L c ntain r and po d to 4.5 L L1 1-M P. Total 1-M P tr atm nt w r p rform d fi r 24 h t 2 0 and 85% r l ativ humidity (RH) ( on single tr atm nt wa for 6 h). Imm di t I [i II ing tr atm nt th fruit were remov d from th tr atm nt chamb r

PAGE 119

106 and then tran ferred to 13 C storage facilities (85% RH). Aft e r 14 d t ra g at 13 (85% RH) half of the 1-MCP-treated fruits were exposed to 100 L L 1 C 2 rLi for 24 h a t 20 and th n returned to l 3 C storage facilities (85% RH). Control fruit ( not e x po se d t o C 2 H 4 and 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). Fruit quality was assessed on the basis of fruit firmness weight loss and CO 2 and C2H4 production. In the third experiment 'Booth 7' avocados (12 fruit) were placed in 18-L containers and exposed to 1-MCP gas. The concentration selected 0.9 L 1 was achieved through addition of 10 mg of the powder to 100 ml ofFloraLife buffer. 1-MCP treatment was performed for 12 hat 20C and 85% relative humidity (RH). Immediately following 1-MCP treatment 1-MCP-treated fruit were removed from the treatment chambers and then transferred to 13 C storage facilities. After 19 d storage at 13 C (85% RH) 1-MCP-treated fruits were transferred to 20C (85% RH). Half of the 1-MCP treated fruits were treated with C 2 rLi (100 L L. 1 for 12 hat 20C) before transferring to 20C (85% RH). Control fruit (not exposed to 1-MCP) were stored for 12 d at 13C (85% RH) and then transferred to 20C. The time for transfer from 13 C to 20C was based on fruit attaining firmness values (whole fruit compression) of 75 to 90 N. 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). Fruit quality was assessed on the basis of fruit firmness weight loss C 2 H4 production and peel color. Mesocarp tissue derived from the equatorial region of selected fruit was stored at -30C and later used for analysis of cell wall enzymes and structural polysaccharides.

PAGE 120

107 Fruit firmne T o dif r nt methods (compression and puncture) were performed to measure avocado fruit firmness. In the compression test, firmness was determined on whole, unpeeled fruits 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 min1 The force was recorded at 2.5 mm deformation and was determined at two equidistant points on the equatorial region of each fruit. In the puncture test, firmness was determined on peeled mesocarp using an Instron Universial Testing Instrument fitted with a 10 mm diameter probe and 50 kg load cell. After establishing zero force contact between the probe and the peeled mesocarp the probe was driven with a crosshead speed of 50 mm min1 The force was recorded at 5 mm puncture and was determined at two equidistant points on the equatorial region of each fruit. The same four fruit of each treatment were measured every other day until they reached the full-ripe stage (10 to 20 Nin the compression test' 5 to 10 N in th puncture test) Re piration and ethylene evolution R sp iration and ethylene production were measured every other day using the same four fruit of each tr atm nt. Fruit were individually sealed for 30 min in 2-L plastic containers prior t ampling. A 0.5 mL gas sampl was withdrawn by syringe from each container thr u gh a rubb r ptum and carbon dioxide in the ample was d t rmined using a ow-Mac gas chromatograph ( eries 580 Bridg water NJ USA) quipped ith a th rmal conduct i v it y d t ct r T D) thyl n was mea ur d by inj cting a 1 0 mL ga

PAGE 121

108 sample from th containers into a HP 5890 gas chromatograph (H e wl tt Packard Avondale PA USA) equipped with a flame ionization detector (FID) Peel color Individual fruit were marked at the equatorial region (2 region s 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 met r was calibrated with a white standard tile. The color was reported as hue angle (H 0 ) with a value of 90 representing a totally yellow color and 180 a totally green color. The results are presented as lightness (L *), chroma (C*) and hue angle (H 0 ) The chroma and hue angle were calculated from the measured a* and b* values using the formulas C* = (a* 2 +b* 2 ) 1 1 2 and H 0 =arctangent (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 4C. 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 4 C. The pellets were transferred to 50 mL of ice-cold acetone for 10 min followed by centrifugation (7840 g, 10 min, 4 C). 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 (5C) and then centrifuged (7840 g 10 min 4 C). The pellets were transferred to 30 mL of 10 mM Na-acetate pH 6.0 containing 1 8 M NaCl for 30 min in ice-cold water bath and centrifuged. The supernatant was analyzed for enzyme activities as described below. Protein content as

PAGE 122

109 m a s ur e d according to the bicinchoninic method (Smith et al. 1985)) with bovine serum a lbumin u d a a standard Enzyme a a s Pol y galacturonase (PG E.C. 3 .2.1.15) activity was assayed reductometrically by incubatin g a 100 L aliquot of the cell-free protein extract with 500 L substrate of pol y galacturonic acid (4 mgs mL1 ) (from orange peel Sigma Chemical Co St. Louis MO USA) dissolved in 30 mM KO Ac, pH 5.5 containing 100 mM KCl. After 30 min at 34 C 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 p e r 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 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 L of the cell-free protein extract adjusted pH 7.5 and the decrease in A 620 a r cord d PM activity was xpressed as flA 6 2 o per mg prot in p r minute. x llula se ( ndo-1 4-~-glucanase ; .C. 3.2 1.4) activity was measured v i s com e tricall y A 100 L aliquot of the cell-free protein extract was added to 1.5 mL of a 2.5 % luti n of carboxymethylcellulose (CMC.7HSP Fisher cientific Co Fair Lawn N J A) in 40 mM NaOAc pH 5 0 with 0 02% NaN 3 and th mixtur wa incubat d at r om t mp ratur for 30 min Th timer quired for th olution t pass through a

PAGE 123

110 alibrat d p rtion of a 1-mL pip tte as record d. Activity was expr ssed as% change m i co ity p r mg protein per min. Alphaand beta-galactosidase activities were measured using modifications of the method of Pharr et al. (1976). p-NO 2 -phenyl aand ~-D-galactopyranosides (Sigma Chemical Co St. Louis MO USA) were used as substrates. Substrates were prepared at 2 mg mL1 in 0.1 M NaOAc pH 5.2. A 200 L aliquot of cell-free protein extract adjusted to pH 5 .2 was added to 200 L of substrate and the reaction mixture incubated at 37C for 15 min The release of p-NO 2 -phenol was measured spectrometrically at 400 nm The specific activity of each protein extract was expressed as moles of NO 2 -phenol equivalents released per minute per kg protein. NO 2 -phenol concentration was determined using free NO 2 -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 Lunzem 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 40C for 5 h and stored in a desiccator at room temperature.

PAGE 124

111 Pectin extraction and analysi Wat rand CDTA(1 2-cyclohexylenedinitrilotetraacetic acid) soluble pectins ere 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. Suspension was filtered under acuum through glass fiber filters (GF/C Whatman). Extractions were carried out at 34C for 4 hand quantified in terms of extractable uronic acid (UA) content. UA were determined b the hydroxydiphenyl assay (Blumenkrantz and Asboe-Hansen 1973) and expressed as g kg1 EIS. Total uronic acids in the EIS preparations were determined using the method of Ahmed and Labavitch (1977). Gel permeation chromatography of polyuronides The waterand CDT A-soluble uronic acids were concentrated using rotary evaporat ion to a final UA concentration of approximately 500 g mL1 Two-mL aliquots ( 1 mg UA equivalents) were passed through a Sepharose CL-2B-300 (Sigma C hemical 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 h1 and aliquots (0.5 ml) were used for the determination of UA content (Blumenkrantz and Asboe-Hansen 1973) The column void (Vo) and total (VT) vo lum es wer id ntified by the elution positions of Blue Dextran (2000 kDa.) and g luco se, respectively. The UA content in each column fraction was expr ss d as a p re ntage f th total U r cov r d. H micellulo c e traction H mic llul i olat d using th m thod of Hub r and N vins ( 1981) as m difi d by V tt n and Hub r (1990). Tor move pectin prior to h mic llulose xtracti n 200 mg f I w r incubat din 100 mL of 40 mM Na-phosphate pH 6.8 m

PAGE 125

112 a boilin g at r bath for 20 min. The suspension wa filt red through Miracloth and a h d ith 1 L of distill d water. The r si due s w r e filt red under vacuum through g las fib r filt r (GF /C Whatman) and washed with 200 mL of 100% acetone The phosphat -bu f r-treated EIS were dried in an oven at 40C for 5 h. For hemic e llulo se extraction 50 mg of buffer-treated EIS were incubated in 5 mL of 4 M KOH includin g 26 mM NaBH 4 for 12 hat room temperature. The suspension was filtered through glass fiber filters (GF / C Whatman). The solution was neutralized over ice with concentrated acetic acid and then dialyzed (molecular weight cut-off of 2000 Da .) overnight against running tap water followed by deionized water (2 x 4 L 12 h total ) Co-extracted acidic pol y mers were removed by passing each sample through a column (1.7 cm wide 18 cm high ) of D EAE -sephadex (Sigma) in 10 mM Na phosphate pH 6 8 containing 20 mM NaCl. The non-binding fraction contained the hemicelluloses and these were quantified by the phenol-sulfuric acid method (Dubois et al. 1956). Compositional analysis of pectins and hemicelluloses The neutral-sugar composition of waterand CDT Asoluble pectins and 4 M alkali-extractable hemicelluloses was analyzed by hydrolysis and alditol acetate derivati za tion (A lbersheim et al. 1967) An internal standard of 200 g m yo -inositol was a ir-dri e d w ith a pproximately 1 mg UA (pectins) or glucose equivalents (hemicelluloses) and h y droly ze d with 1 mL of 2 M trifluoroacetic acid for 1 hat l 20C. The sample was cooled and th e acid removed under a stream of filtered air. The hydrolyzed sugars were then reduced with 1 mL of 0 66 M NaBH 4 in IM N~OH overnight at room temperatur e. The samples were neutralized with Dowex-50W cation exchange resin filtered and dried The samples were washed with methanol (1 mL 3X) ethanol (1 mL) dried aft r each washing and then derivatized with 200 Leach of acetic anhydride and p ridin r

PAGE 126

113 1 hat 100 C. fter drying the alditol acetates were dissolved in methylene chloride and separated b gas chromatography (Hewlett Packard Model 5890 II Atlanta GA) on a capillar y column (25 mm X 0.33 m film thickness) of HP-5 (crosslinked 5% phrnol methol silicone) (Supelco Inc ., Bellafonte PA) at 250 C with flame ionization detection. Composite sugars were identified and quantified in relation to standard quantities of derivatized rh a mnose arabinose xylose mannose galactose and glucose 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 ANOV A 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 Monroe avocado fruit firmness in the first experiment are shown in Figure 6-1 A. E thylene-treated and control fruit softened rapidly and completed ripening (attained firmness val ues of 10 to 20 N) within 12 and 14 d respectively of storage at 13 C. Treatm nt at 4.5 L1 1-MCP for 24 hat 20C following ethylene treatment (100 L-L 1 for 24 hat 20 C) had a significant effect on fruit firmness relative to control and th y l n -tr at d fruit. Firmness decrease of fruit treated with 1-MCP was significantly d lay e d and 1-MCP-tr ated fruit did not reach the full-ripe stag (10 to 20 N) during the storag p riod due to decay incidence After 24 days of storag at l 3C fruit treated with 1-M P ex hibit d firmn ss valu of 39 1 15 5 N.

PAGE 127

114 Changes in Monroe avocado fruit weight loss in the first exp riment are hown in Figure 6-lB. After 12 or 14 d at which time fruit were fully ripe weig ht lo ss of control and ethylene-treated fruit ranged between 7.1 and 7 .6 %, resp ctively whereas fruit treat d with 1-MCP at 4.5 L-L1 for 24 hat 20C showed weight loss values of 11 % after 24 days of storage. Final weight loss values of fruit treated with 1-MCP were significantly higher than those for control and ethylene-treated fruit. Changes in Monroe' avocado fruit firmness in the second experiment are shown in Figure 6-2A. Control fruit softened rapidly and completed ripening (10 to 20 N) within 14 d of storage at 13C. Treatment with 4.5 1 1-MCP for 24 hat 20C had a significant effect on fruit firmness relative to control fruit. The rate of fruit softening was significantly reduced and 1-MCP-treated fruit did not reach the full-ripe stage ( 10 to 20 N) during the storage period. After 22 d of storage at l 3C, fruit treated with 1-MCP exhibited firmness values of 44.6 4.6 N. Treatment of fruit with 100 1 ethylene for 24 hat 20C following the initial 13 d storage at l 3C did not significantly influence the firmness properties of the 1-MCP-treated fruit. Fruit treated with both 1-MCP (prior to storage) and ethylene (after 13 d at 13C) also did not reach the full-ripe stage during the storage period. Firmness values of 1-MCPand ethylene-treated fruit were 42.8 8.3 N after 22 d of storage. Changes in Monroe' avocado fruit weight loss in the second experiment are shown in Figure 6-2B. After 14 d at 13C weight loss of control fruit was 7.1% and that of 1-MCP-treated fruit was about 6.2%. 1-MCP-treated fruit showed weight loss alues of 10.0 % after 22 d at l 3C. No significant differences in the magnitude of weight loss

