Citation
A Rapid, Simple, Inexpensive and Reproducible Endo-Beta-Mannanase Assay Test for Determining Optimal Hydrothermal Timing of Commercial Priming of Lettuce Seed

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Title:
A Rapid, Simple, Inexpensive and Reproducible Endo-Beta-Mannanase Assay Test for Determining Optimal Hydrothermal Timing of Commercial Priming of Lettuce Seed
Creator:
BONINA, JENNIFER R. ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Analysis of variance ( jstor )
Endosperm ( jstor )
Enzymes ( jstor )
Germination ( jstor )
High temperature ( jstor )
Lettuce ( jstor )
Seed germination ( jstor )
Seed priming ( jstor )
Seeds ( jstor )
Tomatoes ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Jennifer R. Bonina. Permission granted to University of Florida to digitize and display 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.
Embargo Date:
5/1/2005
Resource Identifier:
436098695 ( OCLC )

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A RAPID, SIMPLE, INEXPENSIVE AND REPRODUCIBLE
ENDO-BETA-MANNANASE ASSAY TEST FOR DETERMINING OPTIMAL
HYDROTHERMAL TIMING OF COMMERCIAL PRIMING OF LETTUCE SEED
















By

JENNIFER R. BONINA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Jennifer Bonina














Then God said:
"I give you every seed-bearing plant on the face of the whole earth and every tree
that has fruit with seed in it. They will be yours for food."
Genesis 1:29
























I would like to dedicate this thesis to my parents, Jill and Andrew; my sisters, Jessica and
Jolene for their love and support while pursuing this endeavor; and to my boyfriend,
Joshua, for his enduring love and encouragement.















ACKNOWLEDGMENTS

First, I thank God for my passion for science, for allowing me to rely on His

strength and power, and for teaching me patience as I complete this journey. This has

been one of the most challenging but rewarding experiences of my life. I will forever

cherish all of the friendships I have made during my time in Gainesville, FL.

Next, I would like to express the depth of my gratitude to Dr. Daniel J. Cantliffe

(department chairman) committee chair, for the opportunity to pursue this degree, and for

guidance and support throughout my research. I extend my warmest appreciation to Dr.

Donald J. Huber and Dr. Peter J. Stoffella for their patience, invaluable advice, and time

spent serving on my supervisory committee.

I offer sincere thanks to our Lab Biologist, (Nicole Shaw) for her assistance during

my research. I would also like to acknowledge all the present and past members of the

Seed Physiology Lab who fostered an environment of learning in the lab, provided

valuable advice, and even helped me count seeds for my germination experiments on

more than one occasion. Special thanks go to Paulo Campante-Santos, Ivanka Kozareva,

Jeanmarie Mitchell, Amanda Collins and Camille Esmel.

I would like to express my appreciation to Dr. Grace Ju at Educational Concerns

for Hunger Organization (ECHO) Fort Myers, FL. You were an inspiring mentor at

Gordon College and gave me the courage to pursue this degree in science.

Finally, I would like to express my personal gratitude to my mother. Without your

encouragement, love, and faith in me, this research could not have been accomplished.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .............. ................. ............ .......................... vii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

A B STR A C T ................................................. ..................................... .. x

CHAPTER

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

2 LITER A TU R E REV IEW ............................................................. ....................... 4

Therm odorm ancy in L ettuce............................................................................. 4
Techniques to Circumvent Therm odorm ancy ........................................ ...................5
Seed P rim ing T techniques ............................................................. ....................... 6
Factors A affecting Prim ing .................................................. .............................. 9
Lim stations to Prim ing Treatm ents.................................................................. ....... 10
Control of Lettuce Seed Germination at High Temperatures................................12
Endo-Beta-Mannanase Activity and Endospermic Seeds ........................................13
Improving Seed Priming Technology..................................................................... 17

3 ENDO-BETA-MANNANASE ASSAY CORRELATION TO PRIMING
TREA TM EN T ........................................................ .......... ............... 19

M materials and M methods ....................................................................... ..................20
Plant M material and Production ................................... ............................. ....... 20
Seed Dry Weight and Seed Moisture Content.........................................22
Lettuce seed M icroscope Study................................................. ............... 22
S eed P rim in g .................................................... ................ 2 3
G erm nation Experim ents............................................ ............................ 23
Endo-beta-mannanase Enzyme Activity Assay..........................................25
Results and Discussion .................................... ...... .. ...... ............. 27

APPENDIX

A ENDO-BETA-MANNANASE ASSAY ....................................... ............... 55









B ANALYSIS OF VARIANCE TABLES...... ................. ..............56

L IT E R A T U R E C IT E D ............................................................................. ....................60

B IO G R A PH IC A L SK E T C H ...................................................................... ..................68















LIST OF TABLES


Table page

3-1 Production locations and conditions for Bennett and Connick cultivars .................21

3-2 Germination (%) of non-primed lettuce in the light at 200C and 36C
(E xperim ent # 1) ......................................................................36

3-3 Germination (%) of non-primed 'Bennett' and 'Connick' lettuce seed after 8
months storage at 100C and 45% RH (Experiment 2) .................. ................36

3-4 Germination (%) of primed and non-primed lettuce seed in dark and light at
several tem peratures (Experim ent 3 and 4)................................... .................38

3-5 Mean hours to germination (MHG) of primed and non-primed lettuce seed in
dark and light at several temperatures (Experiments 3 and 4)..............................42

3-6 Total percent of seeds with endo-beta-mannanase activity (EBM) in the
endosperm during seed prim ing ........................................ ......................... 43

3-7 Total percent of seeds with endo-beta-mannanase activity (EBM) in the
endosperm after termination of each hydrothermal priming duration-seeds were
assayed at Oh time and after dry back and 14 days of storage ............... ...............46

3-8 Germination (%) and Mean Hour to Germination (MHG) of primed lettuce
seeds (from EBM study) after 14 days of storage in the light at 200C and 36C
(E x p erim en t 5).................................................... ................ 5 0

3-9 Cost analysis for Endo-beta-mannanase assay test for 1000 seeds (100 lots) .........54

A-i Analysis of variance for germination (%) non-primed lettuce in light at 200C and
3 6 C ........................................................................................5 6

A-2 Analysis of variance for germination of non-primed 'Bennett' and 'Connick'
lettuce seed after 8 months storage at 100C and 45% RH........................................56

A-3 Analysis of variance for mean hours to germination (MHG) and total
germination of primed and non-primed lettuce seed in dark and light at a range
of tem peratures .......................... ... ...... ...... .............................. 57









A-4 Analysis of variance for total percent of seeds with endo-beta-mannanase
activity in the endosperm during seed priming ........................................ .......... 58

A-5 Analysis of variance for total percent of seeds with endo-beta-mannanase
activity in the endosperm priming and 14 days of storage................... ..............59

A-6 Analysis of variance for germination of primed lettuce seeds (from EBM study)
after 14 days of storage in Light at 200C and 36 C .............................................. 59















LIST OF FIGURES


Figure page

3-1 Size differences between 'Bennett' and 'Connick' lettuce seed cultivars ...............29

3-2 Seed moisture content (SMC) of lettuce seeds imbibed in water ..........................30

3-3 Radicle protrusion through the micropylar tip of the endosperm of non-primed
lettuce after 15h of imbibition in water at 200C in light. .............. .......... ....31

3-4 Radicle protrusion through the endosperm (view of whole seed) of non-primed
lettuce (pericarp removed) after 14h of imbibition in water at 200C in light...........32

3-5 Radicle protrusion through the endosperm and pericarp (view of whole seed) of
non-primed lettuce (pericarp removed) after 15h of imbibition in water at 200C
in lig h t .............................................................................. 3 3

3-6 Seed moisture content of lettuce seeds in polyethylene glycol. Vertical bars
indicate standard error ........................... ........... .. ... ............ 34

3-7 Micropylar tip of lettuce seed after priming in PEG (no visible radicles) ..............35

3-8 Percent of seeds exhibiting endo-beta-mannanase activity (by seed part) during
prim ing .............................................................................44

3-9 Micropylar tip of lettuce seed after priming in PEG, stored and re-imbibed for
3hrs on ice (no visible radicles)........................................ ............................ 45

3-10 Percent of seed exhibiting endo-beta-mannanase activity (by seed part) after dry
back and re-im bibition ......................... .... ................ ........................ 47

A-i Assay of Endo-beta-mannanase in individual lettuce endosperm seed parts using
a g el d iffu sio n .................................................. ................ 5 5















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

RAPID, SIMPLE, INEXPENSIVE AND REPRODUCIBLE
ENDO-BETA-MANNANASE ASSAY TEST FOR DETERMINING OPTIMAL
HYDROTHERMAL TIMING OF COMMERCIAL PRIMING OF LETTUCE SEED

By

Jennifer R. Bonina

May 2005

Chair: Daniel J. Cantliffe
Major Department: Horticultural Sciences

Extended high temperatures during imbibition of lettuce (Lactuca sativa) can lead

to a phenomenon known as thermoinhibition and thermodormancy, causing non-uniform

germination and emergence. Seed priming was developed to circumvent this problem.

Priming regulates water uptake by using an osmoticum. This hydrates the seed to a

sufficient moisture level, allowing initial stages of germination to occur before radicle

emergence. However, the requirements for (and results from) priming lettuce seed vary

greatly according to age, vigor, and thermosensitivity. Optimal duration of priming time

must be determined for each species, cultivar, and seed lot. The enzyme endo-beta-

mannanase (EBM) is a prerequisite for radicle emergence in some endospermic seeds. In

lettuce, this enzyme has been observed during priming and causes weakening of the

lettuce endosperm. The present investigation determined the optimal hydrothermal

timing for priming, lettuce seeds, independent of cultivar, seed lot, and storage (using an









inexpensive gel assay for endo-beta-mannanase). It is proposed that the assay test can

accurately indicate the optimal hydrothermal priming time by correlating EBM activity in

seed sub-sampled from each priming lot, to correlate with optimal priming time.

Two head-type lettuce cultivars 'Bennett' and 'Connick' and two seed lots of each

(produced both in Australia and California) were used in our study. The seeds were

primed in an aerated polyethylene glycol 8000 (PEG) solution at 150C in constant light,

and were sampled at 24 h, 48 h, and 72 h. A single-seed gel assay for endo-beta-

mannanase was used to determine enzyme activity in each lot at each priming duration.

At 360C, germination of primed seeds was over 90%; while non-primed seeds

germinated at 40%. Priming increased germination rate up to 40% as compared to non

primed seeds at both 20C and 36C under either light or dark conditions. EBM activity

was non-detectable in dry seeds and for seeds primed for 24 h exhibited any EBM

activity. After priming for 48 or 72 h, 30% of the micropylar section or the whole seed

exhibited EBM activity regardless of seed lot or cultivar. Since the percentage of seeds

exhibiting EBM in the whole endosperm after 48 or 72 h of priming were similar and

germination rate and total percent germination were optimal at 48 h prime duration, 48 h

was determined as the optimal hydrothermal priming time. EBM activity was observed

in >25% of all lots at 48 h prime duration. Thus it was proposed if at least 25% of seeds

exhibit EBM activity, optimal hydrothermal time is achieved. Determining priming time

with the EBM test, gives the seed industry a method to better optimize the priming

procedure for endospermic seeds such as lettuce.














CHAPTER 1
INTRODUCTION

Non-uniform seed emergence, especially at high temperatures, can lead to poor

stand establishment in crops such as lettuce (Lactuca sativa). Prolonged emergence

periods can predispose plants to damage by adverse environmental conditions and result

in poor uniformity and a low percentage of seedling emergence. Slow emergence results

in smaller plants and seedlings, that are more vulnerable to soil-borne diseases (Ellis

1963). Rapid field emergence under all conditions is a fundamental prerequisite for

increasing the yield of many species and increasing profits from annual crops.

Methods have been developed to overcome non-uniform seed germination at high

temperatures including priming (Cantliffe 1981), seed coat removal (Guedes and

Cantliffe 1980; Ikuma and Thimann 1963; Keys et al. 1975), and seed-coat puncture

(Sung 1996). Commercial seed priming was introduced in the 1970s and is currently the

most common method used in research and industry (Cantliffe 2000). Seed priming is a

presowing treatment: seeds are placed in an osmotic solution and controlled imbibition

occurs (along with the initial stages of germination before radicle emergence) resulting in

more rapid, uniform seed germination (Heydecker et al. 1973).

The requirements for and results from seed priming of different crops vary greatly

according to the species and seed lot; and vary within lot due to storage. Developing a

new testing methodology that improves the efficiency of the seed priming technique is

the ultimate goal of this study. The proposed investigation is to determine optimal

hydrothermal timing for priming in lettuce seeds, independent of cultivar, seed lot, and









storage using an inexpensive endo-beta-mannanase gel assay (EBM). It is proposed that

this simple rapid test could be used as an indicator of optimal hydrothermal priming time.

Endo-beta-mannanase hydrolyzes mannan-polymers in the endosperm cell walls of

lettuce. The enzyme is produced and secreted by the endosperm (Halmer and Bewley

1979). Dutta et al. (1997) reported that cell-wall-bound endo-beta-mannanase was

expressed in lettuce endosperm prior to radicle emergence, and noted that enzyme levels

were regulated by the same conditions that govern seed germination. Nascimento et al.

(2001) reported a build-up of endo-beta-mannanase both before radicle emergence and

during priming of lettuce. Since endo-beta-mannanase detection indicates readiness for

germination, the assay could indicate when sufficient priming has occurred, rather than

requiring additional time-consuming germination trials. The assay test might accurately

indicate the optimal hydrothermal priming time by correlating EBM activity with the

critical point when optimal priming has occurred, thus seeds will not be over-primed or

under-primed.

By defining optimal hydrothermal priming time with the EBM assay test, the seed

industry will have a method that optimizes the priming procedure resulting in a simple,

more efficient test. In the present work, the test was evaluated for lettuce, to correlate

endo-beta-mannanase build-up prior to radicle emergence as an indicator of the

completion of priming; however, endo-beta-mannanase activity has also been observed

prior to radicle emergence in other vegetable seed species including tomato, pepper,

carrot and celery (Bewley 1997b; Watkins et al. 1985; Williams et al. 2001). Thus, the

test could be used in other seed species thereby streamlining the priming procedure by









not requiring additional germination trials for each seed lot or seed type; resulting in

improved seed performance in many seed species in the greenhouse or field.














CHAPTER 2
LITERATURE REVIEW

Hydrothermal priming is a technique used to circumvent dormancy and improve

seed performance at high temperature in many seed species including lettuce will be

reviewed. An overview will be provided of the mechanisms of lettuce seed germination

including restriction at high temperature and the role of endo-beta-mannanase in breaking

temperature dormancy.

Thermodormancy in Lettuce

Seed dormancy is commonly described as the failure of a viable seed to germinate

under favorable growing conditions (Bewley 1997a). Many factors play a role in

promoting or inhibiting dormancy. The embryo could be restricted by the surrounding

seed coat (seed coating), or the seed may require a specific temperature or light treatment

(Bewley 1997a). In a mature dry seed, uptake of water is triphasic; initially there is a

rapid uptake of water, followed by a plateau phase, and finally increased water uptake

until germination is completed and radicle emergence occurs. When seeds are dormant,

they do not complete germination and do not enter phase III (Bewley 1997a). Certain

seeds (including lettuce) can enter secondary dormancy if temperatures are too high for

an extended length of time, resulting in no germination.

When lettuce seeds are imbibed at high temperature for a period of time below 72 h

one of two phenomena can occur. If imbibition at high temperatures is brief, a reversible

condition known as "thermoinhibition" will occur that can be broken when the

temperature is returned to an optimal range (-200C) (Khan 1980/81). Sung et al. (1998)









reported that when lettuce seeds were exposed to high temperatures (above 350C) for a

period of time less than 72 h and then moved to lower temperatures (200C), normal

germination occurred (Sung et al. 1998). If seeds are exposed to high temperatures for

prolonged periods of time (more than 72 h) before high temperatures are reduced, the

seeds will enter a secondary dormancy referred to as "thermodormancy" and the seeds

will not germinate (Nascimento 1998).

Techniques to Circumvent Thermodormancy

All seeds depend on temperature for optimal germination and stand establishment

(Nascimento 1998). Lettuce seeds will germinate at temperatures ranging from as low as

5C to as high as 330C, optimal temperature ranges are generally between 150C and 22C

but vary by genotype (Cantliffe et al. 2000; Gray 1975). A reduction in germination

occurs at temperatures ranging from 250C to 270C, and if lettuce seeds are allowed to

imbibe water at an elevated temperature (280C to 330C), germination will not occur

(Borthwick and Robbins 1928, Cantliffe et al. 2000).

To circumvent thermoinhibition in lettuce seed, several methods have been

developed including controlled hydration (priming) (Cantliffe 1981), seed-coat removal

(Guedes and Cantliffe, 1980; Keys et al. 1975; Ikuma and Thimann 1963) and seed-coat

puncture (Sung 1996). Hormones such as gibberellic acid (GA) also induce germination

in lettuce, tomato, and many other seeds (Bewley 1997b). Dry storage of freshly

harvested seeds for a certain period of time known as after-ripening also helps to break

dormancy (Atwater 1980; AOSA 1993; Baskin and Baskin 1998).









Hydration treatments promote initial stages of germination by hydrating the seed,

which increases the moisture level, thereby increasing germination rate and uniformity

(Copeland and McDonald 1995; Pazdera and Hosnedl 2002).

Hydration treatments are divided into two categories: pre-hydration and controlled

hydration (Taylor et al. 1998). Pre-hydration does not limit water uptake; rather it is

governed by the seeds' affinity to water. Seeds are imbibed on moistened blotter paper or

in water, and the process is ended at a specific time to prevent radicle emergence

(Pazdera and Hosnedl 2002). Controlled hydration regulates water uptake by osmotic,

drum, and solid matrix priming. Seed priming, as described by Heydecker (1973), is a

presowing treatment in which seeds are placed in an osmotic solution. The amount of

water available for the seed is restricted, thereby hydrating the seed to a sufficient

moisture level; allowing initial stages of germination prior to radicle emergence. The

seeds can be re-dried to the original moisture content for storage, or planted directly

(Parera and Cantliffe 1994). Priming with osmoticum was introduced in the 1970s and is

currently the most common method used in research and industry to overcome

non-uniform seed germination at high temperatures (Cantliffe 2000).

Seed Priming Techniques

To prime with osmoticum, seeds are imbibed with a solution of either inorganic

salts such as KNO3, K3P04 or polyethylene glycol 8000 (PEG) for a period of time at a

constant temperature (Bodworth et al. 1981; Cantliffe et al. 1981; Cantliffe et al. 2000;

Ellis et al. 1963; Hegarty et al. 1977; Heydecker et al. 1973).

In early work, inorganic salts such as NaNO3, MnSO4, and MgCl were used as

osmoticum to prime pepper (Capsicum annum) seed (Kotowski 1926). Later, K3PO4 and

KNO3 were used to the improve germination rate in tomato seed (Ellis 1963).









Controversy occurred over the use of inorganic salts because of conflicting germination

rates in some crop species. This occurred in carrot (Daucus carota) and leek (Allium

porrum) seed primed in KH2PO4 and PEG. Seeds primed in KH2PO4 germinated less

compared to those treated with PEG (Brocklehurst and Dearmam 1984). In contrast,

tomato germination rate increased at 150C in seeds treated with KNO3 compared to PEG

(Alvarado et al. 1987). Pepper germination rates were similar when seeds were primed in

NaC1, MgSO4 or PEG (Aljaro and Wyneken 1985). Currently many seed species are

primed with PEG including celery (Apium graveolens) (Perez-Garcia et al. 1995),

sunflower, (Helianthus annuus) (Mwale et al. 2003), and leek (Bujalski et al. 1993).

In addition to the use of inorganic salt, or PEG, mannitol has also been used as a

priming solution to regulate osmotic potential (Parera et al. 1994). Pepper seeds primed

in mannitol had improved final percent germination over those not primed. (Georghiou et

al. 1987). Other species including pepper, eggplant (Solanum melongena), and melon

(Cucumis melo) seeds primed in mannitol produced larger seedlings than non-primed

seeds (Passam et al. 1989). Sodium polypropionate (Zuo et al. 1988) or glycerol

(Brocklehurst and Dearman 1984) have been used to prime seeds. Synthetic sea water

has also been used on tomato, (Lycopersicon esculentum) and asparagus (Asparagus

officinalis) (Owen and Pill 1994; Pill et al. 1991).

Growth regulators have also been added to the priming solution. Carter and

Stevens (1998) determined that when primed with gibberellic acid (GA3), pepper seeds

had higher germination (91%) as compared to non-primed at high temperatures (400C).

However, the addition of growth regulators is added expense above PEG or salt costs.

Therefore, growth regulators are often added in low concentration to priming solutions









(Lorenz et al. 1988). Priming with PEG and GA4+7 increased early germination in pepper

especially at low temperature (150C), 3.8 days as compared 4.7 days in non-primed seeds

(Watkins and Cantliffe 1983). Lorenz at al. (1988) reported that the addition of GA to

the PEG priming solution increased soybean (Glycine max) emergence rate. In celery,

seeds primed with PEG + GA and ethylene germinated at high temperatures as compared

to non-primed seeds (Brockelhurst et al. 1982). In some cases, fungicides were also

added to prevent pathogen growth (Leskovar and Sims 1987; Szafirowska et al. 1981).

There are benefits and drawbacks to priming with solutions of salt, polyethylene

glycol, mannitol, or others. Using salt as an osmoticum is less expensive and easier to

remove from the seed after treatment, whereas PEG is often viscous and adheres to the

seed, requiring a rinse step (Parera and Cantliffe 1994). Yet, elevated ion concentrations

from salts can affect the embryo and germination, as reported by Brockelhurst and

Dearman (1984). When lettuce seed was primed in K3PO4, more water was imbibed by

the seeds than PEG or PEG+ K3PO4, it was suggested that the difference was due to

natural differences in the seed coat composition (Guedes et al. 1979). This also occurred

in onion (Allium cepa), carrot (Daucus carota), celery and leek (Allium porrum) seeds

primed with KH3PO4 and there was also a reduction in germination than seeds primed

with PEG (Brockelhurst and Dearman 1984). This suggests that ions from salt solutions

can penetrate and build up inside the seed during priming and the accumulation could

reduce the osmotic potential of the seed and induce more water absorption as the

treatment progresses (Parera and Cantliffe 1994). Priming with PEG, which is a

chemically inert osmoticum, does not allow movement of the PEG molecule through the









seed coat due to its large molecular weight allowing for only water movement into the

seed during imbibition (Parera and Cantliffe 1994).

Factors Affecting Priming

Many factors influence the success of lettuce seed priming, including temperature,

aeration, length of priming treatment, water potential of the osmotic solution, as well as

the species, cultivar, seed quality and storage conditions (Cantliffe et al. 2000; Guedes

1979; Heydecker and Gibbins 1978; Parera and Cantliffe 1994). Some lettuce seed

varieties are more heat sensitive than others therefore, and modifications to the priming

process must be made carefully to ensure that the priming treatment will circumvent

thermodormancy for all lettuce varieties.

In thermosensitive 'Minetto' lettuce seed, aeration of a 1% K3PO4 osmotic solution

at 150C increased germination slightly to 33% as compared to seeds that were not aerated

(28%) especially when germinating at high temperatures (350C) (Guedes and Cantliffe,

1980). The study also determined that priming at 150C resulted in higher germination

after 6h (64%) at 350C compared to other priming temperatures, 50C (22%) or 25C

(48%) (Guedes and Cantliffe 1980). Other studies conducted by Khan et al. (1980/81)

confirm that the ideal priming temperature range is 100C to 150C, temperatures above

that do not significantly improve the priming procedure. In another lettuce variety,

germination of 'Mesa 659' improved when primed for three to four days at 150C in -8.4

bar PEG as compared to seeds primed for shorter durations (one to two days), therefore

priming time must be determined to achieve optimal results (Khan et al. 1981). Storage

often affects primed seeds, especially thermosensitive 'DGB' seeds had germination of

99% after priming but had reduced germination (27%) when stored for six months.









However, thermotolerant 'EVE' was not affected by storage (Nascimento and Cantliffe

1998). Consequently, storing 'DGB', a thermosensitive variety, after priming is not

recommended as 'DGB' seeds may be more desiccation-sensitive increasing

susceptibility to deterioration (Nascimento and Cantliffe 1998). In some cases, using both

salt and an osmoticum such as PEG improve germination in lettuce. A thermosensitive

lettuce cultivar, 'Valmaine' had improved germination (71%) with no thermodormancy

when both K3PO4 and PEG were used as compared to priming in either alone (-50-60%)

(Cantliffe 1981).

In conjunction with many factors affecting the success of a priming treatment, there

are limitations to its use as a seed treatment since the treatment conditions are often

varied depending on species, cultivar, and seed lot, these may have to be individually

tested. Identifying optimal hydrothermal priming time is critical to achieve proper

moisture content within the seed. Often the length of time must be adjusted for each seed

lot, cultivar, or species; this can be inefficient but it is currently the industry standard

practice.

Limitations to Priming Treatments

There are limits to the length of seed storage once primed due to a more rapid

reduction in seed viability over time (Alvarado and Bradford 1988; Argerich and

Bradford 1989; Carpenter and Boucher 1991; Maude et al. 1994; Odell and Cantliffe

1986). Standard industry practice for seed priming recommend seed to be primed prior to

the immediate growing season to avoid reductions in viability.

Dearman and Bradford (1987) observed a reduction in seed viability after storage

of primed leek and carrot but it has also been reported in tomato (Alvarado and Bradford

1988; Argerich and Bradford,1989; Odell and Cantliffe 1986), lettuce (Tarquis et al.









1992; Weges, 1987) and wheat (Triticum aestivum L.) (Nath et al. 1991). In one study,

tomato seeds primed in PEG or 3% KNO3 solutions, the seeds germinated under

laboratory and field conditions, then were stored at 100C, 200C, and 300C, for 12 months

or longer. Seeds primed and stored for up to 18 months at 100C and 45% RH maintained

50% more rapid germination than when stored at lower temperatures. At 300C storage,

primed seeds had increased loss of viability over non-primed seeds (Alvarado et al.

1988).

Reduction in seed viability and longevity has been reported in many lettuce studies.

Tarquis et al. (1992) reported a 61% decrease in emergence rate (MTG) but seed

longevity in storage was reduced by as much as 84% compared to non-primed seeds. In

'Dark Green Boston' (DGB) and 'Everglades' (EVE) lettuce, seeds primed with PEG had

a 100% germination rate at high temperature (Nascimento et al. 1998). When the

primed seeds were stored for six months at 100C at 45%RH for 12 months, germination

remained at 100% at 200C and 35C in the EVE cultivar but in DGB, viability decreased

(Nascimento and Cantliffe, 1998). To prevent reductions in vigor and germination, seeds

are often not stored after priming for more than nine months, thereby retaining the

improved germination from the treatment and avoiding the deleterious effects of storage.

Treating seeds with PEG can also be more costly than salt solutions, and it requires

disposal in an environmentally safe manner. It is also more difficult to achieve uniform

aeration during priming with PEG due to the viscosity of the solution. Furthermore, PEG

often adheres to the seed, requiring an additional water rinse step (Parera and Cantliffe

1994).









Control of Lettuce Seed Germination at High Temperatures

The lettuce endosperm consists of a mannose-rich double cell layer, completely

encompassing the embryo (Borthwick and Robbins 1928). Cells in the micropylar region

opposite the radicle tip are more prolific, up to three or four cell layers thick (Dutta et al.

1994). For germination to occur, the radicle must grow and penetrate the micropylar end

of the endosperm (Bewley and Black 1994). Researchers suggested that lettuce

endosperm cell walls acted as a barrier to germination, mechanically restricting radicle

emergence especially at high temperatures (300C) leading to secondary dormancy

(Borthwick and Robbins 1928; Dutta et al. 1994; Halmer et al. 1976; Ikuma and Thimann

1963; Nascimento et al. 2000). To overcome mechanical restriction at high temperature,

two mechanisms have been suggested; pressure exerted on the endosperm by the

developing embryo and endosperm tissue weakening, which may work together leading

to germination (Bewley and Black, 1994; Bradford 1995; Nascimento 1998) However,

the mechanisms that control thermoinhibition and thermodormancy, though widely

studied, remain unclear.

Early studies conducted by Halmer et al. were the first to demonstrate endo-beta-

mannanase synthesis and activity in the lettuce (Lactuca sativa L.) endosperm (Halmer et

al. 1975; Halmer et al. 1976). Enzyme activity was detected "post-germatively" and was

thought to aid in mobilization of endosperm reserves to serve as a nutrient source for

seedling development (Halmer et al. 1979). Weakening of the lettuce endosperm layer

by an enzyme, endo-beta-mannanase, was later determined to be a prerequisite to radicle

emergence, especially at high temperatures (Park et al. 1974, Halmer et al. 1975;

Nascimento 1998; Sung 1996). Oluoch and Welbaum (1996) reported that thinning of

the endosperm occurred at the radicle end prior to germination reducing resistance and









promoting radicle emergence in melon and parsley (Petroselium crispum), a process

which may also occur in lettuce (Olszewski et al. 2004).

The enzyme endo-beta-mannanase is a cell-wall hydrolytic enzyme that is thought

to be synthesized "de novo" once imbibition begins (Bewley 1997b). Endo-beta-

mannanase degrades galactomannan-rich endosperm cells, which produce and secrete the

enzyme (Halmer et al. 1979). Endo-beta-mannanase activity increases initially in the

micropylar region and later increases in the rest of the endosperm cells. (Leviatov et al.

1995; Nomaguchi, et al. 1995; Toorop et al. 1996). Activity is often detected in the

lateral region of the endosperm once radicle emergence occurs (Groot 1988; Nomaguchi

et al. 1995; Nonogaki et al. 1992). Nonogaki and Morohashi (1996) suggested that the

presence of endo-beta-mannanase first in the micropylar and then the lateral may be due

to the different physiological roles of the enzyme. When it is present pre-germinatively

in the micropylar region it is responsible for cell wall weakening and post-germinatively

for galactomannan storage reserve mobilization.

Endo-Beta-Mannanase Activity and Endospermic Seeds

Many endospermic seeds including lettuce contain reserves which are retained

exclusively in the endosperm until maturity. These reserves are degraded by specific

enzymes after the initiation of germination to facilitate the developing seedling until

photosynthesis is initiated (Bewley and Black 1994; Homrichhausen et al. 2003). In most

cases, endo-beta-mannanase degrades endosperm cell walls in endospermic seeds and can

be detected during germination in tomato, carrot, asparagus (Asparagus officinalis), and

pepper (Bewley 1997b, Cantliffe et al. 2000; Homrichhausen et al. 2003; Watkins et al.

1985; Williams et al. 2001). In fenugreek and carob seed, endo-beta-mannanase activity









was not detected until after the completion of germination (Bewley 1997b; Kontos et al.