PAGE 128

115 (9.4 % ) er noted b tween 1-MCP-treated fruit and 1-MCP-treated fruit provided ith an int r ning 24 h 100 1 ethylene treatment. hanges in Booth 7 avocado fruit firmness in the third experiment are shown in Figure 6-3. In the experiment with Booth, both compression and puncture tests were performed to measure fruit firmness. In the compression test, control fruit softened rapidly and completed ripening (10 to 20 N) within 12 d of storage at l 3C followed by 4 d at 20C (Fig. 6-3A). The time for transfer from 13C to 20C was based on fruit attaining firmness val ues of75 to 90 N. Treatment at 0.9 1 1-MCP for 12 hat 20C had a significant effect on fruit firmness relative to control fruit. Firmness decrease of fruit treated with the 1-MCP was significantly delayed and reached the full-ripe stage (10 to 20 N) after about 18 d of storage at l 3C followed by 10 d at 20C. Treatment with 100 L'L1 ethylene for 12 hat 20C following the initial 18 days storage at 13C did not significantly affect the firmness of 1-MCP-treated fruit. Final firmness values of the 1MCP-treated fruit with or without ethylene were 13.1 and 12.5 N, respectively. Firmness of Booth 7" avocado fruit determined via mesocarp puncture analysis is shown in Fig. 63B All fruits showed a significant decrease in firmness values after fruits ere tran fi rred to 20 C. Changes in the kinetics of firmness decline as affected by transfer to 20 C w r much more evident in the puncture analysis. However th trends of all treatm nt w re similar to those evident in compression analysis W ight loss tr nds for Booth 7' avocado fruit in the third experiment ar shown m igur 6-4 After 16 d at l 3C w ight loss value of control fruit wa 8.3% wher a that of fruit tr at d with 1-M Pat 0.9 L'L1 for 12 hat 20 a about 5.2%. 1-M Ptr at e d fruit how d w ight lo ss value of 14 9 % after 28 d of torag Although

PAGE 129

116 treatment at 100 L-L1 ethylene for 12 hat 20C following the initial 18 d stora ge at l 3C did not significantly affi ct the magnitude and rat of weight lo ss of the 1-M treated fruit 1-MCP-treated fruit with ethylene treatment had a lower weight los valu e (12.1 %) than those without ethylene treatment at the full-ripe stage (10 to 20 N) All fruits showed a significant increase of weight loss values after transfer to 20 C. Respiration and ethylene evolution The CO 2 and ethylene production of 'Monroe' avocado fruit in the first experiment are shown in Figure 6-5. The CO 2 production in control fruit and fruit treated with ethylene at 100 1 for 24 hat 20C showed characteristic climacteric patterns during storage at l 3C (Fig. 6-5A) Respiration in control fruit and ethylene-treated fruit began to increase before storage at l 3C and CO 2 production reached maxima of 78.2 and 133.3 mg kt 1 h1 respectively, after 5 to 9 d storage at 13C (Fig. 6-5A). The application of 1-MCP at 4.5 1 for 24 hat 20C significantly suppressed the magnitude of CO 2 production during storage at 13C. The CO 2 production of 1-MCP treated fruit decreased after ethylene and 1-MCP treatment and a clear respiratory peak did not occur during storage at 13C (Fig. 6-5A). Ethylene production in control fruit and fruit treated with ethylene at 100 L-L1 for 24 hat 20C showed characteristic ethylene climacteric patterns during storage at l 3C (Fig 6-5B). Ethylene production in control fruit began to increase after 2 d in storage and reached maximum values of 93.4 L kg1 h1 after 5 d storage at 13C whereas ethylene production in ethylene-treated fruit began to increase soon after ethylen treatment and reached maximum values of 81 3 L kt 1 h1 after 2 d storag at 13 thylene production trends in fruit treated with 1-MCP at 4.5 L1 for 24 hat 20 were atypical. A distinct p ak of ethylene production did not occur during the

PAGE 130

117 storage p riod and maximum ethylene production rates were reduced o er 90 % compared ith all other treatments (Fig. 6-5B). The CO 2 and ethylene productions of Monroe avocado fruit in the second experiment are shown in Figure 6-6. The CO 2 production in control fruit showed characteri tic climacteric patterns during storage at l 3C (Fig. 6-6A). Respiration in control fruit began to increase before storage at 13 C and CO2 production reached maxima of 78.2 mg kg1 h1 after 9 d storage at l 3C (F ig 6-6A). The application of 1MCP at 4.5 L1 for 24 hat 20C significantly suppressed the magnitude of CO 2 production. The CO 2 production of the 1-MCP-treated fruit decreased after 1-MCP treatment and a clear respiratory peak did not occur during storage at l 3C (Fig. 6-6A). Treatment at 100 1 ethylene for 24 hat 20C following the initial 13 d storage at l 3C did not significantly influence on CO 2 production of the 1-MCP-treated fruit. Fruit treated with both 1-MCP (prior to storage) and ethylene (after 13 d storage at 13C) also did not show peak respiratory activity during storage at 13C (Fig. 6-6A). Ethylene production in control fruit showed characteristic ethylene climacteric patterns during storage at l 3C (Fig. 6-6B). Ethylene production in control fruit began to increase after 2 din storage and reached maximum values of 93.4 L kg1 h1 after 5 d storage at 13 whereas ethylene production trends in fruit treated with 1-MCP at 4.5 L-L 1 for 24 hat 20C did not show a distinct peak during the storag p riod and maximum thyl n production rates were r duced over 94 % compar d with control fruit ( i g. 6-6B). thyl n tr atm nt following the initial 13 d storag at 13 also did not ignificantly influ nc on thylene production of the 1-MCP-tr at d fruit and did not s how a di tinct peak of thylene production during torage at 13 (Fig. 6-6B)

PAGE 131

118 Th thyl ne production of 'Boot h 7' avocado fruit in the third experiment is sho n in Figur 6-7. Ethylene production in control fruit and fruit treated with 1-MCP at 0 .9 L-L1 for 12 hat 20C showed characteristic ethylene climacteric patterns during storage (Fig. 67). Ethylene production in control fruit began to increase after 12 d in storage at 13C and reached maximum values of 163.2 L kt 1 h1 2 dafter transfer to 20C. 1-MCP-treated fruit showed a delayed climacteric pattern and maximum ethylene production rates were increased over 35% compared with control fruit (Fig. 6-7). Ethylene production in 1-MCP-treated fruit began to increase after 18 d of storage at 13C followed by 4 d at 20C and reached maximum values of 256.8 L kg1 h1 after 4 additional days at 20C. Treatment at 100 1 ethylene for 12 hat 20C following the initial 18 d storage at 13C did not significantly affect ethylene production of 1-MCP treated fruit. Ethylene production in the 1-MCP-treated fruit subsequently treated with ethylene also began to increase after 18 d of storage at l 3C followed by 4 d at 20C. Maximum values of 244.0 L kg1 h1 were observed after 4 more days storage at 20C (Fig. 6-7). Peel color The pe I color of Booth 7 avocado fruit in the third experiment is shown in Table 6-1. The peel of avocado fruit prior to storage had a moderate green color (hue angle= 128.8 where pure yellow= 90 and pure green= 180). At the full-ripe stage, there were significant differences in the chroma value (C) and hue angle of the peel color between control and 1-MCP-treated fruit (Table 6-1 ). Changes in hue angle constituted the major alt ration of color coordinates of fruit. The decline in hu angle represented th change from green to yellow and the increase in chroma value reflect d increasing inten ity of y llow color. Treatment with 100 L L1 ethylene for 12 hat 20 following

PAGE 132

119 the initial 18 d storage at l 3C did not significantly effect peel color of the 1 MCP-treated fruit. 1-MCP-treated fruit treated with or without ethylene had a lower chroma value and a higher hue angle than control fruit (Table 6-1 ). These data indicate that the peel of 1-MCP-treated a ocado fruit retained moderate green color with low color intensity (light) during storage. Table 6-1. Pe e l color of Booth 7' avocados stored at 13 C after 1-MCP treatment (1 L L1 for 12 hat 20C) and then transferred to 20C. Some fruit were treated with C 2 ~ (100 L L1 for 12 hat 20C) before transfer to 20C. Control fruit were stored at 13C and then transferred to 20C. Peel color was measured at the full-ripe stage (compression firmness from 10 to 20 N). 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 38.1, 20.1 and 128.8 respectively. Treatments Days to fully ripe L* Chroma Hue angle Control (no 1-MCP) 16 44.5 a 33.1 a 120 .3 b MCP (1 l r 1 for 12 h) 28 40.4 a 26.6 b 124 5 a MCP (1 l r 1 for 12 h) & 28 38.9 a 25.1 b 124.6 a 2 H4 ( 100 l r 1 for 12 h) Enzyme activity Booth 7' avocado fruits treated with 0.9 L L1 1-MCP for 24 hat 20C in the third experiment were employed in experiments addressing the effects of ethylene and it antagonist on s 1 cted cell wall enzymes and polysaccharide metaboli m. The activitie of cell wall nzymes in cell-free extracts of avocado fruit treated with 1-MCP at 0.9 L L1 for 24 hat 20C are shown in Figures 6-8 and 6-9. The activity of polygalacturona (P ) wa ry low in fr s hly harv st d preclimacteric fruit incr as d during th c lim act ric p ri d and continu d to increas during th postclimacteric phase to level 9 tim hi g h r in fruit at the full-ripe stag (10 to 20 N) than 1 1 at harvest (Fig. 6A Thi patt e rn of P accum ulation in Booth 7 avocado fruit is con i t nt with

PAGE 133

120 hown for 'Fuerte' avocado (Awad and Young 1979 Zauberman and chiffmann-Nad I 1972) PG activity in 1-MCP-treated fruit during the preclimact e ric period r main d at levels comparable to those detected at harvest increased during the climacteric p riod and continued to increase during the postclimacteric phase to levels 3 .9 time high r in fruit at the full-ripe stage (10 to 20 N) than levels at harvest (Fig 68A). Treatment at 100 L L1 ethylene for 12 hat 20C following the initial 18 d storage at 13C significantly effected PG accumulation of the 1-MCP-treated fruit. PG accumulation in 1-MCP-treated fruit with ethylene treatment increased during the climacteric period and continued to increase during the postclimacteric phase to levels 9.2 times higher in fruit at the full-ripe stage than levels at harvest (Fig. 6-8A). PME activity in control fruit declined from a maximum value at harvest to a low level at the full-ripe stage (day 16 Fig. 6-8B). The trend for 1-MCP treated fruit paralleled that for control fruit although the decline in activity was significantly delayed. The levels of PME in 1-MCP treated fruit after 28 d were similar to those noted for control fruit after 16 d (Fig. 6-8B). Ethylene treatment following the initial 18 d storage at l 3C did not significantly influence PME activity of the 1-MCP-treated fruit (Fig. 68B) C -cellulase activity was not detectable in fruit measured within 24 h of harvest (Fig. 6 9A). C -cellulase levels of control fruit increased significantly after day 6 reaching levels 17 6-fold higher than those noted at day 6 within 10 d C x -cellulase in fruit treated with 1-MCP was not detectable until day 24. C x -cellulase levels increased significantly after day 24, reaching the highest level within 4 d. Ethylene treatment following the initial 18 d storage at l 3C had a significant effect on C x -cellula acti it

PAGE 134

121 of 1-M P-tr ated fruit (Fig. 6-9A). After ethylene treatment Cx-cellulase levels increased ignificantly reaching the highest level within 8 d. Alpha-galactosidase activity of control fruit decreased significantly during storage and reached a minimum (2.4 mole kt 1 min1 x l 0-4) at the full-ripe stage (Day 16 Fig. 69B). a-galactosidase activity in the 1-MCP-treated fruit during the preclimacteric period remained at levels comparable to those detected at harvest decreased during the climacteric period continued to decrease during the postclimacteric phase and reached a minimum (2 5 mole kg1 min1 x 10-4) at the full-ripe stage (Day 28 Fig. 6-9B). Ethylene treatment following the initial 18 d storage at l 3C did not have a significant effect on galactosidase activity of the 1-MCP-treated fruit (Fig. 6-9B). Beta-galactosidase activity of control fruit declined from a maximum value at harvest to a low level at the full-ripe stage (Day 16, Fig. 6-9C). ~-galactosidase activity of control fruit during the preclimacteric period remained at levels comparable to those detected at harvest decreased during the climacteric period continued to decrease during the postclimacteric phase and reached a minimum (7.4 mole kg1 min1 x 10-4) at the full ripe stage (Day 16 Fig. 6-9C). ~-galactosidase activity in the 1-MCP-treated fruit during the pr climacteric period also remained at levels comparable to those detected at harvest decreas d during the climacteric period, continued to decrease during the postclimact ric phas and r ached a minimum (7.3 mole kt 1 min1 x 10-4) at the full-ripe stag (Day 28 Fig. 6-9 ) thyl ne tr atm nt following the initial 18 days torag at 13 did hav a ignificant fD ct on ~-galacto idas activity of 1-MCP-tr ated fruit (Fig. 6-9C). ft r th y I n tr atm nt ~-galacto idase l vels decreased significantly r aching a minimum I v I 7.4 m I k g1 min 1 x 10 4 ) b for the climact ric period. Th reafter activity in 1

PAGE 135

122 MCP-tr ated fruit tr at d with thylene remained con tant through th r mainin g storag period at 20C. Solubility and molecular ma of avocado polyuronides P ctin solubilization wa markedly alt red during ripening and in respon to treatm nt with 1-MCP. Figure 6-10 illustrates changes in waterand 50 mM CDTA solubl UA content, total UA content and EIS levels for both control fruit and fruit treated with 0.9 L L1 1-MCP for 12 hat 20C. By the full-ripe stage in control fruit ( 16 d) total UA in EIS had declined by nearly 19% whereas total UA levels in 1-MCP treated fruit with or without ethylene treatment declined about 16% throughout 28 d storage (Figure 6-1 OA). Treatment with 100 L L1 ethylene for 12 hat 20C following the initial 18 d storage at l 3C did not significantly affect total UA levels of 1-MCP treated fruit. Water-soluble UA in control fruit increased significantly after 6 d at l 3C and reached levels 1.7-fold higher than those at day O (Fig. 6-lOB). The levels of CDTA soluble UA, though much lower than water-soluble levels decreased significantly after 6 d at l 3C (Fig. 6-1 OC) In the 1-MCP treated fruit water-soluble UA increased significantly after 18 d at l 3C and reached levels 1.8-fold higher than those at day 0 (Fig. 6-1 OB) whereas the levels of CDT A-soluble UA slightly increased during 18 d of storage at l 3C and then decreased significantly during 10 d of storage at 20C (Fig. 61 OC). E th y lene treatment did not significantly affect waterand CDT A-soluble UA le els of the 1-MCP-treated fruit. There were no statistical differences in the changes in the EIS levels of control and 1-MCP-treated fruit throughout the storage (Fig. 6-1 OD). By the full-ripe stage in control fruit (16 d) EIS levels in mesocarp tissues had declined by nearly 3% relative to the level at day O (Fig. 6-1 OD). In contrast EIS l vels in 1-M P treated fruit with or without ethylene treatment remained constant throughout 18 d of