1995; Reid and Bewley 1979; Spyropoulos and Reid 1988).

The role of endo-beta-mannanase has been extensively studied in tomato, due to the

presence of high levels of endo-beta-mannanase due to the endosperm, which is the major

storage tissue and serves as a nutrient source for the embryo during seedling development

(Nonogaki and Morohashi 1995). In lettuce however, the endosperm is not major nutrient

source for the embryo and only supplies nutrients to the embryo for a short period of time

until the cotyledons develop thus, lower levels of endo-beta-mannanase have been

detected (Bewley and Black 1994). However, tomato often serves as the model for

further studies of the role of endo-beta-mannanase in lettuce.

In tomato, endo-beta-mannanase activity increases in intact endosperms prior to

radicle emergence, and is initially detected in the thin-walled endosperm (micropylar) cap

opposite the radicle tip prior to germination (Nonogaki et al. 1992; Still and Bradford

1997). Endo-beta-mannanase increases later (-48 h) in the rest of the endosperm,

followed by germination. The presence of endo-beta-mannanase at two separate times in

the same tissue prior to germination indicates that EBM is mobilized prior to germination

and plays a key role during germination (Bewley 1997b; Leviatov et al. 1995; Nonogaki

et al. 1996; Toorop et al. 1996). This indicates that the enzyme builds up prior to

germination, weakens the cell wall and aids radicle emergence (Groot et al. 1987; Groot

and Karssen 1988; Toorop et al. 1996).

In the mutant gibberellin-deficient (gib-1) tomato, there was also an increase in

endo-beta-mannanase activity in the endosperm when the seeds were treated with

gibberellic acid (GA) (Groot et al. 1988). Therefore, it was suggested that endosperm









weakening prior to radicle emergence was mediated by gibberellin-induced enzymatic

degradation of the cell walls (Bewley 1997b; Groot et al. 1988; Nascimento 1998).

However, more recently it has been suggested that germinating tomato radicles produce

or release GA that induces endo-beta-mannanase production in the endosperm.

Consequently, the enzyme causes weakening of the endosperm cell walls, reducing

mechanical resistance, leading to radicle emergence (Bewley, 1997b; Ni and Bradford,

1993). In lettuce, the presence of gibberellin alleviated thermoinhibition and increased

endo-beta-mannanase activity (Dutta et al. 1997; Nascimento 1998).

The presence of endo-beta-mannanase prior to radicle emergence in lettuce has

been debated. Early studies reported no endo-beta-mannanase activity in 'Grand Rapids'

lettuce seeds prior to radicle emergence (Halmer et al. 1975) while others claimed that

endo-beta-mannanase is strictly a post-germative event (Bewley 1997b). However, it

was reported by Dutta et al. (1997) that cell-wall-bound endo-beta-mannanase was

expressed in lettuce endosperm prior to radicle emergence and that enzyme levels were

regulated by the same conditions that govern seed germination.

Endo-beta-mannanase activity varies in the whole endosperm, depending on

genotype (Dirk et al. 1995). In a study of five non-primed lettuce cultivars, endo-beta-

mannanase was not detected in the first 4 hours of imbibition incubated at 200C

(Nascimento et al. 2000). However, after 6 hours activity was detected in all the cultivars

except Dark Green Boston 'DGB', a thermosensitive type. Activity could be detected in

seeds imbibed at 350C but only after radicle emergence, except in two of the

thermotolerant cultivars, PI 251245 'PI' and Everglades 'EVE'(Nascimento et al. 2000).

The increase in endo-beta-mannanase in the thermotolerant genotypes may have been due









to a lower endosperm resistance as compared to thermosensitive genotypes suggesting

that weakening of the endosperm layer was a pre-requisite for radicle emergence

(Nascimento 1998; Sung et al. 1998)

Nascimento et al. (2001) reported an increase of endo-beta-mannanase both before

radicle emergence and after priming in lettuce. Endo-beta-mannanase could be detected

in non-primed and primed 'EVE' and 'DGB'. The study noted that endo-beta-mannanase

activity was higher in seeds incubated at 200C than 350C possibly due to high

temperature which may inhibit endo-beta-mannanase enzyme synthesis (Dutta et al.

1997; Nascimento et al. 2001). However, primed seeds germinated at 350C germinated

faster (-4h) compared to 200C (-12h). This suggested that the presence of endo-beat-

mannanase in lower concentration ensured germination at high temperature.

This may also be the case in stored primed lettuce seed. After three days of dry-

back, seeds imbibed at 350C germinated at 100% and endo-beta-mannanase was 1.3 pmol

min' in DGB and 81 pmol min' in EVE indicating that even if levels of endo-beta-

mannanase are low (1 pmol min' ) at high temperatures, it is adequate for endosperm

weakening (Nascimento 1998; Nascimento et al. 2001).

In a related study, endo-beta-mannanase was detected during priming between 24

and 48 hours in EVE and between 24 and 72 hours in DGB. Enzyme activity in primed

EVE seeds remained highest as compared to DGB and non primed lettuce seeds,

suggesting that priming could overcome the inhibitory effect of elevated temperatures

due to the weakening effect of endo-beta-mannanase enzyme on the endosperm cell walls

(Nascimento 1998). Endo-beta-mannanase activity persisted even after seed drying when

seeds were re-imbibed. However, endo-beta-mannanase activity detected after radicle









emergence was still higher than prior to radicle emergence (Nascimento et al 2001). This

may be due to carbohydrate mobilization in the endosperm (Nascimento et al. 2001).

The mechanisms that control endo-beta-mannanase leading up to germination in lettuce

are still largely unknown. However, much more is known about mechanisms

surrounding endo-beta-mannanase in tomato making tomato and ideal model for the

study of endo-beta-mannanase in lettuce.

Improving Seed Priming Technology

The conventional method for circumventing temperature-related dormancy in many

seeds is priming, yet there are factors that limit the efficiency of this technology. The

optimal duration of priming time must be determined for each cultivar and each seed lot

due to variations in seed age, vigor and thermosensitivity. Adjusting the priming

conditions for each cultivar and lot requires additional time and labor.

Though initially reported as a post-germinative event, Dutta et al. (1997) observed

endo-beta-mannanase in endosperm cell walls prior to radicle emergence and noted that

enzyme levels were regulated by the same conditions that govern seed germination.

Nascimento et al. (2001) reported the build up of endo-beta-mannanase both before

radicle emergence and during priming of lettuce using the gel diffusion assay. Thus, it is

endo-beta-mannanase that has been detected during priming.

If a peak in the number of seeds exhibiting endo-beta-mannanase activity can be

determined during priming using the gel-diffusion assay and correlated to optimal

germination at high temperature then the assay may be used to determine optimal

hydrothermal priming time. The gel-diffusion assay test (Downie et al. 1994; Still and

Bradford 1997) requires minimal training and labor and is simple, rapid and inexpensive

to perform. Using this test to indicate the achievement of optimal hydrothermal time









during priming would provide additional safeguards and quality control for companies

that prime and sell primed lettuce seed.

The endo-beta-mannanase activity assay could be used as an indicator of optimal

priming time, increasing efficiency in priming procedures used for improving seed

quality. By defining optimal hydrothermal priming for different vegetable seed species,

this technology will aid the seed industry by providing a more efficient procedure for

improving the accuracy of priming. As a result, there may be a more consistent

improvement in primed seed performance in the greenhouse or field environment.














CHAPTER 3
ENDO-BETA-MANNANASE ASSAY CORRELATION TO PRIMING TREATMENT

Non-uniform seed emergence, especially at high temperatures, can lead to poor

stand establishment in crops such as lettuce (Lactuca sativa). Rapid field emergence

under all conditions is a fundamental prerequisite to increase yield of many species and

ultimately profits from annual crops. Methods have been developed to overcome non-

uniform seed germination at high temperatures including priming (Cantliffe et al. 1981),

seed coat removal (Guedes and Cantliffe 1980; Ikuma and Thimann 1963; Keys et al.

1975) and seed coat puncture (Sung 1996). Commercial seed priming is currently the

most common method used in research and industry however, there are drawbacks to the

technique (Cantliffe 2000).

The requirements for and results from priming lettuce seed vary greatly according

to variations in seed age, vigor and thermosensitivity. Optimal duration of priming time

must be determined for each cultivar and seed lot. This can take additional time and

labor. Developing a new testing methodology that improves the efficiency of the seed

priming technique is the ultimate goal of this work. The proposed investigation is to

determine optimal hydrothermal timing for priming in lettuce seeds, independent of

cultivar, seed lot, and storage using an inexpensive endo-beta-mannanase gel assay

(EBM). It is proposed that this simple rapid test could be used as an indicator of optimal

hydrothermal priming time.

Nascimento et al. (2001) reported a build up of endo-beta-mannanase both before

radicle emergence and during priming of lettuce. Dutta et al. (1997) reported that cell-









wall-bound endo-beta-mannanase was expressed in lettuce endosperm prior to radicle

emergence and noted that enzyme levels were regulated by the same conditions that

govern seed germination. Since endo-beta-mannanase detection indicates readiness for

germination, the assay indicates when sufficient priming has occurred, rather than

requiring additional time-consuming germination trials. The assay test indicates

accurately the optimal hydrothermal priming time by correlating EBM activity with the

critical point when optimal priming has occurred, thus seeds are not over-primed or

under-primed.

Two cultivars of lettuce, 'Bennett' and 'Connick', Lactuca sativa and two seed lots

(Australia and California) were primed for 0 h, 24 h, 48 h and 72 h. EBM activity was

determined. The EBM assay is a rapid and simple test that can be completed in 32 hours.

Seeds were re-dried, and then germinated at 360C to determine the effectiveness of the

priming process. Germination in all lots was improved to over 90% when the seeds were

primed for 48 h or 72 h. Essentially no EBM activity was detected in non-primed seeds

or seeds primed for 24 h. By using this model developed for lettuce, the seed industry

will benefit by this procedure, improving the efficiency and consistency of results of the

priming process for different vegetable seed species, cultivars and seed lots.

Materials and Methods

Plant Material and Production

Two 'head-type' lettuce (Lactuca sativa L.) cultivars ('Bennett' and 'Connick')

produced by Sun Seed Company (now Nunhems Seeds USA, Parma, ID) were used in

this study. The seeds were stored at 100C, 40% RH for subsequent use in this study

(Table 3-1).






21


Table 3-1. Production locations and conditions for Bennett and Connick cultivars
Cultivar Production Location Harvest Date Avg. max Avg. min
temp temp
Bennett New South Wales, AU February 2002 780F/260C 660F/190C

Bennett Kerman, CA September 2001 910F/32C 560F/120C

Connick New South Wales, AU February 2002 780F/260C 660F/190C

Connick Kerman, CA September 2001 910F/320C 560F/120C









Seed Dry Weight and Seed Moisture Content

Seed dry weight was determined by oven drying four replications of 1 g of seed for

8 h at 1300C. Seeds were removed and placed in a desiccator for 20 minutes and

weighed (AOSA, 1993).

Fresh weight was determined by weighing Ig of seed (per replication) during

priming (seed priming protocol previously referred to above) at 150C with constant light

in an incubator (Precision Scientific, Winchester, VA). Seed weight was recorded at time

intervals of 1 h, 2 h, 3 h, 4 h, 6 h ,12 h, 24 h, 48 h and 72 h. Seeds were removed from

a -1.2 MPa of polyethylene glycol 8000 (PEG) (30 ml of solution per 1 g of seed) and

rinsed once with water using a Buchner funnel and seed weight was recorded. Seed

moisture content (SMC) protocol was repeated in water and fresh weight was recorded at

time intervals of 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, 16 h, 20 h and 24 h. Seed moisture content

as a percentage of weight was calculated using the following formula: (fresh weight dry

weight) / fresh weight x 100. The experimental design was conducted using a split-plot

design. Standard error was calculated at each time interval for each cultivar and seed lot.

Lettuce seed Microscope Study

Non-primed seeds (20) were placed on one layer of 5.0 cm diameter Whatman #1

filter paper moistened with distilled water in a 5.5 cm Petri dish. The dishes were covered

and placed at 200C under constant light in an incubator (Precision Scientific, Winchester,

VA). The pericarp was removed from the seed and photos were taken with a Leica

MZ 16 microscope (Leica Microsystems, Wetzlar, Germany) at each hour until radicle

emergence (approximately 15 h) to trace radicle growth as protrusion through the

endosperm and seed coat.









Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30 ml

of solution per 1 g of seed) of 30% -1.2 MPa PEG 8000 (30 g /100 ml water) (Fischer

Scientific, Philadelphia, PA) for 24, 48 or 72 hours at 15Co with constant light

(fluorescent -30 [nmol m-2 s-1) in an incubator (Precision Scientific, Winchester, VA). At

each priming duration, 0.5 g samples were removed from the PEG priming solution and

rinsed once with 25 ml of distilled water. Following the rinse, the pericarp was removed

using forceps and a surgical blade. Microscope images were immediately recorded

digitally. The remaining primed seeds were damp dried and stored for 7 days in

uncovered 5.5 cm Petri dishes in a storage room at 160C, 50% RH. After 7 days, the

seeds were removed from storage and imbibed on moistened #1 filter paper (2 ml

distilled water) in a 5.5 cm Petri dish on ice for 3 hours. The pericarp was removed and

images were recorded immediately.

Seed Priming

Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30 ml

of solution per 1 g of seed) of 30% -1.2 MPa PEG 8000 (30 g/100 ml water) (Fischer

Scientific, Philadelphia, PA) for 24, 48 or 72 hours at 150C with constant light in an

incubator (Precision Scientific, Winchester, VA). After each priming time, seeds were

removed and placed in a Buchner funnel and rinsed three times with distilled water, damp

dried, and stored in uncovered 5.5 cm Petri dishes in a cold storage room at 160C and

50% RH for four to six days.

Germination Experiments

For each cultivar, seed lot, and priming time, 25 seeds each placed on one layer of

5.0 cm diameter Whatman #1 filter paper, moistened with 2 ml of distilled water in 5.5

cm Petri dishes, and incubated under constant light (fluorescent -26 [mol m-2 s1) in









incubators (Precision Scientific, Winchester, VA). Germination, defined as visible radicle

protrusion through the pericarp, was recorded at three hour intervals beginning at 9 h, 12

h, 15 h, 18 h, 21 h, 24 h, 48 h and 72 hours after initial incubation.

Experiment #1: Non-primed seeds were germinated in each incubator (chamber) at

200C and 360C in the light. Experiment #2: Non-primed seeds were incubated in each

incubator (chamber) at 150C, 200C, 240C, 300C, 360C in the light. Experiment #3:

Primed seeds were germinated in each incubator (chamber) 150C, 200C, 240C,

300C,360C in the light. Seeds were primed according to previously listed protocol for

seed priming. Experiment #4: Primed seeds were germinated in each incubator

(chamber) 150C, 200C, 240C, 300C, 360C in the dark similar to Experiment #3. Dark

germination was monitored under a green safe light. Primed dark and light seed

germination experiments were not conducted concurrently. Experiment #5: Primed seeds

from the endo-beta-mannanase assay test were germinated after 14 days in storage, 16C

and 50% RH. Seeds were primed according to previously listed protocol for seed

priming. The seeds were germinated in each incubator (chamber) 150C, 200C, 240C,

30C,36C in the light.

Total percent germination and mean hours to germination (MHG) were calculated

as a measure of response to the treatment priming time (ptime). The MHG was

calculated according to the formula XTi/INi, where Ni is the number of newly

germinated seeds at hour interval Ti (Maguire, 1962).