PAGE 136

123 storage at 13 and increased by nearly 8 and 13% respectively within 10 d at 20C relati to I ls at day O (Fig. 6-1 OD). G 1 p rmeation profiles of water-soluble polyuronides from control and 1-MCP treated a ocado are shown in Figure 6-11. At the pre-ripe stage (Day 0) water soluble polyuronides representing approximately 33% of total EIS UA eluted as a polydisperse population. As ripening proceeded (Day 6 through 16 ; Fig. 6-11 A) water-soluble polyuronides of control fruit at each developmental stage eluted as one polydisperse peak (Day 6 elution peak= 50 mL ; Day 12 elution peak= 50 mL; and Day 16 elution peak = 58 mL) and 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 were delayed in 1-MCP-treated fruit (Day 9 elution peak = 52 mL; Day 18 elution peak= 52 mL; Day 24 elution peak= 50 mL and Da 28 elution peak= 56 mL) (Fig. 6-11B). 1-MCP-treated at the full-ripe stage (Day 28 Fig. 6-11 B) had slightly less breakdown of water-soluble polyuronides ( elution peak = 56 mL) compared with control fruit (elution peak= 58 mL) at the same developmental tage (day 16 Fig. 6-1 lA). As evident from Figure 6-10 (A B) the levels of water -solubl e pol y uronid s in 1-MCP-treated fruit represented a proportionally similar percentage ( 70 %) of total E I UA com par d with the 71 % for control fruit. Ethylene treatment follo w in g th initial 18 d storag at l 3C significantly affected th polyuronide profil of 1-M P-tr at d fruit at day 24 (Fig 6-11 C); however gel filtration profiles from full-rip 1-M P-tr e at d fruit with ethyl n tr atment (Day 28 Fig 6-11 ) w r n arly indi s tin g ui h a bl from those from full-rip 1-MCP-treat d fruit without ethylen tr a tm e nt D ay 2 8 i g. 6-11 B)

PAGE 137

124 Th m I cular ma di tribution and down hift of D T A-soluble pol y uronid parallel d tho vid nt for wat r-solubl polymers (Fig. 6-12). In control fruit DTAsoluble polyuronides at each de elopmental stage eluted as one polydisperse peak ( D ay 6 elution peak = 50 mL Day 12 elution peak= 50 mL ; and Day 16 elution peak = 56 mL ) and x hibited a gradual molecular mass downshift during ripening (Fig. 6-l 2A) Mol cular m a downshifts were significantly delayed in 1-MCP treated fruit (Day 9 elution p ak = 48 mL ; Day 18 elution peak= 48 mL; Day 24 elution peak= 48 mL ; and Day 28 elution peak= 54 mL) (Fig. 12 B). 1-MCP treated fruit with the peak (elution peak= 54 mL) at the full ripe stage (Day 28, Fig. 6-12B) had limited breakdown of CDT A-soluble polyuronides compared with control fruit with the peak ( elution peak = 56 mL) at the same stage (Day 16 Fig. 6-12A). Ethylene treatment following the initial 18 d storage at l 3C significantly affected the profile of 1 MCP treated fruit (Day 24 elution peak = 52 mL and Day 28 elution peak= 56 mL) (Fig. 6-12C) 1-MCP-treated fruit treated with ethylene at the full-ripe stage (Day 28 Fig. 6-l 2C) had more breakdown of COTA soluble polyuronides (elution peak= 56 mL) compared with 1-MCP-treated fruit without ethylene treatment (elution peak= 54 mL) at the same stage (Day 28 Fig. 612B) and gel filtration profiles of fully ripe 1 MCP-treated fruit with ethylene treatment ( Day 28 Fig. 6-l 2C) were nearly indistinguishable from those of full-ripe control fruit (Day 16 Fig 6 l 2A). Extensive downshifts were evident for ripe control fruit (Day 16 Fig. 6-l 2A) and slightly less pronounced for ripe 1-MCP treated fruit with or ithout ethylene treatment (Day 28, Fig. 6-12 C D). Proportionally how v r CDTA-solubl polyuronid were a relatively minor component of avocado polyuronides representin g

PAGE 138

125 approximate! 6.8% (control) 8.3% (1 MCP treated) or 7.8% (1 -M CP and ethylene treat d) of total EI UA le els at the full -rip e stage. e utral u gar composition of avocado polyuronides and hemicellulo ses N utral ugar analysis of water -soluble pol yuronides from control and 1 MCP-tr ated avocado are shown in Tables 6-2 and 6-3. In control fruit neutral sugar composition significantly changed during fruit ripening (Tab l e 6-2). Table 6-2. Sugar composition of water-solu ble UA from EIS prepared from Booth 7' avocado stored at 13 C for 12 days and then transferred to 20C. Dat a are means standard deviation of 3 replications. Neutral sugar composition (mo l e%) NS/UA Stage (mole Rha Ara Xyl Man Glu Gal ratio) Before 9.8 26.0 21.9 10 .7 6.5 25.1 0.41 storage 3.6 1.2 1.7 0.7 0.7 3. 1 6 days 9.5 26.6 25.6 9.2 7 .8 21.3 0.48 at 13 c 1.1 1.9 6.2 1.7 3.0 2.2 12 days 11.4 23.4 22.5 10.4 9.1 23.3 0.48 at 13 c 0.5 3.6 1.0 2.2 2.7 0.3 12 days at 13 c 9.3 59 6 8 7 3.0 4 5 14 .9 0.97 + 4 days 1.5 1.1 1.8 0.1 1.1 0.3 at 20 Rha rhamnose Ara, arabinose; Xyl xylose; Man mannose G lu glucose; Gal galacto y Mol rati of total neutral sugar (mole) and total uronic acids amo u nt (mole) h maj r neutral u gars were arabinose xy l ose and galactos coll cti e l y compri ing 73% and 83% of th neutral sugar in water solub l e polyuronid s of fruit at harv st and at th full-rip tage (10 to 20 N) resp ctively. During torag there was an ov rail l (ba d on th mol ratio of tota l neutral sugars/uronic acid ) of rhamnose xy l o mann g luco e and ga l actos whereas th proportion of arabino

PAGE 139

126 significantly increased at the full-ripe stage. The major neutral sugar s of polyuronide s from 1-MCP-treated fruit were also arabinose xylose and galactose collectively comprising 73% and 84% of the neutral sugars in water-soluble polyuronides of fruit at harvest and at the full-ripe stage respectively (Table 6-3). Table 6-3. Sugar composition of water-soluble UA released from EIS prepared from Booth 7' avocado treated with 1-MCP or 1-MCP & C21Li. Fruit were treated with 1-MCP (0.09 L L1 for 12 h) and stored at l 3C for 18 d and then transferred to 20 C Half of the 1-MCP treated fruit were exposed to C 2 H 4 (100 L L1 for 12 hat 20 C) before transfer to 20 C. Data are means standard deviation of 3 replications Sugar composition (mole%) NS/UA Stage (mole Rha Ara Xyl Man Glu Gal ratio) Y Before storage 9.8 26.0 21.9 10.7 6.5 25.1 0.41 3.6 1.2 1.7 0.7 0.7 3.1 9 days at l 3 C 11.9 25.9 20.0 8.8 10.2 23.2 0.48 0.8 4.1 2.1 2.2 1.1 3.7 18 days at 13 C 11.7 25.6 21.1 9.1 7.8 24.6 0.50 0.3 4.5 0.1 0.6 1.4 2.1 18daysatl3 C 11.9 28.9 21.1 6.6 5.9 25.7 0.37 + 6 days at 20 C 3.3 .4 0.5 0.3 1.1 2.8 18daysatl3 C 8.2 53.5 9.2 3.8 4.6 20.8 0.82 + 10 days at 0.9 0.5 0.1 0.1 0.8 2.2 20 C 18 days at 13 C 11.8 26 3 21.6 8.8 7.7 23.7 0.50 + 12 h C 2 H 4 + 6 0.2 0.2 0.4 0.2 0.3 1.3 days at 20 C 18 days at 13 C 8.8 51.5 9.7 3.5 5.4 21.0 0.90 + 12 h C 2 H 4 + I 0 0.1 0.3 0.2 0.3 0.3 0.4 days at 20 C Rha rhamnose Ara arabinose Xyl xylose Man mannose ; Glu gluco Gal galactose y Mole ratio of total neutral sugar (mole) and total uronic acids amount (mol )

PAGE 140

127 There as an overall loss of rhamnose xylose mannose glucose and galactose during the storage of 1-MCP-treated fruit. The proportion of galactose in 1-MCP-treated fruit did not change relative to the significant proportional decrease noted for control fruit at the full-ripe stage. As in control fruit the proportion of arabinose in 1-MCP treated fruit significantly increased at the full-ripe stage.Ethylene treatment following the initial 18 d of storage at l 3C did not significantly influence the neutral sugar composition of the water soluble polyuronides of 1-MCP-treated fruit (Table 6-3). Neutral sugar analysis of CDT A soluble polyuronides from control and 1 MCP treated avocado are shown in Tables 6 4 and 6 5. In control fruit neutral sugar composition significantly changed during fruit ripening (Table 6 4). Table 6 4 Sugar composition of CDT A-soluble UA re l eased from EIS prepared from Booth 7' avocado stored at 13 C for 12 d and then transferred to 20 C Data are means standard deviation of 3 replications. Neutral sugar composition (mole%) NS /UA Stage (mole Rha Ara Xyl Man Glu Gal ratio) Y Before 6.9 24.7 17 8 4.2 30.1 16.3 0.79 storage 3.5 3.9 12.4 1.8 10.5 0.4 6 days 9.2 26.1 12.6 5.0 29.0 18.2 0.56 at 13 C 8.7 8.7 1.0 0.5 0.4 9.6 12 days 12.4 22.2 10.0 5 6 32.1 17 7 0 75 at 13 C 2.3 6 1 2.9 0 .3 6.7 12 5 1 2 days at 13 5 9 59.3 3.2 3.3 11.2 17 1 1.34 0.3 12.0 0.2 0.7 2.2 9.2 rhamn s Ara arabinose Xyl xylose Man mannose Glu glucose Gal g a l a t Mol rati f total n utral ugar (mole) and total uronic acids amount (mole)

PAGE 141

128 The major neutral sugar were arabinose xylase glucos and galactose coll cti el c mpri ing 88% and 91 % of then utral sugar in CDT As oluble polyuronid of fruit at harve t and at the full-ripe stage (10 to 20 N) r spectiv ly. During storage th re was an ov rall loss of rhamnose xylose, mannose glucose and galactose. The proportion of arabinose significantly increased at the full-ripe stage. The major neutral sugars of 1-MCP-treated fruit were also arabinose xylase glucose and galactose collectively comprising 73% and 88% of the neutral sugar in CDT A-soluble polyuronides of fruit at harvest and at the full-ripe stage, respectively (Tab l e 6-5). The overall quantities of xylose and mannose decreased during the storage whereas those of arabinose increased. The overall quantities of rhamnose, glucose, and galactose at the full-ripe stage remained relatively constant. Ethylene treatment following the initial 18 d storage at l 3C had a significant effect on the neutral sugar composition of CDT A-soluble polyuronides of 1-MCP-treated fruit (Table 6-5). The quantities of arabinose and rhamnose in 1-MCP-treated fruit with ethylene treatment at the full-ripe stage further increased, whereas those of xylose, mannose, glucose and galactose further decreased relative to those of 1-MCP-treated fruit without ethylene treatment. Neutral sugar analysis of 4 M alkali-soluble hemicellulose from control and 1-MCP-treated avocado are shown in Tables 6-6 and 6-7. In control fruit the major neutral sugars were xylose, glucose, and galactose, collectively comprising 76% and 74% of the neutral sugar in 4 M alkali-soluble hemicellulose of fruit at harvest and at the full ripe stage, respectively (Table 6-6). The quantities (mole% basis) of rhamnose arabinose, and xylose slightly increased during ripening whereas mannose glucos and

PAGE 142

129 galactos decreased. The quantities of rhamnose arabinose and xylose slightly increased during ripening whereas mannose glucose and galactose decreased. Table 6-5. Su g ar composition of CDT A-soluble UA from EIS prepared from Booth 7 a ocado treated with 1-MCP or 1-MCP & C 2 H 4 Fruit were treated with 1-MCP (0.09 L L1 for 12 h) and stored at 13C for 18 d and then transferred to 20 C Half of the 1-MCP treated fruit w re exposed to C 2 ~ (100 L L1 for 12 hat 20C) before transfer to 20 C. Data are means standard deviation of 3 replications. Sugar composition (mole%) Stag e Rha Ara Xyl Man Glu Gal Before storage 6.9 24.7 17.8 4.2 30.1 16.3 3 5 3.9 12.4 1.8 10.5 0.4 9 days at 13 C 18 days at 13 C 6.7 5.2 8.9 6.5 18 days at 13 C 10.8 + 6 days at 20 C 1. 1 18 da y s at 13 C 8.5 + 10 days at 2 6 2o c 18da ysa tl3 C 10 6 + 12 h 2 H 4 + 6 1.3 d ays at 2 0 18daysatl3 C 10 9 + 12hC 2 H 4 + 10 1.5 days at 20 C 23.5 9.4 19.0 5.9 18.8 0.1 34.2 16.2 21.9 5 3 45.3 12.9 8.0 2.1 6 9 0.4 7 8 1.9 5.0 1.8 8.2 .4 3.8 1.1 5.3 1.7 4.7 0.3 3.7 0.2 3.8 0.2 6.0 0.3 2.5 0.8 36.3 9.9 41.3 20.2 8 3 19.3 2 8 10.3 45.0 5.8 31.7 18.6 36.0 .4 22.4 7.2 13 9 .4 16 9 6.7 17.3 5.3 15.2 7.4 NS/UA (mole ratio) v 0.79 0.64 0.50 0 65 0 90 0 55 0.93 Rha rhamnose ; Ara arabinose; Xyl xylose Man mannose Glu glucose Gal ga lacto se v Mol ratio of total n utral sugar (mole) and total uronic acid amount (mole)

PAGE 143

130 Table 6-6. Sugar composition of 4 M alkali-soluble h em icellulo se from EI prepared from Booth 7 avocado stored at 13 C for 12 days and th en tran sferred to 20 Data are mean s s t a ndard d ev iation of 3 replication s. Sugar composition (mole%) tag Rha Ara Xyl Man Glu Ga l B fo re 2 .5 10.9 35.4 10 7 25.3 15.3 torag e 0 1 3.7 3.3 0 8 2 6 1.4 6 days at 3.2 13.2 34.1 11.0 22 9 15.6 13 c 2.1 0.4 0.1 1.5 5.5 1.7 12 days at 3.8 13.8 35.8 11.3 19.1 16 3 13 c 0.6 0.3 2.1 0.2 1.2 0.1 12 days at 13 c + 4 3.4 14.2 36.8 8.9 22.3 14 .5 da ys at 3.7 1.1 2 7 2.4 1.7 0.6 2 o c Rha rhamnose ; Ara, arabinose ; Xyl xylose; Man mannose ; Glu glucose; Gal galactose The major neutral sugar of 1-MCP-treated fruit were also xylose, glucose and galactose collectively comprising 76% and 75% of the neutral sugar in 4 M alkali soluble hemicellulose of fruit at harvest and at the full-ripe stage respectively (Table 6-7). There were an overall loss of mannose and an increase in rharnnose ; however the quantities (mole% basis) of arabinose xylose, glucose and galactose did not change s ignificantl y during storage. Ethylene treatment following the initial 18 d storage at l 3C did not significantly affect the neutral sugar composition of the 4 M alkali-soluble hemicellulos e of 1-MCP-treated fruit (Table 6-7).

PAGE 144

131 Tabl 6-7 ugar composition of 4 M alkali-soluble hemicellulose from EIS prepared from Booth 7 a ocado treated with 1-MCP or 1-MCP & C 2 ~. Fruit were treated with 1-MCP (0.09 L L1 for 12 h) and stored at 13C for 18 d and then transferred to 20C Half of the 1-MCP treated fruit were exposed to C 2 H 4 (100 L L1 for 12 hat 20C) before trans r to 20 C. Data are means standard deviation of 3 replications. Sugar composition (mole%) tage Rha Ara Xyl Man Glu Gal Before storage 2.5 0.1 10.9 3.7 35.4 3.3 10.7 0.8 25.3 2.6 15.3 1 .4 9 days at 13 c 1.3 0.4 13.8 1.9 36.2 2.0 10.1 o.5 22.6 6.3 16.1 1.5 18 days at 13 c 1.2 1.0 13.8 2.9 35.7 3.2 9.2 1.4 23.9.0 16.2 2.6 18 days at 13 c + 6 days at 20 4.5 0.4 10 .0 4.0 32.4 3.8 11 .4 1.8 27.8 2.5 13.9 0.2 c 18 days at 13 c + 10 days at 20 6.9 7.3 11.2 4.0 37.4 3.0 6.4 0.3 23.6 9.1 14.6 0 6 c 18 days at 13 c + 12 h C 2 H 4 + 6 2.6 1.2 days at 20 0 18 days at 13 + 12 9 9 3.3 33.6 1 .4 9.5 0.2 29.7 2.2 14.8 1.3 h 2 H4 + 6.7 6.9 11.6 4 6 37.4 1.0 7.2 1.5 23.3 4.2 13 8 0 5 10 days at 20 Rha rhamno e Ara arabinose Xyl xylose Man mannose Glu glucose Gal galactos

PAGE 145

132 Di cussion In th present study several parameters (firmness weight lo ss r e spiration and C 2 H 4 production peel color selected cell wall enzymes activities structural carbohydrate and neutral sugar composition) were examined to determine the effect of ethylene and 1-MCP in regulating avocado fruit ripening. Avocado firmness wa s ignificantly r tained in response to 1-MCP treatment consistent with the fact that softening is one of the most sensitive ripening processes to ethylene (Lelievre et al. 1997a). Significantly delayed softening by 1-MCP in the present study substantiates that ethylene is involved in augmenting the activity of softening-related enzymes and metabolism Similar effects of 1-MCP in attenuating fruit softening have been observed for Hass avocado (Feng et al. 2000) apricot (Fan et al. 2000) and McIntosh' and Delicious' apple (Rupasinghe et al. 2000) fruits. Monroe avocado fruit treated with a relatively high concentration (4 5 L L" 1 ) of 1-MCP for 24 hat 20 C did not recover ethylene sensitivity and normal respiratory behavior during storage at l 3C. At lower concentrations and exposure periods (0.9 L 1 for 12 h) however 1-MCP significantly delayed but did not prevent the onset of climacteric ethylene production in 'Booth 7' avocado fruit. The influence of 1-MCP at delaying climacteric behavior has been shown previously for apricot (Fan et al. 2000) apple (Fan et al. 1999), banana (Fan and Mattheis 1999; Fang et al. 2000 ; Golding et al. 1998 ; Golding et al. 1999) and avocado ( cv. Hass) (Feng et al. 2000). 1-MCP treatment ( 0 9 1 for 12 h) in 'Booth 7 avocado significantly increa ed the magnitude of the ethylene production peak. Enhanced ethylene production following 1MCP treatm nt has also been reported with mature green banana (Golding et al. 1998) and grapefruit (Mullins et al. 2000). In tomato fruit DACP (Diazocyclopentadiene)

PAGE 146

133 inhibits eth lene production but after the fruits again became sensiti e to ethylene the climact ric of ethylene production is considerably greater than that of control fruit (Sisler and Blankenship 1993). The greater capacity for producing ethylene may have de eloped during period of the receptor inactivation. The recovery of C 2 H 4 production in 1-MCP-treated Booth 7 avocados during storage 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 rapid softening of avocado fruit was accompanied by increases in PG and Cx-cellulase activities (Awad and Young 1979 Christofferson et al. 1984; Pesis et al., 1978) and a decline in the activity of PME (Awad and Young 1979 ; Awad and Young 1980; Zauberman and Schiffmann-Nadel, 1972). PG accumulation in 1-MCP-treated fruit was significantly delayed and failed to recover to levels similar to those attained in the control fruit; however, 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 influenc on fruit softening until the late stages of ripening (Carrington et al. 1993 Kram r et al. 1992) Feng et al. (2000) found that 30 50 or 70 nL L1 1-MCP suppr s d PG acti ity about 10 to 30% in 'Hass' avocado. Exogenous ethylene treatment (100 L L 1 for 12 hat 20 ) enhanced PG accumulation which eventually r ached levels similar to tho ynth b rved in control fruit at the full-ripe stage. This observation indicates that PG may be upr gulated by ethyl n In tomato fruit ethylene al o stimulate PG

PAGE 147

134 app aran and it stimulat s ither transcription of mRNA or subsequ nt translation (J ffi ry t al. 1984). ignificant mol mass downshifts of pectic polysaccharides occurred durin g ripening of Booth 7 avocado fruit, as has been reported in Lula (Huber and O'Donoghue 1993) and Hass avocado (Sakurai and Nevins 1997). Consistent with th marked delay of PG accumulation in 1-MCP-treated Booth 7' avocado fruit th e solubilization and degradation of pectic polysaccharides was significantly dela ye d by 1-MCP treatment. At the pre-ripe stage (Day 0) waterand CDTA-soluble UA constituted 33 and 7 3% of the total EIS UA content respectively. At the full-ripe stage (fi rmness 10 to 20 N) waterand CDT A-soluble polyuronides of control fruit (Day 16) comprised approximately 71 % and 6.8% respectively of the total UA content, while those of 1-MCP treated fruit (Day 28) comprised approximately 70% and 8 3% respectively of the total U A content. In the present study, both water and CDT A-soluble polyuronides of control fruit exhibited the characteristic molecular mass downshifts reported in earlier studies of avocado fruit ( Huber and O Donoghue 1993 ; Wakabayashi et al. 2000) with polyuronides in ripe fruit eluting as low mol mass polymers near the total column volume. Through 24 d of storage (Fig. 6-1 IB, 12B), 1-MCP-treated fruit showed considerably less extensive breakdown of both waterand CDT A-soluble polyuronid e Further downshifts in polyuronide molecular mass were evident in 1-MCP-treated fruit after 28 d of storage. PG activity of control and 1-MCP-treated fruit significantly increas d at the time (Day 12 and Day 24 respectively) as the major downshift in polyuronid polym r size occurred (Fig 68 11 and 12). The deer a in mol cu lar 1 z

PAGE 148

135 of aterand CDT A-soluble polyuronides was apparently the result of polygalacturonase acti ity. These data suggest that PG was mainly responsible for the degradation of polyuronides during avocado fruit ripening consistent with interpretations of the role of PG in tomato (Bra dy 1987 ; Giovannoni et al. 1989). 1-M P treatment delayed the decrease in PME activity by 12 din Booth 7' avocado. PME has been shown to participate in the extensive depolymerization of polyuronides associated with avocado ripening (Wakabayashi et al. 2000). Increases in C x -cellulase activity in 1-MCP-treated fruit were significantly delayed and the enzyme failed to recover to levels similar to those attained in the control fruit. Feng et al. (2000) found that 30 50 or 70 nL L -I 1-MCP delayed increases in cellulase activity by 4 d in Hass' avocado. In response to exogenous ethylene treatment however C x -cellulase activity in 1-MCP-treated fruit eventually recovered to levels measured in control fruit at the full-ripe stage. This observation implies the C x -cellulase may be upregulated by ethy len e. The induction of cellulase in avocado mesocarp discs is inhibited by aminoethoxyvinylglycine (A VG an inhibitor of ethylene synthesis) and 2 5-norbornadiene (NBD an inhibitor of ethylene action) together b speaking the invol vement of wound ethylene in the process (Starrett and Laties 1993) 1-M CP treatment significantly delayed the decrease in total e tractable aand ~-galacto id ase activities in Booth 7 avocado. a-galactosidas activity in 1-MCP-treat d fruit wa n t influ need by exog nous thyl ne tr atment wher as ~-galactosidas activity d er a e d in r e ponse to ethyl ne treatm nt. Although ~-galactosidase decrea d durin g avocado rip ning the use of total prot in xtracts in our assays would have ma s ked diffi r e ntial r pons s of pecific iso zym s of thes prot ins. For example

PAGE 149

136 Pressey (1983) and Carey et al. (1995) reported that total ~-galactosidase acti v it y in tomato remained relatively constant throughout ripening whereas the levels of one isozyme (~-Gal II) increased significantly. Smith and Gross (2000) found TBG4 ( one o f tomato ~-galactosidase genes) transcript accumulation was significantly impaired in rin nor and Nr fruit relative to wild-type accumulation. This finding suggests that specific isozymes of ~-galactosidase in avocado fruit may be upregulated by ethylene. A net loss of pectic-associated neutral sugars occurred during avocado fruit ripening. The neutral sugar composition of water-soluble polyuronides changed in parallel with increases in the quantity of this polyuronide fraction. Arabinose galactose and xylose were the predominant neutral sugars of water-soluble polyuronides with lower quantities of rhamnose glucose and mannose (Tables 62 and 6-3). During storage there was an overall loss in galactose xylose mannose and glucose in control and 1-MCP-treated fruit. Ethylene treatment following the initial 18 d storage at l 3C did not significantly affect the neutral sugar composition in water -solubl e polyuronides of 1MCP-treated fruit. Although the water-soluble uronic acid levels in 1-MCP-treated fruit were similar to those in control fruit the proportional quantity of galactose in 1-MCP treated fruit did not change relative to the significant proportional decrease noted for control fruit at the full-ripe stage. The role of ~-galactosidase in pectin metabolism was not determined but since a marked decrease in galactose content was noted for water soluble polyuronides from control fruit and not in 1-MCP-treated fruit (Tables 6-2 and 63) it is possible that the enzymes contributing to polyuronide degalactosidation ma y b e upregulated by ethylene.