A split-block experimental design was used with temperature as the main block and

cultivar, lots and priming time as the split block. Analysis of variance (ANOVA) was

conducted by the Statistical System (SAS) software (SAS, 2002). Mean separation for









significant main effects or interactions were performed by Least Significant Difference

(LSD p=0.05).

Endo-beta-mannanase Enzyme Activity Assay

A gel-diffusion assay protocol was used (Downie et al. 1994; Still et al. 1997) as

described by Nascimento et al. (2001). The assay was used to detect endo-beta-

mannanase enzyme activity during the priming treatments. Gel plates were prepared by

dissolving 0.05% (w/v) galactomannan (locust bean gum, Sigma Chemical Co., St. Louis,

MO) in incubation buffer (0.1 M citric acid, 0.2M Sodium phosphate, pH 5.0), stirring

and heating for 30 minutes. Afterward, the solution was clarified by centrifugation at

15,000 g for 15 minutes at 40C. Phytagar (Gibco Lab., Grand Island, NY) at 0.7% (w/v)

was added to the clarified solution and stirred while heating to the boiling point. Thirty

ml of solution was dispensed into 150x25 mm disposable Petri dishes (Falcon, Franklin

Lakes, NJ). After solidification, 32 wells per plate were made using a 2-mm disposable

plastic pipette to remove excised gel by aspiration.

Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30ml

of solution per Ig of seed) of 30% -1.2 MPa PEG 8000 (30g/100 ml water) (Fischer

Scientific, Philadelphia, PA) for 0, 24, 48 or 72 hours at 150C with constant light in an

incubator (chamber) (Precision Scientific, Winchester, VA). Seed samples (0.5 g) were

removed from the priming solution and assayed immediately. For each cultivar, seed lot,

and priming time, four replications of 18 seeds were used in each study, assays were

conducted on six whole individual endosperms, six micropylar radiclee tip only) and six

lateral (remaining endosperm) from the lettuce seeds primed 24 h, 48 h, or 72 h. The

remaining 0.5 g of each period of time was rinsed three times in distilled water, damped

dry and stored in uncovered 5.5 cm Petri dishes in a cold storage room at 160C and 50%









RH for 14 days prior to subsequent EBM assay testing. Endosperms were excised by

pressing on the cotyledon end using forceps and the tip of a surgical blade. A portion of

the excised endosperms remained intact (whole) while the others were separated into the

micropylar tip and the lateral endosperm with forceps and a surgical blade. Each seed

part (whole, micropylar tip or lateral) was placed into an individual microtiter plate

(Nalge Nunc, Naperville, IL) well containing 20 pL of sterile incubation buffer (0.1 M

citric acid, 0.2 M sodium phosphate, pH5.0) and incubated in the dark for 2 hours at

250C.

After incubation, 10 tL of buffer from each well was transferred to the gel-

diffusion plates and incubated for 24 h. A serial dilution of endo-beta-d-mannanase A.

Niger enzyme was added as a standard to each plate, 0.5 ul EBM into 2 ml H20=stock,

#1 5 ul/1 ml H20, #2 5 ul/10 ml H20, #3 5 ul/50 ml H20, #4 5 ul/100 ml H20, #5 5

ul/500 ml H20, #6 5 ul/1000 ml H20 (Megazyme International Ireland Ltd., Wicklow,

Ireland). Petri dishes were covered with a lid and aluminum foil and wrapped in Parafilm

(American National Can., Greenwich, CT). Gels were stained by adding 10 mL of Congo

Red (Sigma Chemical Co., St. Louis, MO) in water (0.4% w/v) to each plate. Plates were

shaken for 20 minutes at 60 rpm during staining. The Congo red solution was decanted

and the gel was gently rinsed with distilled water for 1 min, then 10 mL of

citrate-phosphate buffer pH 7.0 was added. After 3 min on the orbital shaker at 60 rpm,

the buffer was decanted. Plates were scanned within 5-10 min using a Scan Jet 3c/T

(Hewlett Packard, Palto Alto,CA). The diameter of the cleared areas on the plates

indicated endo-beta-mannanase activity. Only seed parts with observed activity were

used for analysis using WinRhizo tm (Regent Instruments Inc., Quebec, Canada) software.









Percentage of seeds assayed with endo-beta-mannanase activity per seed part and

calculated; (number of seeds with activity / total number of seeds assayed) / x 100.

A randomized complete block experimental design was used with treatments

replicated four times. Analysis of variance (ANOVA) was conducted for the percentage

data by seed part with the Statistical Analysis System (SAS) software (SAS,2002).

Significant main effects means were reported by least significant difference (LSD), 5%

level.

Results and Discussion

A significant difference in seed size was observed between the seed lots (-4 mm

Australia and -3 mm California) which may have occurred due to the higher maturation

temperatures (-320C) in California during production compared to Australia (-260C)

(Figure 3-1). Lettuce seed size and weight can be affected by seed maturation

temperature. Sung et al. (1998) observed that when lettuce seeds matured at

temperatures above 300C, seeds were smaller in size. Thus, larger seeds were produced

under 260C than 320C but it also may be due to plant growth conditions such as

irrigation, fertilization and sun light. The seed moisture content (SMC) of quiescent (dry)

seeds varied by seed lot, 7.1% SMC for BAU, 7.0% SMC for CAU, 6.7% SMC for BCA

and 6.5% SMC for CCA. This may have been due to a difference in seed size (Figure

3-2).

Water uptake during imbibition followed a classical triphasic pattern in solutions of

either water or PEG (Bewley and Black 1994). In water, rapid water uptake occurred

during the first hour (-60% fresh weight) of imbibition (Figure 3-2). After 6 hours, SMC

increased to -80% and a plateau occurred between 6 h and 12 h with a final rapid uptake

of water and radical emergence (Figure 3-3). Radicles were visible through the









endosperm after 14 h (Figure 3-4) and through the pericarp between 14 h and 15 h

(Figure 3-5). Between 16 hours and 20 hours, all cultivars attained approximately 115%

increase in fresh weight as compared to original dry weight.

In PEG, water uptake occurred rapidly in the first hour (-40%), followed by an

increase in uptake between 2 h and 6 h (-58) (Figure 3-6). A plateau occurred after Hour

8 (Figure 3-6). Seeds primed in PEG remained at 60-62% moisture without visible

radicles when removed from solution at 24 h, 48 h or 72 h (Figure 3-7). High levels of

osmoticum (PEG) inhibited radicle emergence while allowing for metabolic reactions to

occur during the lag phase of water uptake (Heydecker and Gibbins 1978; Karssen et al.

1989). Consequently, total percent germination and germination rate will increase when

seeds are re-imbibed at planting, especially at high temperatures.

Initial germination tests were conducted on non-primed seeds to determine

germination rate and optimal germination temperature (Table 3-2). Therefore, cultivar

and lot means were separated under these temperatures. At 200C, germination was 95%

for Bennett and 73% for Connick cultivars. The California lot had lower germination at

20C, 71% as compared to the Australia lot which indicated extreme thermosensitivity,

though 200C is an optimal temperature for lettuce (Khan, 1980/81).

After eight months of storage at 100C and 50% RH, germination of all lots was

nearly 100% for temperatures up to 250C whereas at 300C germination was above 80%

(Table 3-3). After-ripening during storage may have led to improved germination at

(300C). Thermodormancy persisted at the highest temperature 360C and germination of

all lots was less than 10%. These results are typical for thermosensitive cultivars.
































Figure 3-1. Size differences between 'Bennett' and 'Connick' lettuce seed cultivars from
left to right: Bennett produced in Australia (BAU), Connick produced in
Australia (CAU), Bennett produced in California (BCA), and Connick
produced in California (CCA).












140


120 -


100 -

BAU
80 BCA
SRadicle- CAU
-0- CCA
60 Emergence


40


20 -


20



0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324

Time(hr)



Figure 3-2. Seed moisture content (SMC) of lettuce seeds imbibed in water. Vertical bars
indicate standard errors



































Figure 3-3. Radicle protrusion through the micropylar tip of the endosperm of
non-primed lettuce after 15h of imbibition in water at 200C in light.






























Figure 3-4. Radicle protrusion through the endosperm (view of whole seed) of
non-primed lettuce (pericarp removed) after 14h of imbibition in water at
200C in light





























Figure 3-5. Radicle protrusion through the endosperm and pericarp (view of whole seed)
of non-primed lettuce (pericarp removed) after 15h of imbibition in water at
200C in light













140


120


100
-U- BAU
-*- BCA
80 -- CAU
-*- CCA

660


40


20 -4


0



0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Time(hr)
Figure 3-6. Seed moisture content of lettuce seeds in polyethylene glycol. Vertical bars
indicate standard error.





































Figure 3-7. Micropylar tip of lettuce seed after priming in PEG (no visible radicles). A)
Oh. B) 24h. C) 48h. D) 72h. The pericarp and seed coat were removed, the
endosperm is visible.









Table 3-2. Germination (%) of non-primed lettuce in the light at 200C and 36C
(Experiment #1)
Germination (%)
Cultivar 200C 360C
Bennett 73 4
Connick 95 4
Significance NS
Seed Lot
Australia 97 6
California 71 2
Significance ** *
*,**,Ns Significant at 5%(*) level, 1% (**) level respectively and non-significant (NS)


Table 3-3. Germination (%) of non-primed 'Bennett' and 'Connick' lettuce seed after 8
months storage at 100C and 45% RH (Experiment 2)
Temp (oC) Germination (%)
15 100
20 98
24 98
30 84
36 8
LSDo.05 5.8
ANOVA
Temperature (T) **
Cultivar (C) NS
Lot (SL) NS
C*T NS
L*T NS
C*L NS
C*SL*T NS
**,NS Significant at 1% (**) level and non-significant (NS). Mean separation within
temperature by Least Significant Difference (LSD) test p = 0.05









The various seed lots were primed for 0 h, 24, 48 or 72 hours in order to determine

optimal hydrothermal priming time for the lettuce cultivars. There were no differences in

germination between cultivar or seed lot. Only the main effects of priming time was

significant at 300C and 360C (Table 3-4). Germination increased in all primed seeds at

300C and 360C in light and dark compared to non-primed seeds. At temperatures below

240C, all non-primed and primed seeds germinated over 90% in both light and dark

(Table 3-4).

In light, germination at 300C was 91% in non-primed seeds whereas priming for

24h or longer germination increased to 99%. At 360C, germination of primed seeds was

over 90% while non-primed seeds germinated at 40% or less regardless of light or dark.

Thus, priming overcame thermosensitivity of the' seeds at high temperature (360C).

Germination of primed seeds in the dark at 300C was over 92% whereas non-

primed seed germination was 18% (Table 3-4). Primed seeds germinated over 90% at

360C regardless of priming time, whereas non-primed seed germinated 1%. Germination

in dark at 360C resulted in reduced germination of seeds primed for 72 h compared to

those primed for 24 h or 48 h. Low germination of seeds imbibed in the dark at high

temperatures (300C) was reported for other lettuce cultivars (Sung et al. 1998;

Nascimento et al. 2000). Other studies have also observed an interaction between high

temperature and light and suggested it is due to a phenomenon that occurs during

mediation by the phytochrome system (Taylorson and Hendricks 1972; Fielding et al.

1992). However, priming improved total percent germination, regardless of temperature,

duration of priming time, or germinating in the dark.












Table 3-4. Germination (%) of primed and non-primed lettuce seed in dark and light at several temperatures (Experiment 3 and 4)
Temperature (OC)
Light Dark
Priming time (h) 15 20 24 30 36 15 20 24 30 36
0 98 93 99 91 35 98 95 95 18 1
24 91 96 98 99 91 96 99 97 94 96
48 96 99 100 99 98 100 100 100 99 97
72 98 100 100 99 96 99 100 99 92 88
LSDo.05 4.0 8.3 8.8 8.7
ANOVA
Cultivar (C) NS NS NS NS NS NS NS NS NS NS
Lot (SL) NS NS NS NS NS NS NS NS NS NS
Ptime (PT) NS NS NS ** ** NS NS NS ** **
PT*SL NS NS NS NS NS NS NS NS NS NS
C*SL NS NS NS NS NS NS NS NS NS NS
C*PT NS NS NS NS NS NS NS NS NS NS
C*SL*PT NS NS NS NS NS NS NS NS NS NS


-,NS Significant at 1% (**)
0.05.


level and non-significant (NS) Mean separation within temperature by Least Significant Difference test p-









Priming decreased mean hour to germination (MHG), and improved germination

rate at each temperature in light or dark when compared to non-primed seeds (Table 3-5).

The increases in uniformity, synchrony and rate confirm findings reported in other

studies of seed priming (Argerich and Bradford 1989; Brocklehurst et al. 1984). In the

light, seeds primed for 48 or 72 h germinated most rapidly, 50% faster as compared to the

non-primed seeds at 30 to 360C. The three priming treatments had similar germination

rates at the lower temperatures (150C to 240C), but germinated approximately 30% faster

as compared to the non-primed seeds. In the dark, all priming treatments had similar

MHG and germination rate was -65% faster as compared to control with the exception of

the 24 h treatment at 15C which was slower, 51%. Therefore, the 24 h priming

treatment led to slower germination and priming was not as complete as 48 h or 72 h. In

the dark, germination was slower at 360C (-50%) as compared to the control. MHG in

the dark at 72 h of priming was less than 48 h, thus, priming may have gone too far.

EBM activity was observed in a low number of seeds or non-detectable levels in

the control or seeds primed for 24 hours (Figure 3-8). Studies conducted by Nascimento

et al. (1998) also noted that EBM activity was not detected in 'dry' (not imbibed) non-

primed lettuce seeds but suggested that EBM synthesis occurred during priming hence

EBM was observed. After priming for 48 h or 72 h, 30% of the micropylar and whole

seeds exhibited EBM activity regardless of location, seed lot, or cultivar (Figure 3-8).

The percentage of seeds exhibiting EBM after 72 h were slightly higher as compared to

the 48 h prime duration, however the values were not significantly different (Table 3-6).

In the lateral seed part there was a fluctuation in activity at both 48 h and 72 h prime

durations. After 48 h of priming EBM activity was in 48% of the seeds in the Australia









seed lot and 2% in the California seed lot, respectively. At 72 h EBM activity was

observed in higher percentages (54%) in the Australia seed lot as compared to 36% in the

California seed lot. It is also important to note that in the Connick California seed lot, a

lower percentage of seeds exhibited EBM activity in the micropylar region (Table 3-6).

The difference in observed EBM in the lateral endosperm and the micropylar

region of the Connick California seed lot may have occurred for several reasons. When

the micropylar and lateral endosperm were separated during sampling, the tissues were

disrupted. This may have caused lower levels of EBM to be detected in the lateral region

at 24 h and 48 h, as EBM may have leached out. Nascimento (1998) observed a

correlation between higher EBM activity in primed seeds incubated at 200C and faster

germination rate compared to non-primed seeds. It was suggested that the elevated

amount of EBM in the primed seeds caused endosperm cell wall weakening leading to

more rapid germination. MHG was 35% slower at 200C in the California seed lot after

48 h of priming than the Australia seed lot when the primed seeds were germinated

(Table 3-8). This may be why lower EBM was observed in the Connick California lot.

Thus, lower levels of EBM were observed when germination rate was slower.