PAGE 150

137 Th neutral sugar composition of CDT A-soluble polyuronides also significantly changed during storage (Tab les 6-4 and 6-65) The major neutral sugars in this pol y uronide fraction were arabinose glucose galactose, and xylose with lower quantities of rhamnose and mannose (Tables 6-4 and 6-5) During ripening of control fruit there was a proportional loss in rhamnose galactose xylose, mannose and glucose although pol y uronides from ripe fruit exhibited a significant increase in the quantity of pol y uronide-associated neutral sugars. In 1-MCP-treated fruit the proportional quantities of rhamnose and arabinose increased during storage. Ethylene treatment further increased the quantities of rhamnose and arabinose and decreased the quantities of galactose xylose mannose and glucose in the COTA-soluble polyuronides of 1-MCP-treated fruit. Previous experiments (chapter 5) showed that 1-MCP treatment did not significantly affect the extractable quantities of 4 M alkali-soluble hemicelluloses during ripening whereas it did suppress the magnitude of molecular mass downshifts in these pol y mers Glucose and xylose were the predominant neutral sugars of hemicelluloses consistent with the relatively high xyloglucan content of avocado (Table 6-6). In control fru it the overall quantities of arabinose rhamnose and xylose slightly increased whereas those of glucose, galactose and mannose decreased during storage. O Donoghue ( 1992) also reported that glucose and xylose are the major neutral sugars of hemicelluloses in 'L ula avocado and glucose increased and xylose remained constant during ripening. The changes in avocado hemicelluloses are similar to those reported for oth e r fruits. Glucose and galactose decreased and xylose increased during ripening of mu km 1 n (Mc ollum et al. 1989) whereas th r w r major lo of galactos and and xy lo e content during kiwifruit ripening (Redgwell et al. 1990

PAGE 151

138 1991 ). 1-MCP or ethylene tr atm nt did not hav a significant eff e ct on th e n e utral s u ga r compo ition in 4 M alkali-soluble hemicellulose of avocado fruit. Thi s implie s that th br akdown of hemicelluloses is influenced by ethylene but that the products are not lo s t from the cell wall. Therefore, 1-MCP or ethylene may not change the composition. og nou ethylene treatment before or after 1-MCP treatment did not influence fruit firmness weight loss respiration or C 2 H 4 production. These observations indicate that ethylene responses were completely suppressed by 1-MCP. Yueming et al. (1999 ) investigated the affinity of 1-MCP for ethylene-binding sites using Lineweaver-Burk plots (Whitaker 1972). The Km for 1-MCP was low (17 nL L1 ) in comparison to that estimated for ethylene (96 nL L1 ). Yueming s data indicated that 1-MCP has greater affinity than ethylene for the ethylene-binding sites and concluded that inhibition by 1-MCP is noncompetitive. In ripening fruit, even high concentrations of ethylene ( e.g. 1 000 L L1 ) do not give a response after 1-MCP treatment (Sisler and Serek 1999). Although exogenous ethylene cannot overcome the 1-MCP-induced inhibition of ripening avocado fruit treated with 1-MCP do become sensitive to ethylene after a certain period. It is possible that new receptors are synthesized or that 1-MCP is either eventually metabolized or dissociates from the receptor (Sisler and Serek 1999). These results indicate substantial evidence for ethylene to bind ethylene receptors and then to elicit subsequent signal transduction and translation which is required for normal fruit npenmg. Summary In this study preclimacteric avocado (Per sea americana Mill. cv. Monro and Booth 7') fruit were used to investigate the effects of 1-MCP and ethyl neon rip nin g

PAGE 152

139 chara t ri tics in a ocado fruit. Firmness weight loss respiration and C2H4 production p I color I cted cell wall enzymes (polygalacturonase, pectinrnethylesterase a~galactosidase and C -cellulase) and compositional and mol mass properties of polyuronides and hemicelluloses were monitored during storage. Application of 1-MCP delayed ripening of avocado fruit as evidenced by a significant delay in fruit softening and in the onset of the ethylene climacteric. Avocado fruit treated with 1-MCP retained more green color than control fruit at the full-ripe stage ( 10 to 20 N). 1-MCP treatment affected the activity trends of all cell wall enzymes measured and completely suppressed the appearance of polygalacturonase activity for up to 24 d. The loss of fruit firmness during avocado fruit ripening coincided with modifications of polyuronides and a net loss of non-cellulosic neutral sugars. An increase in solubility and a decrease in molecular size of polyuronides occurred relatively later during ripening. 1-MCP treatment significantly delayed the solubilization and degradation of polyuronides. The decrease in molecular size of waterand CDT A-soluble polyuronides was apparently the result of polygalacturonase activity. This decrease in mol mass of polyuronides was accompanied by changes in neutral sugar composition 1MCP treat d fruit significantly retained the quantity (mole% basis) of galactose in wat so lubl polyuronides relative to the significant proportional decrease noted for control fruit at th full-ripe stage. 1-M P or ethylene treatment did not have a significant effi ct on the neutral ugar composition of hemicelluloses. xog 11 us ethyl 11 tr atm nt b fore or after 1-M P treatment did not influ nc fruit firmn w ight lo re piration and C 2 H 4 production howev r the acti iti of

PAGE 153

140 polygalacturonase ~-galactosidase and Cx-cellulase in 1-MCP-treated fruit respond d to exogenous ethylene.

PAGE 154

z ti') ti') Q,) C: E a.. t;: ...., ::J a.. u.. 0 ti') ti') 0 1 4 1 220 ---------------------. 200 180 160 140 120 100 80 60 40 20 I I I I I I I I I I I I I I I 1-MCP --Control (no C2H4 no 1-MCP) A -oC2H4 -bC2H4 & 1-MCP o_...., ___________________ ,..... 14 ---------------------12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Storage period (days) igur 6-1. ruit firmn (N) and weight l oss(%) of 'Monroe avocado gassed imm diat ly with 2 1-Li (100 L L 1 ) for 12 hat 20 C th n ither stor d at 13 or c ntinu u l y treated with 1-M P ( 4.5 L L1 ) for 24 hat 20 and th n transfi rr d to 13 V rti I bars r pr nt standard deviation of 6 ind p nd nt amp l

PAGE 155

240 200 z "' 160 "' C1) C: E 120 ... t;: :::I ... LL 80 40 1-MCP 142 ____ Control (no 1-MCP, no C 2 H 4 ) --o--1-MCP ----6---1-MCP & C2H4 A o..._ ___________________ 12 ---------------------~ 10 8 "' "' 0 6 4 2 0 1-MCP B 0 2 4 6 8 10 12 14 16 18 20 22 Storage period (days) Figure 6-2. Fruit firmness (N) and weight loss(%) of 'Monroe avocados treated with 1 MCP (4 5 L L1 for 24 hat 20C) and stored at 13C. Half of the 1-MCP-treated fruit were treated with C 2 H4 (100 L L1 for 24 hat 20 C) following the initial 13 d at 13 C. Vertica l bars represent standard deviation of 6 independent samples.

PAGE 156

143 250 ------------------------. 1-MCP (12 h) 200 z 150 (1) C: E L.. .;: 100 50 0 300 --------------------z en en (1) C: E L.. .;: -250 200 150 100 50 B 0 0 1-MCP (12 h) ----C ontro I -o--1-MCP 1-MCP & C 2 H 4 5 10 15 20 25 30 Storage period (days) F i g ur 6 3 ~ ruit firmn e s (N) of Booth 7 avocados treated with 1-M P (0 9 L L 1 for 1 2 h a t 20 ) s t r d a t 1 3 for 19 d and th e n trans rred in 20 Half of th 1 M t r a t d fr uit w r x po d to C 2 H 4 (100 L L 1 for 12 hat 20 C) befor transfer to 20 ntr I fr uit ( not x p ose d to 1-M C P and 2 ~) were stored at 13 C for 12 d and th e n t ra n s rr d t o 2 0 V rtical bar s r pr s nt standard deviation. ompr ssion t st (A) and pun c tur t t ( B ). A rrow ) r present tran s ferrin g to 20

PAGE 157

144 18 -----------------------, 0 16 14 12 "' 10 ti) 0 :c 8 C) (1) 3: 6 ---Control (no 1-MCP, no C 2 H 4 ) --o1-MCP ---&1-MCP & C 2 H 4 4 1-MCP (12 h) 2 j 0 0 5 10 15 20 Storage period (days) 25 30 Figure 6-4 Weight loss(%) of Booth 7' avocados treated with 1-MCP (0.9 L L1 for 12 hat 20 C) stored at l 3 C for 19 d and then transferred in 20C. Half of the 1-MCP treated fruit were exposed to C 2 ~ (100 L L1 for 12 hat 20C) before transfer to 20 C Control fruit (not exposed to 1-MCP and C 2 H 4 ) were stored at 13C for 12 d and then transferred to 20 C. Arrow marks represent transferring to 20 C. Vertical bars represent standard deviation.

PAGE 158

160 140 120 I .c: 100 I O') 80 O') E N 60 0 (..) 40 20 0 140 120 100 I .c: I 80 O') _J ::l. 60 J: N (..) 40 20 0 145 A 1-MCP C2H4 I I I -+Control (no C2H4, no 1-MCP) B -o-C2H4 1 1-MCP I C2H4 & 1-MCP I I I I I I I I I I I I I I I I I I I I I I I I I I 0 2 4 6 8 10 12 14 16 18 20 22 24 Storage period (days) igur 6-5. arb n dioxid (mg kg1 h-) and thylen (L kg1 h1 ) prod u ction of M nr a cado gas d immediat ly with 2 H 4 (100 L L1 ) for 12 hat 20 th n ith r t r d at 13 r c ntinuou ly tr at d with 1-M P (4.5 L L1 ) for 24 hat 20 and th n tran fi rr d t 13 V rtical bar r present tandard d viation of 6 ind p nd nt amp

PAGE 159

146 100 A 80 1-MCP T"" I .s:: T"" 60 I C) ::it:. C) E 40 N 0 (.) 20 0 140 ------Control (no 1-MCP no C 2 H 4 ) 8 120 -o--1-MCP ----b--1-MCP & C2H4 100 T"" I .s:: T"" 80 I C) 1-MCP ::it:. ...J I :::i. 60 I I ...,. I :::c I I N 40 I (.) I I I I C2H 4 (24 h ) I I 20 I I I I I 0 0 2 4 6 8 10 12 14 16 1 8 20 2 2 Storage period (days) Figure 6-6. Carbon dioxide (mg kg1 h1 ) and ethylene (L kg1 h1 ) production of Monroe avocados stored at 13 C after 1 MCP treatment (4.5 L L1 for 24 hat 20 C ) Half of the 1 MCP treated fruit were treated with C 2 H 4 (100 L L1 for 24 hat 20 C) following the initial 13 d at 13 C Vertica l bars represent standard de v iation of 6 independent samp l es.

PAGE 160

147 350 300 ----Control ( no 1 MCP no C2H4 ) --0--1-MCP 250 --6--1-MCP & C 2 H 4 ";.r:. .... 200 0) :::s:,, ..J :::1. 150 v J: N (.) 100 50 1 MCP ( 12 h ) 0 0 5 10 15 20 25 30 Storage period (days) igure 67. thylene production (L kg1 h1 ) of Booth 7 avocados treated with 1-MCP (0.9 L L1 for 12 hat 20C), stored at l3C for 19 d and then transferred in 20 Half of the 1-M P-tr at d fruit were exposed to C 2 H 4 (100 1 for 12 hat 20 C) before tran fir to 2 0 ontrol fruit (not exposed to 1-MCP and C 2 ~) were stored at 13 for 12 d and th n transferred to 20C. Arrow marks (----~ r present transfer to 20C. Vertical bar repr ent standard deviation.

PAGE 161

50 40 II) 0 >< >-30 ~.,.. > C u E co '7 (!) C, 20 a..~ Q) 0 E 10 0 12 10 .,.. 8 Z' C > E :.:; .,.. 6 (J C, co E w 0 N 4 a.. (0
PAGE 162

1 49 35 Contorl (no 1-MCP no C 2 H 4 ) >, -30 > ---0--1 MCP 25 1-MCP & C 2 H 4 (.) .... ----6,C'O c: (1) E (/) 20 C'O .... 15 'C) :::, E 10 (1) (.) 0 I 5 >< (_) 0 6 ~'st 0 > T"" 5 >< ns 'r"Q) c: (/) 4 ns E "C u; ":" 0 C) 3 +J 0 ns a, ns o 2 9> E ts ~ 'st 10 '> 0 T"" C 0 >< 9 ns \ r Q) ":" (/) = 8 ~E 'r" 1-MCP (12 h) (/) I 7 0 C) +J C 2 H 4 ( 12 h) 0 Q) ns 6 0 ~E ca. 5 0 4 8 12 16 20 24 28 Storage period ( days) igur 6-9. x-c llul as and aand ~-ga l actosidas activities of Booth 7' avocados tr at d ith 1-M P (0.9 L L1 for 12 hat 20C) tored at 13 for 19 d and then tran rr d in 20 Half of th 1-M P-tr ea ted fruit w re expo d to C 2 rLi (100 L L1 for 12 hat 20 ) before tr a n s r to 20 Control fruit (not xpos d to 1-MCP and 2 fLt r tor d at 1 3 for 1 2 d and th e n transferred to 20C. Arrow marks ( ----r pr nt t ran r to 20 V rtical bars repr s nt tandard deviation

PAGE 163

C: Q.) C: u '7 C) <(.::s:. ::::> C) n, 0 IC: Q.) C: 0 u <( ::::> Q.) '7 0) j:i .::s:. :::, C) c5(/) I <( o (.) 1 50 260 --------------~~~ __ Control (no 1 MCP no C 2 H 4 ) 240 -o1-MCP 220 -----61-MCP & C2H4 200 180 160 C: 140 ------------------------------. 140 c:: B J 0 u 120 <( ::::> Q.) '7 0) 100 j:i .::s:. :::, C) 80 I 'Q.) 22 ...,_ ______________ 60 20 C 18 16 14 12 10 -----------------------------. 1 300 D 1200 1100 1000 900 --------------800 0 3 6 9 12 15 18 21 24 27 30 Storage period (days) a. 'ti) n, "'C u 0 (/) 0 Q.) (/) E .c :t :::, .c: 0 (/) (/) Q.) C: 'I 0 C) C: 0 n, N .c: C: w C) E Figure 6 10. The changes on the amount of EIS in the mesocarp tissue and on th changes in water CDT A so l uble UA and total UA in EIS from Booth 7' avocado treated with 1 MCP (0.9 L L1 for 12 hat 20 C) stored at 13C for 19 d and th n transferred i n 20 C. Ha l f of the 1 MCP-treated fruit were expos d to 2 H 4 (100 L L 1 for 12 hat 20 C) before transfer to 20 C. Control fruit (not expo d to 1-M P and 2 ~ were stored at 13 C for 12 d and then transferred to 20 C. Arro mark (----~ r pr nt transfer to 20 C Vertical bars represent standard d viation.