EBM was also detected in re-dried seeds (Table 3-7) as well as during priming

(Table 3-6). Radicles were also not visible in any of the seeds during priming (Figure

3-9) and after three hours of imbibition of re-dried primed stored seeds (14 days). EBM

activity was observed in a low number of seeds or non-detectable levels in the control or

seeds primed for 24 hours (Figure 3-10). EBM was observed in the micropylar tip in all

the seed lots, except in the Connick California seed lot (Table 7). This may be due to

disruption during sampling. At 48h prime duration, EBM increased to 20% or higher in









all seed parts (Figure 3-10). The difference in observed EBM in the lateral endosperm

was similar to that of seeds removed directly from the priming solution (Table 3-6). At

48h prime duration 'Bennett' had a higher percentage of seeds that exhibited EBM

activity than 'Connick', 61 and 22%. After 72h prime duration, 'Connick' was higher

with 39% and 22% seeds with activity, respectively. This may also have been due to the

separation of the micropylar and lateral endosperm leading to tissue disruption causing

EBM to leach out. Also, it is likely that the EBM enzyme may be inactive in portions of

the endosperm cell walls, thus EBM could not be detected. Which may be the case in the

lateral region of the endosperm as EBM mobilizes storage reserves from the lateral

endosperm immediately prior to radicle emergence.

Similar amounts of seeds exhibited EBM activity (<25%) in whole seeds after 48 h

or 72 h of priming. Since there was no significant increase in EBM in the total percent of

the seeds exhibiting EBM activity it is proposed that 48 h prime duration is the optimal

hydrothermal priming time. Therefore, if 25% of seeds in the priming solution exhibit

EBM activity optimal hydrothermal time has been reached.

Also as noted, the 24 h priming treatment led to slower germination and 72 h of

priming led to reduced total percent germination (Table 3-4 and Table 3-5). Thus,

priming for 24 h was not as complete as 48 h and priming for 72 h may have gone too far.

Therefore, it is proposed that the optimal hydrothermal priming time is 48 h as it led to

the highest total percent germination and increase in uniformity and rate for all lots in

both light conditions.












Table 3-5. Mean hours to germination (MHG) of primed and non-primed lettuce seed in dark and light at several temperatures
(Experiments 3 and 4)
Temperature (C)


Light


Priming time (h)


24 28
48 17
72 16
LSDo.05 6.4
ANOVA
Cultivar (C) NS
Lot (SL) *
Priming time (PT) **
PT*SL NS
C*L NS
C*PT NS
C*SL*PT NS
*,**,NS Significant 5% (*), 1%
Significant Difference test p


(**) confidence levels,
=0.05.


20 24


Dark
30 36


and non-significant (NS) Mean separation within temperature by Least









Table 3-6. Total percent of seeds with endo-beta-mannanase activity (EBM) in the
endosperm during seed priming. (seeds were assayed at Oh time and after
termination of each hydrothermal priming duration)
Seed Part
Micropylar Lateral Whole
Lot
Priming Time 'AU' 'CA'
(h)
Percent of Seeds Exhibiting EBM Activity
0 ND ND ND ND
24 2 8 ND 2
48 30 48 2 25
72 41 54 36 31
LSDo.05 12.8 13.3 10.0

Cultivar Seed Lot
Bennett Australia 16
California 22
Connick Australia 29
California 5
LSDo.05 9.5
ANOVA
Cultivar (C) NS NS NS
Lot (SL) NS ** *
Priming time (PT) ** ** **
C*SL NS NS
C*PT NS NS NS
SL*PT NS NS
C*SL*PT NS NS NS
*,**,NS Significant 5% (*), 1% (**) confidence levels, and non-significant (NS). Endo-beta-
mannanase enzyme activity was not detected (ND).

















50


m 40
w

S30





10
a-


0 24 48 72
Priming Time (hr)


Figure 3-8. Percent of seeds exhibiting endo-beta-mannanase activity (by seed part)
during priming. Means pooled for cultivar, lot and rep. n= 16. Micropylar
LSD=12.8, Lateral LSD=13.3, Whole LSD=10.0.


Smicropylar
lateral
- w hole





































Figure 3-9. Micropylar tip of lettuce seed after priming in PEG, stored and re-imbibed for
3hrs on ice (no visible radicles). A) Oh. B) 24h. C) 48h. D) 72h. The pericarp
and seed coat were removed, the endosperm is visible.


100 UM 115X









Table 3-7. Total percent of seeds with endo-beta-mannanase activity (EBM) in the
endosperm after termination of each hydrothermal priming duration-seeds
were assayed at Oh time and after dry back and 14 days of storage
Seed Part
Micropylar Lateral Whole
Cultivar
Priming Time Bennett Connick
(h)
Percent of Seeds Exhibiting EBM Activity
0 ND1 ND ND ND
24 4 3 3 6
48 22 61 22 36
72 14 22 39 38
LSDo.05 10.0 17.0 16.0

Cultivar Seed lot
Bennett Australia 15
California 11
Connick Australia ND
California 14
LSDo.05 9.4
ANOVA
Cultivar (C) NS NS NS
Lot (SL) NS NS *
Priming time (PT) ** ** **
C*SL NS NS
C*PT NS NS
SL*PT NS NS NS
C*SL*PT NS NS NS
*,**,Ns Significant 5% (*), 1% (**) confidence levels, and non-significant (NS). Endo-beta-
mannanase enzyme activity was not detected (ND) Mean separation within priming treatment by
Least Significant Difference test p=0.05 Mean separation for cultivar x priming time interaction
by Least Significant Difference test p=0.05.


















50


I 40
w
L.U

S30


20

C
10
a-


Smicropylar

lateral

w hole


0 24 48 72

Priming Time (hr)

Figure 3-10. Percent of seed exhibiting endo-beta-mannanase activity (by seed part) after
dry back and re-imbibition. Means pooled for cultivar, lot and rep. n= 16.
Micropylar LSD=10.0, Lateral LSD=17.0, Whole LSD=16.0









The ability of seed lots to germinate at high temperatures after priming, dry-back

and subsequent storage correlated with the occurrence of percent EBM activity detected

in a seed lot. Primed seeds were germinated after 14 days in storage (from EBM assay).

At 200C, non-primed seeds germinated 92%, whereas primed seeds germinated 99%

(Table 3-8). Germination at 360C in primed seeds was 100% while non-primed seed

germination was 8%. Priming overcame thermosensitivity after 24 hours of priming

duration with no significant improvement to total percent germination when seeds were

primed for longer durations. The MHG of non-primed seeds was approximately 26 hours

at 200C and decreased to 12 hours after 72 h prime duration, thereby improving

germination rate of lettuce. The 72 h prime duration led to the most rapid MHG,

however total percent germination did not significantly increase as compared to he 48 h

prime duration. Thus, the 48 h prime duration was considered the optimal hydrothermal

priming time as there would be no further advantages of priming another 24 h to 72 h

priming time.

Variation in MHG occurred in the seed lots at 200C. The Australia lot germinated

more rapidly than the California lot after 48 h prime duration, yet after 72 h prime

duration, MHG were similar between seed lots (Table 3-8). The mean hour to

germination was similar in non-primed and primed seeds at 360C, but total percent

germination was significantly lower in non-primed seeds.

It is clear that although a short 24 h prime duration significantly improved both

germination rate and total germination in light and dark at 360C as compared to the

control, the total percent germination and consistency of germination (MHG) were

improved in seeds primed for 48 h. The 72 h prime duration did not significantly









increase total percent germination or germination rate as compared to 48 h prime duration

in the light. In the dark, germination rate decreased at 360C. This indicated that a 72 h

prime duration may not be needed, therefore priming for 48 h may be sufficient.

Endo-beta-mannanase activity varied within the lots and seed parts in seeds

removed directly from priming solution and those primed and stored. Variation in endo-

beta-mannanase activity in the whole endosperm, have been observed in tomato and

datura (Dirk et al. 1995; Bewley 1997a; Still and Bradford 1997). Variations in EBM

depend on the cultivar and the threshold amount needed for germination has not been

determined (Dirk et al. 1995). Therefore, EBM activity may be observed in varying

amounts with in the endosperm but as reported by Nascimento et al. (2001), low amounts

of EBM seem to be adequate for germination to occur, even at high temperature.

EBM activity was observed in low number of seeds or non-detectable levels in the

control or seeds primed for 24 hours. However, in seeds primed for 48 h or 72 h, EBM

was detected in up to 30% of all seeds regardless of location, seed lot, or cultivar.

Therefore, if similar amounts of EBM activity occurred between 48 h or 72 h prime

duration, and if germination rate and total percent germination is optimal at 48 h prime

duration, then potentially the optimal hydrothermal priming time has been determined.

The EBM activity observed in the whole endosperm at 48 h prime duration for all lots

was <25 % while total percent germination and rate were optimal. Thus, it is proposed

that 25% of seeds in the priming solution should exhibit EBM activity, in order to

achieve optimal hydrothermal time.









Table 3-8. Germination (%) and Mean Hour to Germination (MHG) of primed lettuce
seeds (from EBM study) after 14 days of storage in the light at 200C and 36C
(Experiment 5)
Germination Temp C
20 36
Priming time (hr) Germ MHG Germ MHG
Lot
% AU CA % h
0 92 28 25 8 17
24 99 20 21 100 17
48 99 13 20 100 17
72 100 12 12 100 17
LSDo.05 2.5 11.1 2.8
ANOVA
Cultivar (C) NS NS NS NS
Lot (SL) NS NS NS NS
Priming time (PT) ** ** ** NS
PT*SL NS NS NS
C*SL NS NS NS NS
C*PT NS NS NS NS
C*SL*PT NS NS NS NS
**,NS Significant at 1% (**) level and non-significant (NS) Mean separation within priming time by Least Significant
Difference test p= 0.05.









Though the average percentage of seeds exhibiting EBM activity was relatively

low, (30%) that does not mean that the EBM enzyme was not present. The EBM assay

selected for this study was chosen because it is rapid, simple, and inexpensive. However,

it is likely that more activity would be observed if a more precise test were developed. It

has been suggested that there may be more than one isoform of EBM which this test may

not presently detect. Also, EBM may be inactive in parts of the endosperm, particularly

the lateral region as the enzyme may be cell wall bound prior to mobilization of storage

reserves. Currently, a more precise and specific test is not available and if there was one

it would be likely that the test would require more time, labor and would not be as cost

efficient.

The objective of this investigation was to determine if this cost-efficient EBM

assay test could be used as an indicator of optimal priming time in lettuce. EBM activity

was associated with hydrothermal priming time in all lettuce seed lots tested. Using this

assay, seeds can periodically be removed directly from the priming solution and assayed

for EBM activity. Rather than priming seeds and using germination tests to determine

optimal hydrothermal time, the assay will serve as a more rapid indicator of proper

hydrothermal timing. Results of the EBM assay test can be obtained rapidly, within 12

hours. Whole endosperms have been determined to be an accurate indicator of total

percent EBM activity per seed. The extraction technique is rapid and simple since the

seeds do not need to be separated by seed part for assay. The EBM assay could be

conducted by one person. Under research laboratory conditions, the test costs

approximately $11.56 US per 1000 seeds. By defining optimal hydrothermal priming

through the EBM assay, the lettuce protocol might possibly be useful for other species









including tomato, pepper, carrot and celery. By using this model developed for lettuce,

the seed industry can improve the efficiency and consistency of results of the priming

process for different vegetable seed species, cultivars, and seed lots.

Cost analysis of the endo-beta-mannanase assay test was determined for 1000

primed seeds (10 seeds per 100 seed lots). The analysis was based on chemical and

material expenses for a research lab based on bulk pricing (Table 3-9). The gel plates

cost $165.00 per 100 plates (35 will be used for this study). However, this test does not

require sterile conditions so the plates can be washed and re-used up to ten times thus, the

price per plate is $.165 ($5.77). The cost of the test in 2005 is estimated to be $11.56US

and does not include the cost of lab equipment.

The labor required to conduct this assay was determined for each step of the EBM

test. The skill level to conduct this test would require a high school education. To extract

1000 whole endosperms it would take 8 hours (30 seconds per seed), to incubate the

endosperms in the buffer it would take two hours, to transfer the buffer to the gel plates it

would take 2 hours and to scan and stain 35 plates it would take two hours. The analysis

of the test was done by hand using scanned copies of the gel plates and it is estimated that

this method would two hours. However, a computer program could be developed to scan

and analyze the plates to determine the percentage of seeds with EBM (diameter of the

cleared areas), thus improving the efficiency of the analysis.

In summary, one of the primary drawbacks of priming is that the optimal duration

of priming time must be determined for each cultivar and seed lot due to variations in

seed age, vigor and thermosensitivity. This can take additional time and labor and add

subsequent cost. The objective of this research was to determine if an endo-beta-









mannanase (EBM) activity assay could be used as an indicator of optimal priming time

thereby providing an efficient priming procedure for improving seed quality rather than

the conventional priming technique with subsequent germination trials.

Two cultivars of lettuce, 'Bennett' and 'Connick', and two seed lots (Australia and

California) were primed for 0 h, 24 h, 48h and 72 h. EBM activity was determined. The

EBM assay is a rapid and simple test that can be completed in 12 hours. Seeds were re-

dried, and then germinated at 360C to determine the effectiveness of the priming process.

Germination in all lots was improved to over 90% when the seeds were primed for 48 h

or 72 h. Essentially no EBM activity was detected in non-primed seeds or seeds primed

for 24 h. By testing for EBM in the whole seeds, it was suggested that priming for 48 h

would be optimal for all seed lots used in this study and there is no advantage to priming

for a period beyond (48 h).









Table 3-9. Cost analysis for Endo-beta-mannanase assay test for 1000 seeds (100 lots)

Material Amount Total
Disposable Petri Plates 35 $5.77
Citric acid monohydrate 25g $0.61
Sodium phosphate 32g $1.52

Galactomannan 4g $0.36
Phytagar 7g $1.05
EBM Standard 1U $0.27
Congo red 1.58g $0.44
Parafilm 560 sq ft $1.01
Aluminum foil 36 sq ft $0.54


$11.56 or $0.01 /seed


Total Cost















APPENDIX A
ENDO-BETA-MANNANASE ASSAY


Standards


Micropylar

Lateral

Whole


Figure A-1. Assay of Endo-beta-mannanase in individual lettuce endosperm seed parts
using a gel diffusion. The four wells at the top represent enzyme dilutions.
(1 to r) #5, #4, #2, #1 dilutions of Endo-beta-D-mannanase A. niger.

















APPENDIX B
ANALYSIS OF VARIANCE TABLES

Table A-1. Analysis of variance for germination (%) non-primed lettuce in light at 200C
and 36C
20'C Source DF MSE(Tgerm)
Cultivar (C) 1 2025*
Lot (SL 1 2601*
C*SL 1 961


Error b


264


36C Source
Cultivar (C)
Lot (SL)
C*SL
Error b


MSE (Tgerm)
1
81*
9
10


Table A-2. Analysis of variance for germination of non-primed 'Bennett' and 'Connick'
lettuce seed after 8 months storage at 100C and 45% RH
Source DF MSE (Tgerm)
Temperature (T) 4 24363*
Error a 15 97
Cultivar (C) 1 28
Lot (SL) 1 132
C*T 4 79
C*SL 1 39
SL*T 4 58
C*SL*T 4 88
Error b 45 96








57



Table A-3. Analysis of variance for mean hours to germination (MHG) and total
germination of primed and non-primed lettuce seed in dark and light at a
range of temperatures


15'C Source

Rep

Cultivar (C)

Lot (SL)

Priming Time (PT)

PT*SL

C*SL

C*PT

C*SL*PT

Error


20'C Source

Rep

Cultivar (C)

Lot (SL)

Priming Time (PT)

PT*SL

C*SL

C*PT

C*SL*PT

Error




24'C Source

Rep

Cultivar (C)

Lot (SL)

Priming Time (PT)

PT*SL

C*SL

C*PT

C*SL*PT

Error


Light

MSE(MHG)

287.1

2.6

523.3*

2122.1**

41.5

123.8

31.0

78.3

117.3


Light

DF MSE(MHG)

3 402.9**

1 58.1

1 153.1

3 529.3**

3 43.4

1 21.3

3 5.1

3 35.3

45 38.6


Light

DF MSE(MHG)

3 423.8**

1 3.5

1 23.7

3 692.5**

3 45.5

1 21.4

3 8.3

3 21.0

45 39.3


MSE(Tgerm)

95.6

506.2

30.2

178.2

78.9

210.2

66.9

208.2

209.7


MSE(Tgerm)

108.9

12.2

156.2

157.6

93.0

6.2

106.2

22.9

90.3




MSE(Tgerm)

26.9*

20.3

0.3

12.9

5.6

20.2

3.0

11.0

8.42


Dark

MSE(MHG)

246.5*

10.5

33.0

2712.3**

25.2

36.0

52.6

25.2

44.5



Dark

MSE(MHG)

73.2

87.9

1.3

1807**

17.6

26.3

76.2

78.1

41.0


Dark

MSE(MHG)

67.1

60.0

22.6

2131.7**

2.0

5.0

32.9

29.7

59.6


MSE(Tgerm)

108.3

9.0

4.0

45.6

60.6

64.0

36.3

20.6

42.7


MSE(Tgerm)

76.9

90.2

72.2

74.9

97.6

90.2

92.9

90.3

93.2




MSE(Tgerm)

102.9

132.3

42.3

101.6

170.3

0.3

54.9

134.9

122.4













Table A-3. Continued.


30 C Source D

Rep

Cultivar (C)

Lot (SL)

Priming Time (PT)

PT*SL

C*SL

C*PT

C*SL*PT

Error




36 C Source DI

Rep 3

Cultivar (C) 1

Lot (SL) 1

Priming Time 3
(PT)

PT*SL 1

C*SL 1

C*PT 6

C*SL*PT 45


Light

MSE(MHG)

129.0**

12.2

110.2*

740.8**

78.8*

42.2

39.0

56.8

25.4


Light

MSE(MHG)

91.9

256.0

33.0

1189.9**


49.0

26.8

9.4

66.5


MSE(Tgerm)

34.0

36.0

121.0

278.0**

13.6

1.0

55.3

11.0

45.7





MSE(Tgerm)

267.0

484.0

144.0

14436.3**


1.0

56.6

83.1

193.4


Dark

MSE(MHG)

142.9

115.6

56.2

4012.2**

31.8

81.0

18.6

69.1

144.7



Dark

MSE(MHG)

12.7

0.4

21.4

550.5**


0.8

3.1

3.0

12.6


MSE(Tgerm)

532.9

600.2

6.3

23706.9**

50.9

132.5

60.9

38.3

220.3





MSE(Tgerm

256.6

12.2

56.2

34221.6**


6.2

128.9

138.6

216.0


Table A-4. Analysis of variance for total percent of seeds
activity in the endosperm during seed priming
Source DF MSE(micro)

Rep 3 340.6

Cultivar (C) 1 68.0

Lot (SL) 1 1387.6

Priming Time (PT) 3 6615.2**

PT*L 3 1225.0

C*SL 1 3422.3*

C*PT 3 287.0

C*SL*PT 3 845.4

Error 45 463.7


with endo-beta-mannanase


MSE(lateral)

511.0

435.8

5643.8**

6542.0**

1591.0*

276.4

530.7

466.2

247.4


MSE(whole)

85.8

729.0

1560.2*

3845.2**

685.0

351.6

269.0

190.7

303.2








59



Table A-5. Analysis of variance for total percent of seeds with endo-beta-mannanase
activity in the endosperm priming and 14 days of storage
Source DF MSE(micro) MSE(lateral) MSE(whole)

Rep 2 384.9 220.2 156.3
Cultivar (C) 1 475.0 374.0 50.0
Lot (SL) 1 285.2 200.0 2537.5*
Priming Time (PT) 3 1193.7** 5072.0** 4704.6**
PT*SL 3 404.4 207.4 1300.9
C*SL 1 981.0* 833.3 50.0
C*PT 3 163.3 1674.0* 670.7
C*SL*PT 3 266.9 334.6 20.7
Error 30 191.3 310.4 538.3




Table A-6. Analysis of variance for germination of primed lettuce seeds (from EBM

study) after 14 days of storage in Light at 200C and 360C
20C 36C

Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm)
Rep 3 32.1 1.4 59.6 6.6
Cultivar (C) 1 3.0 0.4 2.6 66.0
Lot (SL) 1 20.2 9.8 21.3 9.8
Priming Time (PT) 3 587.6** 190.0** 60.8 34148.3**
PT*SL 3 78.8* 3.5 37.8 5.6
C*SL 1 1.0 31.7 252.0 19.1
C*PT 3 14.2 10.8 63.4 80.6*
C*SL*PT 3 29.5 25.3 101.0 12.8
Error 45 17.9 18.0 92.8 23.5
















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67


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BIOGRAPHICAL SKETCH

Jennifer Rita Bonina was born on October 6, 1979 (one of triplets, along with

Jessica and Jolene) in Gloucester, Massachusetts to Jill and Andrew Bonina. She was

raised in Gloucester, MA, until she enrolled in the Biology Department at Gordon

College, Wenham, MA. She worked as a teaching assistant and tutor for the department.

She is a member of the American Society of Plant Biology and attended several meetings

as an undergraduate and won The American Society of Plant Biology Educational

Exhibit Award for the best educational booth. She graduated in May 2002 with a

Bachelor of Science degree in biology. She was accepted into the graduate program at

the University of Florida in Fall 2002 in the Horticultural Sciences Department. She was

a teaching assistant in the department, and the founding president of the Horticultural

Sciences Graduate Student Club. She is also a member of Gamma Sigma Delta Honor

Society.




Full Text

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A RAPID, SIMPLE, INEXPENSIVE AND REPRODUCIBLE ENDO-BETA-MANNANASE ASSAY TEST FOR DETERMINING OPTIMAL HYDROTHERMAL TIMING OF COMMERCIAL PRIMING OF LETTUCE SEED By JENNIFER R. BONINA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Jennifer Bonina

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Then God said: I give you every seed-bearing plant on the face of the whole earth and every tree that has fruit with seed in it. They will be yours for food. Genesis 1:29 I would like to dedicate this thesis to my parents, Jill and Andrew; my sisters, Jessica and Jolene for their love and support while pursuing this endeavor; and to my boyfriend, Joshua, for his enduring love and encouragement.

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ACKNOWLEDGMENTS First, I thank God for my passion for science, for allowing me to rely on His strength and power, and for teaching me patience as I complete this journey. This has been one of the most challenging but rewarding experiences of my life. I will forever cherish all of the friendships I have made during my time in Gainesville, FL. Next, I would like to express the depth of my gratitude to Dr. Daniel J. Cantliffe (department chairman) committee chair, for the opportunity to pursue this degree, and for guidance and support throughout my research. I extend my warmest appreciation to Dr. Donald J. Huber and Dr. Peter J. Stoffella for their patience, invaluable advice, and time spent serving on my supervisory committee. I offer sincere thanks to our Lab Biologist, (Nicole Shaw) for her assistance during my research. I would also like to acknowledge all the present and past members of the Seed Physiology Lab who fostered an environment of learning in the lab, provided valuable advice, and even helped me count seeds for my germination experiments on more than one occasion. Special thanks go to Paulo Campante-Santos, Ivanka Kozareva, Jeanmarie Mitchell, Amanda Collins and Camille Esmel. I would like to express my appreciation to Dr. Grace Ju at Educational Concerns for Hunger Organization (ECHO) Fort Myers, FL. You were an inspiring mentor at Gordon College and gave me the courage to pursue this degree in science. Finally, I would like to express my personal gratitude to my mother. Without your encouragement, love, and faith in me, this research could not have been accomplished. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES ...........................................................................................................ix ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Thermodormancy in Lettuce.........................................................................................4 Techniques to Circumvent Thermodormancy..............................................................5 Seed Priming Techniques.............................................................................................6 Factors Affecting Priming............................................................................................9 Limitations to Priming Treatments.............................................................................10 Control of Lettuce Seed Germination at High Temperatures.....................................12 Endo-Beta-Mannanase Activity and Endospermic Seeds..........................................13 Improving Seed Priming Technology.........................................................................17 3 ENDO-BETA-MANNANASE ASSAY CORRELATION TO PRIMING TREATMENT............................................................................................................19 Materials and Methods...............................................................................................20 Plant Material and Production.............................................................................20 Seed Dry Weight and Seed Moisture Content.....................................................22 Lettuce seed Microscope Study...........................................................................22 Seed Priming.......................................................................................................23 Germination Experiments....................................................................................23 Endo-beta-mannanase Enzyme Activity Assay...................................................25 Results and Discussion...............................................................................................27 APPENDIX A ENDO-BETA-MANNANASE ASSAY....................................................................55 v

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B ANALYSIS OF VARIANCE TABLES.....................................................................56 LITERATURE CITED......................................................................................................60 BIOGRAPHICAL SKETCH.............................................................................................68 vi

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LIST OF TABLES Table page 3-1 Production locations and conditions for Bennett and Connick cultivars.................21 3-2 Germination (%) of non-primed lettuce in the light at 20C and 36C (Experiment #1)........................................................................................................36 3-3 Germination (%) of non-primed Bennett and Connick lettuce seed after 8 months storage at 10C and 45% RH (Experiment 2).............................................36 3-4 Germination (%) of primed and non-primed lettuce seed in dark and light at several temperatures (Experiment 3 and 4)..............................................................38 3-5 Mean hours to germination (MHG) of primed and non-primed lettuce seed in dark and light at several temperatures (Experiments 3 and 4).................................42 3-6 Total percent of seeds with endo-beta-mannanase activity (EBM) in the endosperm during seed priming...............................................................................43 3-7 Total percent of seeds with endo-beta-mannanase activity (EBM) in the endosperm after termination of each hydrothermal priming duration-seeds were assayed at 0h time and after dry back and 14 days of storage..................................46 3-8 Germination (%) and Mean Hour to Germination (MHG) of primed lettuce seeds (from EBM study) after 14 days of storage in the light at 20C and 36C (Experiment 5)..........................................................................................................50 3-9 Cost analysis for Endo-beta-mannanase assay test for 1000 seeds (100 lots).........54 A-1 Analysis of variance for germination (%) non-primed lettuce in light at 20C and 36C..........................................................................................................................56 A-2 Analysis of variance for germination of non-primed Bennett and Connick lettuce seed after 8 months storage at 10C and 45% RH........................................56 A-3 Analysis of variance for mean hours to germination (MHG) and total germination of primed and non-primed lettuce seed in dark and light at a range of temperatures.........................................................................................................57 vii

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A-4 Analysis of variance for total percent of seeds with endo-beta-mannanase activity in the endosperm during seed priming........................................................58 A-5 Analysis of variance for total percent of seeds with endo-beta-mannanase activity in the endosperm priming and 14 days of storage.......................................59 A-6 Analysis of variance for germination of primed lettuce seeds (from EBM study) after 14 days of storage in Light at 20C and 36C.................................................59 viii

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LIST OF FIGURES Figure page 3-1 Size differences between Bennett and Connick lettuce seed cultivars...............29 3-2 Seed moisture content (SMC) of lettuce seeds imbibed in water............................30 3-3 Radicle protrusion through the micropylar tip of the endosperm of non-primed lettuce after 15h of imbibition in water at 20C in light..........................................31 3-4 Radicle protrusion through the endosperm (view of whole seed) of non-primed lettuce (pericarp removed) after 14h of imbibition in water at 20C in light...........32 3-5 Radicle protrusion through the endosperm and pericarp (view of whole seed) of non-primed lettuce (pericarp removed) after 15h of imbibition in water at 20C in light......................................................................................................................33 3-6 Seed moisture content of lettuce seeds in polyethylene glycol. Vertical bars indicate standard error..............................................................................................34 3-7 Micropylar tip of lettuce seed after priming in PEG (no visible radicles)...............35 3-8 Percent of seeds exhibiting endo-beta-mannanase activity (by seed part) during priming.....................................................................................................................44 3-9 Micropylar tip of lettuce seed after priming in PEG, stored and re-imbibed for 3hrs on ice (no visible radicles)................................................................................45 3-10 Percent of seed exhibiting endo-beta-mannanase activity (by seed part) after dry back and re-imbibition.............................................................................................47 A-1 Assay of Endo-beta-mannanase in individual lettuce endosperm seed parts using a gel diffusion...........................................................................................................55 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RAPID, SIMPLE, INEXPENSIVE AND REPRODUCIBLE ENDO-BETA-MANNANASE ASSAY TEST FOR DETERMINING OPTIMAL HYDROTHERMAL TIMING OF COMMERCIAL PRIMING OF LETTUCE SEED By Jennifer R. Bonina May 2005 Chair: Daniel J. Cantliffe Major Department: Horticultural Sciences Extended high temperatures during imbibition of lettuce (Lactuca sativa) can lead to a phenomenon known as thermoinhibition and thermodormancy, causing non-uniform germination and emergence. Seed priming was developed to circumvent this problem. Priming regulates water uptake by using an osmoticum. This hydrates the seed to a sufficient moisture level, allowing initial stages of germination to occur before radicle emergence. However, the requirements for (and results from) priming lettuce seed vary greatly according to age, vigor, and thermosensitivity. Optimal duration of priming time must be determined for each species, cultivar, and seed lot. The enzyme endo-beta-mannanase (EBM) is a prerequisite for radicle emergence in some endospermic seeds. In lettuce, this enzyme has been observed during priming and causes weakening of the lettuce endosperm. The present investigation determined the optimal hydrothermal timing for priming, lettuce seeds, independent of cultivar, seed lot, and storage (using an x

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inexpensive gel assay for endo-beta-mannanase). It is proposed that the assay test can accurately indicate the optimal hydrothermal priming time by correlating EBM activity in seed sub-sampled from each priming lot, to correlate with optimal priming time. Two head-type lettuce cultivars Bennett and Connick and two seed lots of each (produced both in Australia and California) were used in our study. The seeds were primed in an aerated polyethylene glycol 8000 (PEG) solution at 15C in constant light, and were sampled at 24 h, 48 h, and 72 h. A single-seed gel assay for endo-beta-mannanase was used to determine enzyme activity in each lot at each priming duration. At 36C, germination of primed seeds was over 90%; while non-primed seeds germinated at 40%. Priming increased germination rate up to 40% as compared to non primed seeds at both 20C and 36C under either light or dark conditions. EBM activity was non-detectable in dry seeds and for seeds primed for 24 h exhibited any EBM activity. After priming for 48 or 72 h, 30% of the micropylar section or the whole seed exhibited EBM activity regardless of seed lot or cultivar. Since the percentage of seeds exhibiting EBM in the whole endosperm after 48 or 72 h of priming were similar and germination rate and total percent germination were optimal at 48 h prime duration, 48 h was determined as the optimal hydrothermal priming time. EBM activity was observed in >25% of all lots at 48 h prime duration. Thus it was proposed if at least 25% of seeds exhibit EBM activity, optimal hydrothermal time is achieved. Determining priming time with the EBM test, gives the seed industry a method to better optimize the priming procedure for endospermic seeds such as lettuce. xi