PAGE 164

151 16 14 A r 12 10 ----+Day 0 j \ 0 Day 6 I 8 .,_ Day 12 6 --vDay 16 4 "C 2 Q,) 'Q,) 0 > 0 16 (.) Q,) 14 ----+Day 0 B '12 0 Day 9 "' ,,_ Day 18 .... 0 10 --vDay 24 .... '+8 -Day 28 0 6 0 fl) 4 "C (.) 2 "' (.) 1Q C: 0 14 ----+Day 0 C '=> 12 0 Day 9 .,_ Day 18 10 --vDay 24 8 -Day 28 6 4 2 0 ~ 10 20 30 40 50 60 70 80 Elution volume (ml) Figure 6-11 Mol cular mass profiles of water-soluble polyuronid s from EI prepared from Booth 7' avocado. Control fruit were stored at 13C for 12 d and then transferred to 20 (A). ruit were tr at d with 1-MCP (0.09 L L1 for 12 hat 20 ) stor d at 13 for 19 d and then tran ferred to 20 (B). Half of th 1-M P-tr ated fruit wer x po e d to 2 H 4 (100 L L1 for 12 hat 20 ) before tran fi r to 20 ( ). Polyuonid ( :::::: 0 5 m g ga l ac turonic acid quivalent ) in 2 mL of wat rolubl fraction wer appli d t th phar L-2B-300 and individual fraction w r mea ured for UA content. a t a fi r ac h fr a ction x pr d as a p re ntag of th total lut d UA. Tick marks at th t p f th fi g ur indic a t V V id vol um (I ft) Vt total vo l ume (right).

PAGE 165

152 12 10 A DayO 8 0 Day 6 ~ Day 12 6 -<;/Day 16 4 2 "'C Cl) ... Cl) 0 > 12 0 0 DayO Cl) 10 8 ... Day 9 0 co ~ Day 18 ... 8 0 -<;/Day 24 ... 't6 -Day 28 0 0 4 "' "'C 2 0 co 0 0 C: 12 0 DayO ... C :::> 10 0 Day 9 8 ~ Day 18 -<;/ Day 24 6 -Day 28 4 2 0 10 20 30 40 50 60 70 80 Elution volume (ml) Figure 6-12. Molecular mass profiles of CDT A-soluble polyuronides from EIS prepared from Booth 7 avocado. Control fruit were stored at l 3C for 12 d and then transferred to 20 C ( A). ruit were treated with 1 MCP (0 09 L L1 for 12 hat 20 C) stored at 13 C for 19 d and then transferred to 20 C (B). Half of the 1 MCP-treated fruit were exposed to C 2 H 4 (100 L L1 for 12 hat 20C) before transfer to 20 C (C). Polyuonides ( :=::; 0.5 mg galacturonic acids equivalents) in 2 mL of CDT A soluble fraction were applied to the Sepharose CL-2B 300 and individual fractions were measured for UA content. Data for each fraction expressed as a percentage of the tota l eluted UA. Tick mark at th top of the figur indicate Vo Void volume (left) ; Vt total volum (right).

PAGE 166

CHAPTER 7 SUMMARY AND CONCLUSIONS The primary objectives of this research were to examine the effects of postharvest application of ethylene on ripening uniformity and fruit quality, to characterize physiological and biochemical responses of avocado fruit to 1-MCP treatment during avocado fruit ripening and to evaluate the ability of 1-MCP as a postharvest tool for regulating the ripening of avocado fruit, to investigate effects of 1-MCP and waxing on ripening characteristics in avocado fruit, and 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 neutral sugar composition. The overall hypothesis was treatment of avocado fruit with ethylene and 1-MCP will influence the rate of ripening Ethylene plays a vital role in the ripening of climacteric fruits and whether applied exogenously or produced naturally, initiates ripening and softening. Ethylen treatm nt of d tached mature avocado fruit promoted the ons t of rip ning. A ocado fruit fr m immediate ethylen treatment had more uniform rip ning and better pulp quality than c ntrol fruit (not xpo d to ethylene) and fruit from d layed ethylene tr atm nt. B t fruit quality was obtain d with immediate ethylen treatments (100 L 1 1 at 20 fi r 12 h or at 12 for 24 h for Booth 7 and 100 1 at 13C for 24 h for Monr ) Aft r ub qu nt torag fruit from th se treatment rip n d normally and 153

PAGE 167

154 uniformly. Ethylene treatment did not affect fruit quality as determined by peel color dry matter content and oil content. The importance of ethylene in regulating fruit ripening has been clearly demonstrated from analyses of fruits exhibiting suppressed ethylene biosynthesis or action. In addition to the use of fruit lines with suppressed ethylene synthesis or perception the application of compounds that block ethylene action has provided a facile approach for examining relationships among ethylene fruit ripening and senescence in a range of horticultural commodities. 1-methylcyclopropene (1 -M CP) blocks ethylene receptors preventing ethylene effects in plant tissues for extended periods. The delay in avocado ripening was significantly influenced by 1-MCP concentration exposure duration, and exposure temperature. 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. 1-MCP treatment at 20C delayed avocado fruit ripening longer than when treated at l 2C. 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 firmness was significantly retained in response to 1-MCP treatment, consistent with the fact that softening is one of the most sensitive ripening processes to ethylene. Significantly delayed softening by 1-MCP substantiates that ethylene is involved in augmenting the activity of softening-related metabolism. Avocado treated with 1-MCP also showed significantly less weight loss and retained more green peel color than control (not exposed to 1-MCP) fruit at the full-ripe stage (10 to 20 N). The gradual recovery of C 2 H 4 production in 1-MCP-treated avocados dming

PAGE 168

155 torag sugg ts either the synthesis of new receptor proteins metabolism of the 1-MCP receptor-prot in complex or dissociation of 1-MCP from the receptor sites. The loss of fruit firmness during avocado fruit ripening was related temporall to modification of pectic polysaccharides and a net loss of non-cellulosic neutral sugar An incr ase in olubility and a decrease in molecular size of polyuronides occurred relative! later during fruit ripening. The decrease in molecular size of waterand CDT A-soluble polyuronides was apparently the result of polygalacturonase activity. This decrease was also accompanied by changes in neutral sugar composition. PG exhibited the strongest response to 1-MCP, showing little or no recovery over the storage period. Although PG activity did not fully recover in 1-MCP-treated avocado the firmness ultimately reached values comparable to those of control (not exposed to 1-MCP) fruit indicating that PG is not required for the extensive softening of avocado fruit. This conclusion is consistent with reports for tomato fruit wherein antisen suppression of PG activity had minimal influence on fruit softening until the very late stages of ripening. onsistent with the marked suppression of PG levels in 1-MCP-treated avocado fruit, th solubilization and degradation of polyuronides was significantly delay d and reduc d in 1-MCP-treated fruit. 1-MCP-treated fruit showed consid rably 1 ss exten i breakdown of both waterand CDT A-soluble polyuronides. Additionally limited mol cular mass down hifts in avocado polyuronid s as evident from g l filtration ana l y can be br u g ht about by d t rification independent! of P action. n equ ntly th r lativ ly normal l ls of PM in th fruit might have influenc d th g I filtrati n b havi r f polyuronid s from 1-M P-tr at d fruit.

PAGE 169

156 During rip ning of avocado hemicelluloses at each developm ntal t ag x hibit d a gradu a l but limit d mol cular mas downshift 1-M P treatm e nt did not i g nificantl affi ct th quantity or composition of the neutral sugar in a 4 M alkalis oluble hemicellulos extract during ripening. 1-MCP treatment however significantly reduced molecular mass downshifts in 4 M alkali-soluble hemicelluloses and x yloglucan. In addition to PG, 1-MCP treatment significantly delayed the activities of C x -cellulase PME, and total extractable aand p-galactosidase during avocado fruit ripening. Selected cell wall enzymes (polygalacturonase p-galactosidase and C x -cellul ase) were upregulated by ethylene. The role of P-galactosidase in pectin metabolism was not determined but since a marked decrease in galactose content in water soluble polyuronides and significant difference quantity between control and 1MCP-treated fruit during fruit ripening, it is possible that this enzyme could be involved in pectin degradation in avocado fruit. Exogenous ethylene treatment before or after 1-MCP treatment did not influence fruit firmness weight loss, respiration, or C 2 H 4 production implying that ethylene response was completely suppressed by 1-MCP. It also suggests that inhibition by 1-MCP is noncompetitive and thatl-MCP has a greater affinity than ethylene for the binding sit s. After an ethylene treatment much of the ethylene diffuses rapidly from the receptor whereas 1-MCP remains bound for long periods. Although exogenous ethylene treatment cannot induce 1-MCP-treated fruit ripening avocado fruit treated with 1-MCP do become sensitive to ethylene after a certain period. It is possible that new receptor ar made or the same receptor is becoming active again by diffusion of 1-MCP. Thes r ult also indicate substantial evidence for ethylene to bind ethylene r ceptor and th n to Ii it

PAGE 170

157 ub qu nt ignal transduction and translation which is required for normal fruit rip ning 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 bett r green peel color and further depressed ethylene production of avocado fruit. Additionally waxing is known to increase internal CO 2 level and to reduce internal 02 concentrations of avocado during storage. Thus, extension of shelf life by wax treatment can be attributed to a combination of reduced water loss and modified internal atmosphere. Inhibition of ethylene action with 1-MCP during the early stages of climacteric produces changes in subsequent ripening behavior. 1-MCP profoundly blocked C 2 H 4 action and delayed C 2 ~ 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 appropriat harvesting and environmental conditions will extend the storage period of avocado and other fruit.1-MCP therefore has provided a valuable tool to investigate ethylen m tabolism in ripening climacteric fruit and has the potential to extend the storage lifi of horticultural products.

PAGE 171

REFERENCES Abdi N. W. 8 McGlasson P. Holford M. William, and Y. Mizrahi. 1998. Responses of climacteric and suppressed-climacteric plums to treatment with propylene and 1m thylcyclopropene. Postharvest Biol. Technol. 14:29-39. Ab les F. B. P. Morgan, and M. E. Saltiveit. 1992. Ethylene in Plant Biology 2nd ed. Academic Press Inc., San Diego, CA. Adato I. and S. Gazit. 1974. Water-deficit stress ethylene production and ripening in avocado fruits. Plant Physiol. 53 :45-46. Ahmed A. E. and J.M. Labavitch. 1977. A simplified method for accurate determination of cell wall uronide content. J. Food Biochem. 1:361-365 Ahmed A. E., and J.M. Labavitch. 1980. Cell wall metabolism in ripening fruit I: cell wall changes in ripening 'Batlett' pears. Plant Physiol. 65: 1009-1013. Albersheim P., D. J. Nevins, P. D. English, and A. Karr. 1967. A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5:430-445. Alonso J.M. T. Hirayama, G. Roman, S. Nourizadeh, and J. R. Ecker. 1999. EIN2 a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284:2148-2152. Anderson J. D. F. C. Cardinale J.C. Jennings H. A. Norman A. Avni and B. A Bailey 1996 Involvement of ethylene in protein elicitor-induced plant responses Biology and Biotechnology of the Plant Hormone Ethylene. NATO Advanced Research Workshop June 9-13 Crete Greece:Abstact p.35 Awad M 1977. Variation in cellulose content of Fuerte avocado fruit after harvest. HortScience. 12:406. Awad M. and R. E Young 1979. Postharvest variation in cellulase polygalacturonase and pectinmethylesterase in avocado (Persea Americana Mill. cv Fuerte) fruits in relation to respiration and ethylene production. Plant Physiol. 64:306-308 Awad M. and R. E. Young. 1980. Avocado pectinmethylesterase activity in relation to temperature, ethylene and ripening. J. Am. Soc. Hort. Sci 105:638-641. 158

PAGE 172

159 ub R M Guis M. Ben Amor L. Gillot J.P. Roustan A. Latche M. Bouzayen and J. P ch. 1996 Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotechnol. 14:862-866. Barmor C. R. 1975 Ethylene preripening of mangos prior to shipment. Proc Fla. tate. Hort. oc. 8 8 :469-4 71. Bartley I. M. 1974 .B-Galactosidase activity in ripening apples. Phytochemistry 13 :21072111. Bergh B.O 1976. Avocado In: N V. Simmonds (ed) Evolution of crop plants. Longman London B ey er E M ., Jr. 1976 A potential inhibitor of ethylene action in plants Plant Physiol. 58 :268-271. Biale J. B 1960 The postharvest biochemistry of tropical and subtropical fruits. Adv. Food Res 10:293-354. Biale J.B. and R. E. Young. 1971. The avocado pear, p. 1-63 In: A C. Hurne (ed) The biochemistry of fruits and their products. Academic Press Inc. New York. Biggs M. S. and A. K. Handa. 1988. Tempera! regulation of polygalacturonase gene expression in fruits of normal mutant and heterozygous tomato genotypes. Plant Physiol. 89:117-125. Blumenkrantz N. and G. Asboe-Hansen. 1973. New method for quantitative determination fo uronic acids. Anal. Biochem. 54:484-449. Bow r J.P. and J. G. Cutting. 1988. Avocado fruit development and ripening phy iology. Hort. Rev 10:229-271. Brad y, J. 1987. Fruit ripening. Anal. Rev. Plant Physiol. 38:155-173 Brad y J. G MacAlpine W B. McGlasson, and Y. Ueda. 1982. Polygalacturonas m tomato fruits and induction of ripening. Aust. J. Plant Physiol. 9: 171-178. Burg P. and E. A. Burg 1965. R lationship between ethylene and ripening in banana s. Bot. Gaz. 126:200-204 Burn s J. K. 1990. a and .B-Galactosidase activities in juice v sicl of stored Val ncia o r a n g s Phytoch mistry 29:2425-2429. ardin a l e . J . J nnin g s and J. Anderson 1995 U of th thylen acti n inhibit r 1-m thylcycloprop n to study the role of thyl n in licitor-induc d e th y l n e bi o y nth i int mato 1 aves. Plant Physiol. ( uppl.) 108: 140.