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CHAPTER 1 INTRODUCTION Non-uniform seed emergence, especially at high temperatures, can lead to poor stand establishment in crops such as lettuce (Lactuca sativa). Prolonged emergence periods can predispose plants to damage by adverse environmental conditions and result in poor uniformity and a low percentage of seedling emergence. Slow emergence results in smaller plants and seedlings, that are more vulnerable to soil-borne diseases (Ellis 1963). Rapid field emergence under all conditions is a fundamental prerequisite for increasing the yield of many species and increasing profits from annual crops. Methods have been developed to overcome non-uniform seed germination at high temperatures including priming (Cantliffe 1981), seed coat removal (Guedes and Cantliffe 1980; Ikuma and Thimann 1963; Keys et al. 1975), and seed-coat puncture (Sung 1996). Commercial seed priming was introduced in the 1970s and is currently the most common method used in research and industry (Cantliffe 2000). Seed priming is a presowing treatment: seeds are placed in an osmotic solution and controlled imbibition occurs (along with the initial stages of germination before radicle emergence) resulting in more rapid, uniform seed germination (Heydecker et al. 1973). The requirements for and results from seed priming of different crops vary greatly according to the species and seed lot; and vary within lot due to storage. Developing a new testing methodology that improves the efficiency of the seed priming technique is the ultimate goal of this study. The proposed investigation is to determine optimal hydrothermal timing for priming in lettuce seeds, independent of cultivar, seed lot, and 1

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2 storage using an inexpensive endo-beta-mannanase gel assay (EBM). It is proposed that this simple rapid test could be used as an indicator of optimal hydrothermal priming time. Endo-beta-mannanase hydrolyzes mannan-polymers in the endosperm cell walls of lettuce. The enzyme is produced and secreted by the endosperm (Halmer and Bewley 1979). Dutta et al. (1997) reported that cell-wall-bound endo-beta-mannanase was expressed in lettuce endosperm prior to radicle emergence, and noted that enzyme levels were regulated by the same conditions that govern seed germination. Nascimento et al. (2001) reported a build-up of endo-beta-mannanase both before radicle emergence and during priming of lettuce. Since endo-beta-mannanase detection indicates readiness for germination, the assay could indicate when sufficient priming has occurred, rather than requiring additional time-consuming germination trials. The assay test might accurately indicate the optimal hydrothermal priming time by correlating EBM activity with the critical point when optimal priming has occurred, thus seeds will not be over-primed or under-primed. By defining optimal hydrothermal priming time with the EBM assay test, the seed industry will have a method that optimizes the priming procedure resulting in a simple, more efficient test. In the present work, the test was evaluated for lettuce, to correlate endo-beta-mannanase build-up prior to radicle emergence as an indicator of the completion of priming; however, endo-beta-mannanase activity has also been observed prior to radicle emergence in other vegetable seed species including tomato, pepper, carrot and celery (Bewley 1997b; Watkins et al. 1985; Williams et al. 2001). Thus, the test could be used in other seed species thereby streamlining the priming procedure by

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3 not requiring additional germination trials for each seed lot or seed type; resulting in improved seed performance in many seed species in the greenhouse or field.

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CHAPTER 2 LITERATURE REVIEW Hydrothermal priming is a technique used to circumvent dormancy and improve seed performance at high temperature in many seed species including lettuce will be reviewed. An overview will be provided of the mechanisms of lettuce seed germination including restriction at high temperature and the role of endo-beta-mannanase in breaking temperature dormancy. Thermodormancy in Lettuce Seed dormancy is commonly described as the failure of a viable seed to germinate under favorable growing conditions (Bewley 1997a). Many factors play a role in promoting or inhibiting dormancy. The embryo could be restricted by the surrounding seed coat (seed coating), or the seed may require a specific temperature or light treatment (Bewley 1997a). In a mature dry seed, uptake of water is triphasic; initially there is a rapid uptake of water, followed by a plateau phase, and finally increased water uptake until germination is completed and radicle emergence occurs. When seeds are dormant, they do not complete germination and do not enter phase III (Bewley 1997a). Certain seeds (including lettuce) can enter secondary dormancy if temperatures are too high for an extended length of time, resulting in no germination. When lettuce seeds are imbibed at high temperature for a period of time below 72 h one of two phenomena can occur. If imbibition at high temperatures is brief, a reversible condition known as thermoinhibition will occur that can be broken when the temperature is returned to an optimal range (~20C) (Khan 1980/81). Sung et al. (1998) 4

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5 reported that when lettuce seeds were exposed to high temperatures (above 35C) for a period of time less than 72 h and then moved to lower temperatures (20C), normal germination occurred (Sung et al. 1998). If seeds are exposed to high temperatures for prolonged periods of time (more than 72 h) before high temperatures are reduced, the seeds will enter a secondary dormancy referred to as thermodormancy and the seeds will not germinate (Nascimento 1998). Techniques to Circumvent Thermodormancy All seeds depend on temperature for optimal germination and stand establishment (Nascimento 1998). Lettuce seeds will germinate at temperatures ranging from as low as 5C to as high as 33C, optimal temperature ranges are generally between 15C and 22C but vary by genotype (Cantliffe et al. 2000; Gray 1975). A reduction in germination occurs at temperatures ranging from 25C to 27C, and if lettuce seeds are allowed to imbibe water at an elevated temperature (28C to 33C), germination will not occur (Borthwick and Robbins 1928, Cantliffe et al. 2000). To circumvent thermoinhibition in lettuce seed, several methods have been developed including controlled hydration (priming) (Cantliffe 1981), seed-coat removal (Guedes and Cantliffe, 1980; Keys et al. 1975; Ikuma and Thimann 1963) and seed-coat puncture (Sung 1996). Hormones such as gibberellic acid (GA) also induce germination in lettuce, tomato, and many other seeds (Bewley 1997b). Dry storage of freshly harvested seeds for a certain period of time known as after-ripening also helps to break dormancy (Atwater 1980; AOSA 1993; Baskin and Baskin 1998).

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6 Hydration treatments promote initial stages of germination by hydrating the seed, which increases the moisture level, thereby increasing germination rate and uniformity (Copeland and McDonald 1995; Pazdera and Hosnedl 2002). Hydration treatments are divided into two categories: pre-hydration and controlled hydration (Taylor et al. 1998). Pre-hydration does not limit water uptake; rather it is governed by the seeds affinity to water. Seeds are imbibed on moistened blotter paper or in water, and the process is ended at a specific time to prevent radicle emergence (Pazdera and Hosnedl 2002). Controlled hydration regulates water uptake by osmotic, drum, and solid matrix priming. Seed priming, as described by Heydecker (1973), is a presowing treatment in which seeds are placed in an osmotic solution. The amount of water available for the seed is restricted, thereby hydrating the seed to a sufficient moisture level; allowing initial stages of germination prior to radicle emergence. The seeds can be re-dried to the original moisture content for storage, or planted directly (Parera and Cantliffe 1994). Priming with osmoticum was introduced in the 1970s and is currently the most common method used in research and industry to overcome non-uniform seed germination at high temperatures (Cantliffe 2000). Seed Priming Techniques To prime with osmoticum, seeds are imbibed with a solution of either inorganic salts such as KNO 3 K 3 PO 4 or polyethylene glycol 8000 (PEG) for a period of time at a constant temperature (Bodworth et al. 1981; Cantliffe et al. 1981; Cantliffe et al. 2000; Ellis et al. 1963; Hegarty et al. 1977; Heydecker et al. 1973). In early work, inorganic salts such as NaNO 3 MnSO 4 and MgCl were used as osmoticum to prime pepper (Capsicum annum) seed (Kotowski 1926). Later, K 3 PO 4 and KNO 3 were used to the improve germination rate in tomato seed (Ellis 1963).

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7 Controversy occurred over the use of inorganic salts because of conflicting germination rates in some crop species. This occurred in carrot (Daucus carota) and leek (Allium porrum) seed primed in KH 2 PO 4 and PEG. Seeds primed in KH 2 PO 4 germinated less compared to those treated with PEG (Brocklehurst and Dearmam 1984). In contrast, tomato germination rate increased at 15C in seeds treated with KNO 3 compared to PEG (Alvarado et al.1987). Pepper germination rates were similar when seeds were primed in NaCl, MgSO 4 or PEG (Aljaro and Wyneken 1985). Currently many seed species are primed with PEG including celery (Apium graveolens) (Perez-Garcia et al. 1995), sunflower, (Helianthus annuus) (Mwale et al. 2003), and leek (Bujalski et al. 1993). In addition to the use of inorganic salt, or PEG, mannitol has also been used as a priming solution to regulate osmotic potential (Parera et al. 1994). Pepper seeds primed in mannitol had improved final percent germination over those not primed. (Georghiou et al. 1987). Other species including pepper, eggplant (Solanum melongena), and melon (Cucumis melo) seeds primed in mannitol produced larger seedlings than non-primed seeds (Passam et al. 1989). Sodium polypropionate (Zuo et al. 1988) or glycerol (Brocklehurst and Dearman 1984) have been used to prime seeds. Synthetic sea water has also been used on tomato, (Lycopersicon esculentum) and asparagus (Asparagus officinalis) (Owen and Pill 1994; Pill et al. 1991). Growth regulators have also been added to the priming solution. Carter and Stevens (1998) determined that when primed with gibberellic acid (GA 3 ) pepper seeds had higher germination (91%) as compared to non-primed at high temperatures (40C). However, the addition of growth regulators is added expense above PEG or salt costs. Therefore, growth regulators are often added in low concentration to priming solutions

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8 (Lorenz et al. 1988). Priming with PEG and GA 4+7 increased early germination in pepper especially at low temperature (15C), 3.8 days as compared 4.7 days in non-primed seeds (Watkins and Cantliffe 1983). Lorenz at al. (1988) reported that the addition of GA to the PEG priming solution increased soybean (Glycine max) emergence rate. In celery, seeds primed with PEG + GA and ethylene germinated at high temperatures as compared to non-primed seeds (Brockelhurst et al. 1982). In some cases, fungicides were also added to prevent pathogen growth (Leskovar and Sims 1987; Szafirowska et al. 1981). There are benefits and drawbacks to priming with solutions of salt, polyethylene glycol, mannitol, or others. Using salt as an osmoticum is less expensive and easier to remove from the seed after treatment, whereas PEG is often viscous and adheres to the seed, requiring a rinse step (Parera and Cantliffe 1994). Yet, elevated ion concentrations from salts can affect the embryo and germination, as reported by Brockelhurst and Dearman (1984). When lettuce seed was primed in K 3 PO 4, more water was imbibed by the seeds than PEG or PEG+ K 3 PO 4, it was suggested that the difference was due to natural differences in the seed coat composition (Guedes et al. 1979). This also occurred in onion (Allium cepa), carrot (Daucus carota), celery and leek (Allium porrum) seeds primed with KH 3 PO 4 and there was also a reduction in germination than seeds primed with PEG (Brockelhurst and Dearman 1984). This suggests that ions from salt solutions can penetrate and build up inside the seed during priming and the accumulation could reduce the osmotic potential of the seed and induce more water absorption as the treatment progresses (Parera and Cantliffe 1994). Priming with PEG, which is a chemically inert osmoticum, does not allow movement of the PEG molecule through the

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9 seed coat due to its large molecular weight allowing for only water movement into the seed during imbibition (Parera and Cantliffe 1994). Factors Affecting Priming Many factors influence the success of lettuce seed priming, including temperature, aeration, length of priming treatment, water potential of the osmotic solution, as well as the species, cultivar, seed quality and storage conditions (Cantliffe et al. 2000; Guedes 1979; Heydecker and Gibbins 1978; Parera and Cantliffe 1994). Some lettuce seed varieties are more heat sensitive than others therefore, and modifications to the priming process must be made carefully to ensure that the priming treatment will circumvent thermodormancy for all lettuce varieties. In thermosensitive Minetto lettuce seed, aeration of a 1% K 3 PO 4 osmotic solution at 15C increased germination slightly to 33% as compared to seeds that were not aerated (28%) especially when germinating at high temperatures (35C) (Guedes and Cantliffe, 1980). The study also determined that priming at 15C resulted in higher germination after 6h (64%) at 35C compared to other priming temperatures, 5C (22%) or 25C (48%) (Guedes and Cantliffe 1980). Other studies conducted by Khan et al. (1980/81) confirm that the ideal priming temperature range is 10C to 15C, temperatures above that do not significantly improve the priming procedure. In another lettuce variety, germination of Mesa 659 improved when primed for three to four days at 15C in .4 bar PEG as compared to seeds primed for shorter durations (one to two days), therefore priming time must be determined to achieve optimal results (Khan et al. 1981). Storage often affects primed seeds, especially thermosensitive DGB seeds had germination of 99% after priming but had reduced germination (27%) when stored for six months.

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10 However, thermotolerant EVE was not affected by storage (Nascimento and Cantliffe 1998). Consequently, storing DGB, a thermosensitive variety, after priming is not recommended as DGB seeds may be more desiccation-sensitive increasing susceptibility to deterioration (Nascimento and Cantliffe 1998). In some cases, using both salt and an osmoticum such as PEG improve germination in lettuce. A thermosensitive lettuce cultivar, Valmaine had improved germination (71%) with no thermodormancy when both K 3 PO 4 and PEG were used as compared to priming in either alone (~50-60%) (Cantliffe 1981). In conjunction with many factors affecting the success of a priming treatment, there are limitations to its use as a seed treatment since the treatment conditions are often varied depending on species, cultivar, and seed lot, these may have to be individually tested. Identifying optimal hydrothermal priming time is critical to achieve proper moisture content within the seed. Often the length of time must be adjusted for each seed lot, cultivar, or species; this can be inefficient but it is currently the industry standard practice. Limitations to Priming Treatments There are limits to the length of seed storage once primed due to a more rapid reduction in seed viability over time (Alvarado and Bradford 1988; Argerich and Bradford 1989; Carpenter and Boucher 1991; Maude et al. 1994; Odell and Cantliffe 1986). Standard industry practice for seed priming recommend seed to be primed prior to the immediate growing season to avoid reductions in viability. Dearman and Bradford (1987) observed a reduction in seed viability after storage of primed leek and carrot but it has also been reported in tomato (Alvarado and Bradford 1988; Argerich and Bradford,1989; Odell and Cantliffe 1986), lettuce (Tarquis et al.

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11 1992; Weges, 1987) and wheat (Triticum aestivum L.) (Nath et al. 1991). In one study, tomato seeds primed in PEG or 3% KNO 3 solutions, the seeds germinated under laboratory and field conditions, then were stored at 10C, 20C, and 30C, for 12 months or longer. Seeds primed and stored for up to 18 months at 10C and 45% RH maintained 50% more rapid germination than when stored at lower temperatures. At 30C storage, primed seeds had increased loss of viability over non-primed seeds (Alvarado et al. 1988). Reduction in seed viability and longevity has been reported in many lettuce studies. Tarquis et al. (1992) reported a 61% decrease in emergence rate (MTG) but seed longevity in storage was reduced by as much as 84% compared to non-primed seeds. In Dark Green Boston (DGB) and Everglades (EVE) lettuce, seeds primed with PEG had a 100% germination rate at high temperature (Nascimento et al. 1998). When the primed seeds were stored for six months at 10C at 45%RH for 12 months, germination remained at 100% at 20C and 35C in the EVE cultivar but in DGB, viability decreased (Nascimento and Cantliffe, 1998). To prevent reductions in vigor and germination, seeds are often not stored after priming for more than nine months, thereby retaining the improved germination from the treatment and avoiding the deleterious effects of storage. Treating seeds with PEG can also be more costly than salt solutions, and it requires disposal in an environmentally safe manner. It is also more difficult to achieve uniform aeration during priming with PEG due to the viscosity of the solution. Furthermore, PEG often adheres to the seed, requiring an additional water rinse step (Parera and Cantliffe 1994).