PAGE 173

160 Carey A T. K Holt S Picard R. Wilde G A. Tucker C. R. Bird W Schuch and G B. eymour. 1995. Tomato exo-(1-4)-~-D-galactanase. Isolation chang s durin g ripening in normal and mutant fruit and characterization of related cDNA clon Plant Physiol. 108: 1099-1107. Carrington C. M. S ., L. C. Greve and J.M. Labavitch. 1993. Cell wall metabolism in ripening fruit. VI. Effect of the anti sense polygalacturonase gene on cell wall changes accompanying ripening in transgenic tomatoes Plant Physiol. 103 :429434. hang S F. Kwok A B. Bleecker and E. M. Meyerowitz. 1993 Arabidopsis ethylene-reponse gene ETRJ: Similarity of product to two-component regulators Science. 262:539-544. Chang C. and E M. Meyerowitz. 1995. The ethylene hormone response in Arabidopsis : A eukaryotic two-component signalling system. Proc. Natl. Acad. Sci USA 92:4129-4133. Chang C. and J. A. Shockey. 1999. The ethylene-response pathway: signal perception to gene regulation. Current Opinion in Plant Biol. 2:352-358. Chen P. M. S. R. Drake, D. M. Varga and L. Puig. 1996. Precondition of 'D'anjou' pears for ear l y marketing by ethylene treatment. J. Food Quality. 19:375-390 Christofferson R.E. M. L. Tucker and G. G. Laties. 1984. Cellulase gene expression in ripening avocado (Persea americana cv Hass) fruit. The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridisation and immunodetection. Plant Mol. Biol. 3:385-392. Clark K. L. P. B. Larsen X. Wang, and C. Chang. 1998. Association of the Arabidopsis CTRl Raf-like kinase with ETRl and ERS ethylene receptors. Proc. Natl. Acad. Sci. USA 95:5401-5406. Crane J. H. C. F. Balerdi, and C. W. Campbell. 1996. The avocado. Circular 1034. F lorida Coop. Extn. Service, IF AS, Univ. of Florida Gainesville FL. Dellapenna D. D. C. Alexander, and A. B. Bennett. 1986. Molecular cloning of tomato fruit polygalacturonase: analysis of polygalacturonase mRNA levels during ripening. Proc. Natl. Acad. Sci. USA 83 :6420 -6424 De Yetten N. C. and D. J. Huber. 1990. Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals. Physiol. Plant. 78:447454.

PAGE 174

161 Dolendo A L. B S Luh and H. K. Pratt 1966. Relation of pectic and fatty acid chan g s to respiration rate during ripening of avocado fruits. J Food Sci 31 :3323 36. Dubois M K. A. J. K. Hamilton P A. Rebers and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. Durand B. J. L Orcan U. Yanko G Zauberman and Y. Fuchs. 1984 Effects of waxing on moisture loss and ripening of 'Fuerte' avocado fruit. HortScience. 19:421-422 Eaks I. L. 1966 The effect of ethylene upon ripening and respiration rate of avocado fruit. Calif. Avocado Soc Yrbk. 50:128-133 Eaks I. L. 1978. Ripening, respiration, and ethylene production of Hass avocado fruit at 20 to 40 C. J. Am. Soc. Hort. Sci. 103:576-578. Eaks I. L. 1980 Respiratory rate ethylene production and ripening response of avocado fruit to ethylene or propylene following harvest at different maturities. J. Am. Soc. Hort. Sci. 105:744-747. Eaks I. L. 1985 Effects of calcium on ripening respiratory rate ethylene production and quality of avocado fruit. HortScience. 110: 145-148. Erickson L. C. and T. Yamashita. 1964. Effect of temperature on the ripening of Hass avocado Calif. Avocado Soc. Yrbk. 48:92-94. Fan X. and J. P. Mattheis. 1999. Methyl Jasmonate promotes apple fruit degreening independently of ethylene action. HortScience. 34:310-312. Fan X L. Argenta and J.P Mattheis. 2000. Inhibition of ethylene action by 1methylcyclopropene prolongs storage life of apricots Postharvest Biol. Technol. 20 : 135-142. Fan g, X L. Ar g enta and J. P Mattheis. 2000. Inhibition of ethylene action by 1m e th y lc y clopropene prolongs storage life of apricots. Postharvest Biol. Technol. 2 0 : 1 3 5-142 Fe n g X. A. Ap e lbaum E C Sisler and R. Goren. 2000 Control of ethylene responses in a v ocado fruit with 1-methylcyclopropene. Postharvest Biol. Technol. 20: 143150 i s h r R L. and A. B Bennett. 1991 Role of cell wall hydrolases in fruit ripening. nn R v Plant. Physiol. Plant. Mol. Biol 42:675-703 F lch J. M L and G. H Sloane tanly. 1957 A imple m thods for the isolation and purifi ca ti n of total lipid from animal tissues. J. Biol. h m 226:497-503

PAGE 175

162 Gazit ., and A. Blumenfeld. 1970. Response of matur e a v ocado fruit s to e th y l e n e b efore and after harvest. J. Am Soc. Hort Sci. 95 : 229-231. Giovannoni J. J. D. Dellapenna A B. Bennett and R. L. Fi s h e r. 198 9. Ex pr ess i o n of a chimeric polygalacturonase gene in transgenic rin (Rip e nin g inhibitor ) t o m a t o fruit results in polyuronids degradation but not fruit soft e nin g Plant C e ll 1 :53 63. Golding J B ., D Shearer W. B. McGlasson and S G. Wyllie. 1999 Relationship s between respiration ethylene and aroma production in ripening banana J. Ag. Food Chem. 47:1646 1651. Goldin g, J. B ., D. Shearer S. G. Wyllie and W. B. McGlasson. 1998 Application o f 1MCP and propylene to identify ethylene dependent ripening process in matur e banana fruit. Postharvest Biol. Technol. 14:87 98. Gross K. C. 1983. Changes in free galactose myo inositol and other monosaccharides in normal and non-ripening mutant tomatoes. Phytochemistry 22: 1137-1139 Gross K C. and C. E. Sams. 1984. Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23:2457 2461. Gross K C. A. E. Watada M. S. Kang S. D. Kim K. S Kim and S. W. Lee. 1986 Biochemical changes associated with the ripening of hot pepper fruit. Ph y siol. Plant. 66:31-36. Guis M. R. Botondi M. Ben Amor R. Ayub L. M. Bouzayen J.C. Pech and A. Latche. 1997. Ripening associated biochemical traits of cantaloupe Charwntais melon expressing an ACC oxidase transgene. J. Am. Soc. Hortic. Sci 122 : 748751. Ha g erman A. E. and P J. Austin. 1986. Continuous spectrophotometric assay for plant p e ctin methyl esterase. J. Ag. Food Chem. 34:440 444. Handenburg R. E. A. E. Watada and C. Y. Wang. 1986. The commercial storage of fruits vegetables and nursery stocks. USDA ARS Handbook. 66. Hansen E ., and G.D Blanpied 1968. Ethylene induced ripening of pears in relation to maturit y and length of treatment. Proc. Am. Soc. Hort. Sci. 93:807 812. Hatfield R ., and D. J. Nevins. 1986. Characterization of the hydrol y tic activit y of avocado cellulase. Plant Cell Physiol. 27:541 552. Hatton T. T. Jr. and C. W. Campbell. 1960. Evaluation of indices for Florida a v oc a do maturit y Fla. State Hort. Soc. Proc. 72:349-353. Hatton T. T. Jr. P L. Harding and W. F Reeder. 1964 Seasonal changes in Florida avocados U.S Dept. Ag Tech. Bui. 1310:47.

PAGE 176

163 Hatton T. T. Jr. and W. F Reeder. 1965. Maturity of minor varieties of Florida a ocados 1964-1965. Proc. Fla. State. Hort. Soc. 78:327-330. Hatton T. T. Jr. and W. F. Reeder. 1972. Quality of 'Lula' avocados stored in controll d atmo pheres with or without ethylene J Am. Soc. Hort. ci. 97:339 -341. Hob on G. 1962. Determination of polygalacturonase in fruits. Nature 195:804-805. Hua J. Chang Q Sun and E. M. Meyerowitz. 1995. Ethylene insensitivity conferred by Arabidopsis ERS gene. Science. 269: 1712-1714. Hua J. and E. M. Meyerowitz. 1998. Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94:261-271. Huber D. J. 1983a. Polyuronide degradation and hemicellulose modifications in ripening tomato fruit. J. Amer. Soc. Hort Sci. 108:405-409. Huber D. J. 1983b. The role of cell wall hydrolases in fruit softening. Hort. Rev. 5: 169219 Huber D.J. 1984. Strawberry fruit softening: the potential roles of polyuronides and hemicelluloses. J. Food Sci. 49:1310-1315. Huber D. J and D J. Nevins. 1981. Partial purification of endoand exo-~-Dglucanase enzymes from Zea mays L. seedlings and their involvement in cell wall autohydrolysis. Planta. 151 :206-214. Huber D. J. and E. M. O'Donoghue. 1993. Polyuronides in avocado (Persea Americana) and tomato (Lycopersicon esculentum) fruits exhibit markedly different pattern of molecular weight downshifts during ripening. Plant Physiol. 102:473-480. Ian DeVeau E. J. K C. Gross D .. J. Huber, and A. E. Watada. 1993. Degradation and solubilization of pectin by /3-galactosidases purified from avocado mesocarp Phy iol. Plant. 87:279-285. Inaba A and R Nakamura. 1986. Effect of exogeneous ethylen concentration and fruit t mp rature on the minium treatment time necessary to induc rip ning in banana fruit. J Jpn. oc Hort ci 55 : 348-354. Jahn . 1975 Rip ning r pons of green tomatoes to ethyl n cone ntration and t mp ratur itru V g. Mag. 39: 18-24. J ffi r y mith P. o d nough I. Prosser and D. Grierson. 1984. Ethylen ind e p e nd nt and thyl n -d p nd nt biochemical chang in rip ning tomato Pl nt Phy iol. 74:32-38

PAGE 177

164 Joyce D. C. A. J. Shorter and P N. Jones. 1995. E ffect of d e la ye d film wrapping an d waxing on the shelf life of avocado fruit. Aust. J Ex p. Agr. 35:657-659 Kader A. A. 1992. Postharvest technology of horticultural crops 2nd ed. University of California Division of Agriculture and Natural Resources. Davis. Kieber J J. M. Rothenberg G. Roman K. A. Feldmann and J.R. Ecker. 1993 CTRl a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raffamily of protein kinases. Cell 72:427-441. Knee I. M. 1978. Metabolism of polymethylgalacturonate in apple fruit cortical ti ss u e during ripening. Phytochemistry 17: 1261 1264 Knight R Jr. 1980. Origin and world importance of tropical and subtropical fruit crops In : S. Nagy and P.E. Shaw (eds) Tropical and subtropical fruits. A VI Publishing Inc. Westport CT. Kooiman P 1960. A method for the determination of amyloid in plant seeds. Reel. Trav Chim. Pays-Bas. 79:675-678. Kramer M ., R. Sanders H. Bolkan C. Waters R.E. Sheehy and W.R. Hiatt. 1992. Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: Processing firmness and disease resistance. Postharvest Biol. Technol. 1: 241 255. Ku V. V. V. and R. B. H. Wills. 1999. Effect of 1-Methylcyclopropene on the storage life of broccoli. PostharvestBiol. Technol. 17:127-132. Ku V. V. V. R. B. H. Wills and S. Ben Yehoshua. 1999. 1-Methylcyclopropene can differentially affect the postharvest life of strawberries exposed to ethylene HortScience. 34: 119-120. Lanahan, M B. H. C. Yen J. J. Giovannoni and H.J. Klee. 1994. The never-ripe mutation blocks ethylene perception in tomato. Plant Cell 6:521-530. Lazan, H. and Z. M. Ali. 1993. Cell wall hydrolases and their importance in the manipulation of ripening of tropical fruits. ASEAN Food J. 8:47-53. Lelievre J.M. A. Latche B. Jones M. Bouzayen and J.C. Pech. 1997a Ethylene and fruit ripening. Physiol. Planta. 101:727-739. Lelievre J. M. L. Tichit P. Dao L. Fillion Y. W. Nam J. C. Pech and A. Latche 1997b. Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits. Plant Mol. Biol. 33:847-855. Leopold A C., and P. E. Kriedemann. 1975. Plant growth and d eve lopment. 2 nd d McGraw-Hill NY.

PAGE 178

165 Littmann M. D 1972 Effect of water loss on the ripening of climacteric fruits Qu e n s land journal of Agriculture and Animal Science. 29: 103-113. Lunt R. E. H. Smith and M. M. Darvas. 1981. A comparison between waxing andcellophane wrapping of avocados for export. Yearbook of South African Avocado Grower Association 4:57-62. McColl um T. G. D. J. Huber and D. J. Cantliffe 1989. Modification of polyuronides and hemicelluloses during muskmelon fruit softening. Physiol. Plant. 76:303-308. McGuire R G. 1992 Reporting of objective color measurements HortScience. 27:12541255 McNeil M. A. Darvill P. Albersheim and S. Fry. 1984. Structure and function of the primary cell walls of plants. Ann. Rev. Biochem. 53:625-663. Meir S. M. Akerman Y Fuchs and G. Zauberman. 1994. Further studies on the controlled atmosphere storage of avocados. Postharvest Biol. Technol. 5:323-330 Meir S. D. Naiman M. Akerman J. Y. Hyman G. Zauberman and Y. Fuchs 1997. Prolonged storage of 'Hass' avocado fruit using modified atmosphere packaging Postharvest Biol. Technol. 12:51-60. Milner Y. and G. Avigad. 1967. A copper reagent for the determination ofhexuronic acids and certain ketohexoses. Carbohydr. Res. 4:359-361. Mort A J. B. M Moerschbacher M L. Pierce and N 0 Maness. 1991. Problems encountered during the extraction purification and chromatography of pectin fra g ments and some solutions to them. Carbohydr. Res. 215 :219-227. Mull e r R. M. Serek E. C. Sisler and A. S. Anderson. 1997. Post storage quality and rooting ability of Epipremnum pinnatum L. cuttings after treatment with ethylene action inhibitors. J. Hort. Sci. 72:445-452. Mullin s E. D. T. G. McCollum and R E McDonald. 2000. Consequ nces on thylene m e taboli s m of inactivating the ethylene receptor sites in diseas d non-climacteric fruit. Postharvest Biol. Technol. 19: 155-164. Nakat uka A ., hiomi Y. Kubo and A. Inaba 1997 Expr ssion and internal fi dba k r g ulation of A yntha e and A oxida e g n s in ripening tomato fruit. Pl a nt II Phy i 1. 38 : 1103-1110 n g hu M. 1992. 11 wall chang and the role of x-cellula during avocado fruit ri p nin g Univ r ity f Florida Gaine vill

PAGE 179

166 O'Donoghue E. M. and D. J. Huber. 1992. Modification of matri x p o l ysacc harid es during avocado (Per sea Americana) fruit ripening: an assessment of the role of C x cellu l as Physiol. Planta. 86:33-42. Oell r P. W ., L. M. Wong L P Taylor D. A. Pike and A. Theologis. 1991. Reversible inhibition of tomato fruit ripening. Science. 254:437-439. O e tik e r J. H. and S. F. Yang. 1995. The role of ethylene in fruit ripening. Acta Hort. 398 : 167-178. O'Neil M. P. Albersheim and A. Darvill. 1990. The pectic polysaccharides of prim ary cell walls p. 415-441 In: P. M. Dey (ed), Methods in Plant Biochemistr y. Academic Press New York NY. Payton S. R Fray S. Brown and D. Grierson. 1996. Ethylene receptor expression is regulated during fruit ripening flower senescence and abscission. Plant Mol. Biol. 31: 1227-1231. Pesis E. Y Fuchs and G. Zauberman. 1978a. Cellulase activity and fruit softening in avocado. Plant Physiol. 61 :416-419. Pesis E Y. Fuchs and G. Zauberman. 1978b. Starch content and amylase activity in avocado fruit pulp. J. Amer. Soc. Hort. Sci. 103:673-676. Pharr D. M. H. N. Sox and W. B. Nesbitt. 1976. Cell wall bound Nitrophenylglycosidase of tomato fruits. J. Am. Soc. Hort. Sci 101:397-400. Platt K. A. and W W. Thomson. 1992. Idioblst oil cells of avocado : distribution isolation ultrastructure histochemistry, and biochemistry Int. J. Plant Sci. 153:301-310. Porat R. A.H. Halevy M. Serek and A. Borochov. 1995 An increase in ethylene sensitivity following pollination is the initial event triggering an increase in ethylene production and enhanced senescence of phala e nopsi s orchid flowers. Physiol. Plant. 93 :778-784. Porat R ., B. Weiss L. Cohen, A. Daus R. Goren and S Droby. 1999. Effects of ethylene and 1-methylcyclorpropene on the postharvest qualities of 'Shamouti' orang es. Postharvest Biol. Technol. 15 : 155-163 Pressey R. 1983. 13-Galactosidases in ripening tomatoes. Plant Ph ys iol. 71: 132-13 5 Raymond D and H. J. Phaff. 1965. Purification and certain properti s of avocado polygalacturonase. J. Food Sci. 30:266 -27 3. Redgwell R. J. L. D. Melton and D. J. Brasch 1990. Cell wall chang sin kiwifruit following postharvest ethylene treatment. Phytochemistr y. 29:399-407.

PAGE 180

167 R dg 11 R. J. L. D Melton and D J. Brasch. 1991. Cell-wall polysaccharides of ki ifruit (Actinidia deliciosa): effect of ripening on the structural features of cell all materials Carbohydr. Res. 209: 191-202 Redg ell R. J. L. D. Melton and D. J. Brasch. 1992. Cell wall dissolution in ripening kiwifruit (Actinidia deliciosa). Solubilization of the pectic polymers Plant Physiol. 98:71-81. Rodriguez F L. J. J. Esch A. E. Hall B. M. Binder G .E. Schaller and A. B. Bleecker. 1999. A copper cofactor for the ethylene receptor ETRl from Arabidopsis Science 283 :996-998. Roe B. and J.H. Bruemmer. 1981. Changes in pectic substance and enzymes during ripening and storage of'Keitt' mangos. J. Food Sci. 46:186-189. Rose J. K C. K. A. Hadfield J. M. Labavitch and A B. Bennett. 1998 Tempera! sequence of cell wall disassembly in rapidly ripening melon fruit. Plant Physiol. 117:345-361. Rupasinghe H.P. V. D. P. Murr G. Paliyath and L. Skog. 2000. Inhibitory effect of 1MCP on ripening and superficial scald development in 'McIntosh' and 'Delicious' apples. J. Hort. Sci. Biotechnol. 75 :271-276. Ryall A. L. and W. T. Pentzer, (eds.) 1982. Handling transportation storage of fruits and vegetables. Vol. 2. AVI publishing Inc., Westport CT. Sakurai N and D. J Nevins. 1997. elationship between fruit softening and wall polysaccharides in avocado (Persea americana Mill) mesocarp tissues. Plant C 11 Ph y siol. 38:603-610. A -In ititute. 1985. SAS/STAT guide for personal computers. Version 6. SA Inst. ary N .. chroed r A. 1953 Growth and development of Fuerte avocado fruit. Proc. Am. Soc Hort. ci. 61:103-109 rek M and M. Reid 1993. Anti-ethylene treatments for potted flowering plants R lative efficacy of inhibitors of ethylene action and biosynth sis Hort c1 nc 28 : 1180-1181. R id. 1993 Commercial pro p cts for mod rating the ff e ct of thy! ne in pott d flow ring plants p. 423-426 In : Au rala ian P tharv t H rticultur onfi renc Proce ding pt mb r 20-24 Gatton Au tralia Th niv r ity f Qu n land Gatten olleg La

PAGE 181

168 Serek M. E. C Sisler and M. S. Reid. 1994a. Novel gaseous ethyl e n e bindin g inhibit o r prevents ethylene effects in potted flowering plants. J Am e r. oc. Hort ci 119:1230-1233. Serek M. E. C. Sisler and M. S. Reid. 1994b. A volatile ethylene inhibitor impro ves th e postharvest life of potted roses. J. Am. Soc Hort. Sci. 119 : 572-577 Serek M ., E. C. Sisler and M. S. Reid. 1995a. Effect of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regul. 16:93-97 Serek M. G. Tamari E. C. Sisler and A. Borochov. 1995b. Inhibition of ethylene induced senescence symptoms by 1-methylecyclopropene a new inhibitor of ethylene action. Physiol. Plant. 94:229-232. Seymour G. B. and G. A. Tucker. 1993. Avocado p. 53-81 In: G. B. Seymour J. Tayler and G.A. Tucker ( eds) Biochemistry of fruit ripening. Chapman & Hall London. Sisler E. C. M. S. Reid and S. F. Yang. 1986. Effect of antagonists of ethylene action on binding of ethylene in cut carnations. Plant Growth Regul. 4:213-218 Sisler E. C. and S. M. Blankenship. 1993a. Diazocyclopentadiene a light sensitive reagent for the ethylene receptor. Plant Growth Regul. 12: 125-132. Sisler E. C and S. M. Blankenship. 1993b. Effect of diazocyclopentadiene on tomato ripening. Plant Growth Regul. 12:155 160. Sisler E. C. S M. Blankenship, M. Fearn C. Jeffrey, and R. Hanes. 1993. Effect of diazocyclopentadiene (DACP) on cut carnations, In: J. C. Pech A. Latche and C Balaque (eds) Cellular and molecular aspects of the plant hormone ethylene. Kluwer Academic Publishers. Dordrecht The Netherlands. Sisler E. C ., and N. Lallu. 1994. Effect of diazocyclopentadiene (DACP) on tomato fruits harvested at different ripening stages. Postharvest Biol. Technol. 4:245-254. Sisler E C ., M. Serek and E. Dupille. 1995. Comparison of cyclopropene 1methylcyclopropene and 3 3-dimethylcyclopropene as ethylene antagonists in plants Plant Growth Regul. 17: 1-6. Sisler E. C. and S. M. Blankenship. 1996. Patent No. 5518988. USA 1996. Sisler E C ., E. Dupille and M. Serek. 1996a. Comparison of cyclopropene 1methylcyclopropene and 3 3-dimethylcyclopropene as ethyl e ne antagonists in plants. Plant Growth Regul. 18: 169-174.

PAGE 182

169 i 1 r E . E. Dupille and M Serek. 1996b. Effect of 1-methylcyclopropene and meth lenecyclopropane on ethylene binding and ethylene action on cut carnations. Plant Growth Regul. 18:79-86. i 1 r E. C. and M. Serek. 1997. Inhibitors of ethylene responses in plants at the receptor level Recent developments. Physiol. Plant. 100:577-582. Sisler E C and M. Serek. 1999. Compounds controlling ethylene receptor. Botanical Bulletin of Academia Scienicia 40: 1-7. Smith P K. R. I. Krohn G. T. Hermanson, A. K. Mallia, F. H. Gartner M. D. Provenzano E. K. Fujimoto N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85 Smith C. J. S ., C. F Watson J. Ray C.R. Bird P. C. Morris W Schuch and D Grierson. 1988. Antisense RNA inhibition of polygalaturonase gene expression in transgenic tomatoes. Nature 334:724726. Starrett D. and G. Laties. 1993. Ethylene and wound-induced gene expression in the preclimacteric phase of ripening avocado fruit and mesocarp discs. Plant Physiol. 103 : 227-234. Tong C. B. S. and K. C. Gross. 1988. Glycosyl-linkage composition of tomato fruit cell wall hemicellulose fractions during ripening. Physiol. Plant. 74:365-370. Valmayor R. V. 1967. Cellular development of avocado from blossom to maturity. Philippine Agriculturist. 50:907-976. Veen H. 1983. Silver thiosulfate: an experimental tool in plant science. Sci. Hort. 20 : 211-224. Wakaba y a s hi K. J. P. hun and D. J. Huber. 2000. Extensive olubilization and d polym e rization of cell wall polysaccharides during avocado (Persea americana) rip ning involve concert d action of polygalacturonase and pectinmethylest ra e Ph y iol. Planta 108:345-352. Watkin B J. M. Haki and C. Frenk 1. 1988. Activities of polygalacturonase a-Dmanno s ida e and a-Dand 13-D-galactosidases in ripening tomato. Hort c1 nc 2 3: 19 2 -194. Watkin B ., J. F. Nock and B. D Whitaker. 2000. Respons of arly mid and lat a on appl cultivars to postharvest application of 1-m thylcycloprop ne M P) und r air and controlled atmosphere torage condition Postharv st Biol. T c hn 1. 19: 17-32

PAGE 183

170 Wegr zy n T. and E. MacRae. 1992. Pectinesterase polygalacturona se, and 13galactosidase during softening of ethylene-treated kiwi fruit. HortSci e nce 27:900902. Whitaker J R. 1972. Principles of enzymology for the food sciences Marcel D ekker Inc. New York. Wilkinson J. Q. M. B. Lanahan H. C. Yen J. J. Giovannoni and H.J. Klee. 1995. An ethylene-inducible component of signal transduction encoded by Never-ripe Science 720:1807-1809. Wills R. B. H. and S. I. H Tirmanzi. 1982. Inhibition of ripening of avocado with calcium. Sci. Hort. 16:323-330 Yang S. F ., and N. E. Hoffman. 1984. Ethylene biosynthesis and its regulation in higher plants Ann. Rev. Plant Physiol. 35:155-189. Yueming J. C. J. Daryl and J.M. Andrew. 1999. Responses of banana fruit to treatment with 1-methylcyclopropene. Plant Growth Regul. 28:77-82. Yueming J. and F. Jiarui. 2000. Ethylene regulation of fruit ripening: Molecular aspects. Plant Growth Regul. 30: 193-200. Zauberman G ., and M. Schiffmann-Nadel. 1972. Pectin methylesterase and polygalacturonase in avocado fruit at various stages of development. Plant Physiol. 49:864-865. Zauberman, G. and Y. Fuchs. 1973. Effect of ethylene on respiration rate and softening of avocado fruit at various stages of development. Intl. Inst. Refrig. Commission C2. l 07-109. Zauberman G. Y. Fuchs U. Yanko and M. Akerman. 1988. Response of mature avocado fruit to postharvest ethylene treatment applied immediately after harvest. HortScience. 23:588-589

PAGE 184

BIOGRAPHICAL SKETCH Ji on Jeong was born in Korea in 1968. He obtained a Bachelor of c1ence degree in horticultural science from Seoul National University in 1993 and a Master of cience degre in plant science from California State University Fresno in 1997. 171

PAGE 185

l c rtif y th a t I ha r ad this tudy and that in my opini n it con~ rm s to ac pt a bl tandard of c hol a rly pre entation and i s fully ad quat in sco pe and qualit y r a a di rt a ti n fo r th d t:-r e of Doctor of Philo r Donald J. Huber Chair Professor of Horticultural Science l certify that I have read this study and that in my opinion it conforms to acceptable standards of cholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Steve A. Sargent C chair Professor of Horticultural Science I certif y that I ha ve r e ad this study and that in my opinion it conforms to acce pt a ble standards of sc holarly presentation andrtis f JJy adequate in scope and quality as a di sse rtation for the d eg ree of Doctor of Philoso y J LvJ L Charles A. Sims Professor of Food Science and Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ssociate Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of sc holarly presentation and is fu ly adequate in scope and qualit y, as a dissertation for the degree of Doctor of Philosop Professor of Horticultural c1 nc

PAGE 187

Thi di rtation wa ubmitted to the Graduate Faculty of the College of Agricultural and Life ci nces and to the Graduate School and was accepted as partial fulfillm nt of the requirements for the degree of Doctor of Philosophy December 2001 Dean Graduate School

PAGE 188

19BF10 1 7740 03/18/02 34760

PAGE 190

LO 1780 20 ....... Q l UNIVERSITY OF FLORIDA 111 11 1111 11 111 11 I I II II I I III I I II I I II I I II II I I II II II II I I II II IIIII I I 3 1262 08554 4533