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12 Control of Lettuce Seed Germination at High Temperatures The lettuce endosperm consists of a mannose-rich double cell layer, completely encompassing the embryo (Borthwick and Robbins 1928). Cells in the micropylar region opposite the radicle tip are more prolific, up to three or four cell layers thick (Dutta et al. 1994). For germination to occur, the radicle must grow and penetrate the micropylar end of the endosperm (Bewley and Black 1994). Researchers suggested that lettuce endosperm cell walls acted as a barrier to germination, mechanically restricting radicle emergence especially at high temperatures (30C) leading to secondary dormancy (Borthwick and Robbins 1928; Dutta et al. 1994; Halmer et al. 1976; Ikuma and Thimann 1963; Nascimento et al. 2000). To overcome mechanical restriction at high temperature, two mechanisms have been suggested; pressure exerted on the endosperm by the developing embryo and endosperm tissue weakening, which may work together leading to germination (Bewley and Black, 1994; Bradford 1995; Nascimento 1998) However, the mechanisms that control thermoinhibition and thermodormancy, though widely studied, remain unclear. Early studies conducted by Halmer et al. were the first to demonstrate endo-beta-mannanase synthesis and activity in the lettuce (Lactuca sativa L.) endosperm (Halmer et al. 1975; Halmer et al. 1976). Enzyme activity was detected post-germatively and was thought to aid in mobilization of endosperm reserves to serve as a nutrient source for seedling development (Halmer et al. 1979). Weakening of the lettuce endosperm layer by an enzyme, endo-beta-mannanase, was later determined to be a prerequisite to radicle emergence, especially at high temperatures (Park et al. 1974, Halmer et al. 1975; Nascimento 1998; Sung 1996). Oluoch and Welbaum (1996) reported that thinning of the endosperm occurred at the radicle end prior to germination reducing resistance and

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13 promoting radicle emergence in melon and parsley (Petroselium crispum), a process which may also occur in lettuce (Olszewski et al. 2004). The enzyme endo-beta-mannanase is a cell-wall hydrolytic enzyme that is thought to be synthesized de novo once imbibition begins (Bewley 1997b). Endo-beta-mannanase degrades galactomannan-rich endosperm cells, which produce and secrete the enzyme (Halmer et al. 1979). Endo-beta-mannanase activity increases initially in the micropylar region and later increases in the rest of the endosperm cells. (Leviatov et al. 1995; Nomaguchi, et al. 1995; Toorop et al. 1996). Activity is often detected in the lateral region of the endosperm once radicle emergence occurs (Groot 1988; Nomaguchi et al. 1995; Nonogaki et al. 1992). Nonogaki and Morohashi (1996) suggested that the presence of endo-beta-mannanase first in the micropylar and then the lateral may be due to the different physiological roles of the enzyme. When it is present pre-germinatively in the micropylar region it is responsible for cell wall weakening and post-germinatively for galactomannan storage reserve mobilization. Endo-Beta-Mannanase Activity and Endospermic Seeds Many endospermic seeds including lettuce contain reserves which are retained exclusively in the endosperm until maturity. These reserves are degraded by specific enzymes after the initiation of germination to facilitate the developing seedling until photosynthesis is initiated (Bewley and Black 1994; Homrichhausen et al. 2003). In most cases, endo-beta-mannanase degrades endosperm cell walls in endospermic seeds and can be detected during germination in tomato, carrot, asparagus (Asparagus officinalis), and pepper (Bewley 1997b, Cantliffe et al. 2000; Homrichhausen et al. 2003; Watkins et al. 1985; Williams et al. 2001). In fenugreek and carob seed, endo-beta-mannanase activity

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14 was not detected until after the completion of germination (Bewley 1997b; Kontos et al. 1995; Reid and Bewley 1979; Spyropoulos and Reid 1988). The role of endo-beta-mannanase has been extensively studied in tomato, due to the presence of high levels of endo-beta-mannanase due to the endosperm, which is the major storage tissue and serves as a nutrient source for the embryo during seedling development (Nonogaki and Morohashi 1995). In lettuce however, the endosperm is not major nutrient source for the embryo and only supplies nutrients to the embryo for a short period of time until the cotyledons develop thus, lower levels of endo-beta-mannanase have been detected (Bewley and Black 1994). However, tomato often serves as the model for further studies of the role of endo-beta-mannanase in lettuce. In tomato, endo-beta-mannanase activity increases in intact endosperms prior to radicle emergence, and is initially detected in the thin-walled endosperm (micropylar) cap opposite the radicle tip prior to germination (Nonogaki et al. 1992; Still and Bradford 1997). Endo-beta-mannanase increases later (~48 h) in the rest of the endosperm, followed by germination. The presence of endo-beta-mannanase at two separate times in the same tissue prior to germination indicates that EBM is mobilized prior to germination and plays a key role during germination (Bewley 1997b; Leviatov et al. 1995; Nonogaki et al. 1996; Toorop et al. 1996). This indicates that the enzyme builds up prior to germination, weakens the cell wall and aids radicle emergence (Groot et al. 1987; Groot and Karssen 1988; Toorop et al. 1996). In the mutant gibberellin-deficient (gib-1) tomato, there was also an increase in endo-beta-mannanase activity in the endosperm when the seeds were treated with gibberellic acid (GA) (Groot et al. 1988). Therefore, it was suggested that endosperm

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15 weakening prior to radicle emergence was mediated by gibberellin-induced enzymatic degradation of the cell walls (Bewley 1997b; Groot et al. 1988; Nascimento 1998). However, more recently it has been suggested that germinating tomato radicles produce or release GA that induces endo-beta-mannanase production in the endosperm. Consequently, the enzyme causes weakening of the endosperm cell walls, reducing mechanical resistance, leading to radicle emergence (Bewley, 1997b; Ni and Bradford, 1993). In lettuce, the presence of gibberellin alleviated thermoinhibition and increased endo-beta-mannanase activity (Dutta et al.1997; Nascimento 1998). The presence of endo-beta-mannanase prior to radicle emergence in lettuce has been debated. Early studies reported no endo-beta-mannanase activity in Grand Rapids lettuce seeds prior to radicle emergence (Halmer et al. 1975) while others claimed that endo-beta-mannanase is strictly a post-germative event (Bewley 1997b). However, it was reported by Dutta et al. (1997) that cell-wall-bound endo-beta-mannanase was expressed in lettuce endosperm prior to radicle emergence and that enzyme levels were regulated by the same conditions that govern seed germination. Endo-beta-mannanase activity varies in the whole endosperm, depending on genotype (Dirk et al.1995). In a study of five non-primed lettuce cultivars, endo-beta-mannanase was not detected in the first 4 hours of imbibition incubated at 20C (Nascimento et al. 2000). However, after 6 hours activity was detected in all the cultivars except Dark Green Boston DGB, a thermosensitive type. Activity could be detected in seeds imbibed at 35C but only after radicle emergence, except in two of the thermotolerant cultivars, PI 251245 PI and Everglades EVE(Nascimento et al. 2000). The increase in endo-beta-mannanase in the thermotolerant genotypes may have been due

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16 to a lower endosperm resistance as compared to thermosensitive genotypes suggesting that weakening of the endosperm layer was a pre-requisite for radicle emergence (Nascimento 1998; Sung et al. 1998) Nascimento et al. (2001) reported an increase of endo-beta-mannanase both before radicle emergence and after priming in lettuce. Endo-beta-mannanase could be detected in non-primed and primed EVE and DGB. The study noted that endo-beta-mannanase activity was higher in seeds incubated at 20C than 35C possibly due to high temperature which may inhibit endo-beta-mannanase enzyme synthesis (Dutta et al. 1997; Nascimento et al. 2001). However, primed seeds germinated at 35C germinated faster (~4h) compared to 20C (~12h). This suggested that the presence of endo-beat-mannanase in lower concentration ensured germination at high temperature. This may also be the case in stored primed lettuce seed. After three days of dry-back, seeds imbibed at 35C germinated at 100% and endo-beta-mannanase was 1.3 pmol min -1 in DGB and 81 pmol min -1 in EVE indicating that even if levels of endo-beta-mannanase are low (1 pmol min -1 ) at high temperatures, it is adequate for endosperm weakening (Nascimento 1998; Nascimento et al. 2001). In a related study, endo-beta-mannanase was detected during priming between 24 and 48 hours in EVE and between 24 and 72 hours in DGB. Enzyme activity in primed EVE seeds remained highest as compared to DGB and non primed lettuce seeds, suggesting that priming could overcome the inhibitory effect of elevated temperatures due to the weakening effect of endo-beta-mannanase enzyme on the endosperm cell walls (Nascimento 1998). Endo-beta-mannanase activity persisted even after seed drying when seeds were re-imbibed. However, endo-beta-mannanase activity detected after radicle

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17 emergence was still higher than prior to radicle emergence (Nascimento et al 2001). This may be due to carbohydrate mobilization in the endosperm (Nascimento et al. 2001). The mechanisms that control endo-beta-mannanase leading up to germination in lettuce are still largely unknown. However, much more is known about mechanisms surrounding endo-beta-mannanase in tomato making tomato and ideal model for the study of endo-beta-mannanase in lettuce. Improving Seed Priming Technology The conventional method for circumventing temperature-related dormancy in many seeds is priming, yet there are factors that limit the efficiency of this technology. The optimal duration of priming time must be determined for each cultivar and each seed lot due to variations in seed age, vigor and thermosensitivity. Adjusting the priming conditions for each cultivar and lot requires additional time and labor. Though initially reported as a post-germinative event, Dutta et al. (1997) observed endo-beta-mannanase in endosperm cell walls prior to radicle emergence and noted that enzyme levels were regulated by the same conditions that govern seed germination. Nascimento et al. (2001) reported the build up of endo-beta-mannanase both before radicle emergence and during priming of lettuce using the gel diffusion assay. Thus, it is endo-beta-mannanase that has been detected during priming. If a peak in the number of seeds exhibiting endo-beta-mannanase activity can be determined during priming using the gel-diffusion assay and correlated to optimal germination at high temperature then the assay may be used to determine optimal hydrothermal priming time. The gel-diffusion assay test (Downie et al. 1994; Still and Bradford 1997) requires minimal training and labor and is simple, rapid and inexpensive to perform. Using this test to indicate the achievement of optimal hydrothermal time

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18 during priming would provide additional safeguards and quality control for companies that prime and sell primed lettuce seed. The endo-beta-mannanase activity assay could be used as an indicator of optimal priming time, increasing efficiency in priming procedures used for improving seed quality. By defining optimal hydrothermal priming for different vegetable seed species, this technology will aid the seed industry by providing a more efficient procedure for improving the accuracy of priming. As a result, there may be a more consistent improvement in primed seed performance in the greenhouse or field environment.

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CHAPTER 3 ENDO-BETA-MANNANASE ASSAY CORRELATION TO PRIMING TREATMENT Non-uniform seed emergence, especially at high temperatures, can lead to poor stand establishment in crops such as lettuce (Lactuca sativa). Rapid field emergence under all conditions is a fundamental prerequisite to increase yield of many species and ultimately profits from annual crops. Methods have been developed to overcome non-uniform seed germination at high temperatures including priming (Cantliffe et al. 1981), seed coat removal (Guedes and Cantliffe 1980; Ikuma and Thimann 1963; Keys et al. 1975) and seed coat puncture (Sung 1996). Commercial seed priming is currently the most common method used in research and industry however, there are drawbacks to the technique (Cantliffe 2000). The requirements for and results from priming lettuce seed vary greatly according to variations in seed age, vigor and thermosensitivity. Optimal duration of priming time must be determined for each cultivar and seed lot. This can take additional time and labor. Developing a new testing methodology that improves the efficiency of the seed priming technique is the ultimate goal of this work. The proposed investigation is to determine optimal hydrothermal timing for priming in lettuce seeds, independent of cultivar, seed lot, and storage using an inexpensive endo-beta-mannanase gel assay (EBM). It is proposed that this simple rapid test could be used as an indicator of optimal hydrothermal priming time. Nascimento et al. (2001) reported a build up of endo-beta-mannanase both before radicle emergence and during priming of lettuce. Dutta et al. (1997) reported that cell19

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20 wall-bound endo-beta-mannanase was expressed in lettuce endosperm prior to radicle emergence and noted that enzyme levels were regulated by the same conditions that govern seed germination. Since endo-beta-mannanase detection indicates readiness for germination, the assay indicates when sufficient priming has occurred, rather than requiring additional time-consuming germination trials. The assay test indicates accurately the optimal hydrothermal priming time by correlating EBM activity with the critical point when optimal priming has occurred, thus seeds are not over-primed or under-primed. Two cultivars of lettuce, Bennett and Connick, Lactuca sativa and two seed lots (Australia and California) were primed for 0 h, 24 h, 48 h and 72 h. EBM activity was determined. The EBM assay is a rapid and simple test that can be completed in 32 hours. Seeds were re-dried, and then germinated at 36C to determine the effectiveness of the priming process. Germination in all lots was improved to over 90% when the seeds were primed for 48 h or 72 h. Essentially no EBM activity was detected in non-primed seeds or seeds primed for 24 h. By using this model developed for lettuce, the seed industry will benefit by this procedure, improving the efficiency and consistency of results of the priming process for different vegetable seed species, cultivars and seed lots. Materials and Methods Plant Material and Production Two head-type lettuce (Lactuca sativa L.) cultivars (Bennett and Connick) produced by Sun Seed Company (now Nunhems Seeds USA, Parma, ID) were used in this study. The seeds were stored at 10C, 40% RH for subsequent use in this study (Table 3-1).

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21 Table 3-1. Production locations and conditi ons for Bennett and Connick cultivars Cultivar Production Location Harvest Date Avg. max temp Avg. min temp Bennett New South Wales, AU February 2002 78F/26C 66F/19C Bennett Kerman, CA September 2001 91F/32C 56F/12C Connick New South Wales, AU Fe bruary 2002 78F/26C 66F/19C Connick Kerman, CA September 2001 91F/32C 56F/12C

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22 Seed Dry Weight and Seed Moisture Content Seed dry weight was determined by oven drying four replications of 1 g of seed for 8 h at 130C. Seeds were removed and placed in a desiccator for 20 minutes and weighed (AOSA, 1993). Fresh weight was determined by weighing 1g of seed (per replication) during priming (seed priming protocol previously referred to above) at 15C with constant light in an incubator (Precision Scientific, Winchester, VA). Seed weight was recorded at time intervals of 1 h, 2 h, 3 h, 4 h 6 h ,12 h, 24 h 48 h and 72 h. Seeds were removed from a -1.2 MPa of polyethylene glycol 8000 (PEG) (30 ml of solution per 1 g of seed) and rinsed once with water using a Buchner funnel and seed weight was recorded. Seed moisture content (SMC) protocol was repeated in water and fresh weight was recorded at time intervals of 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, 16 h, 20 h and 24 h. Seed moisture content as a percentage of weight was calculated using the following formula: (fresh weight dry weight) / fresh weight x 100. The experimental design was conducted using a split-plot design. Standard error was calculated at each time interval for each cultivar and seed lot. Lettuce seed Microscope Study Non-primed seeds (20) were placed on one layer of 5.0 cm diameter Whatman #1 filter paper moistened with distilled water in a 5.5 cm Petri dish. The dishes were covered and placed at 20C under constant light in an incubator (Precision Scientific, Winchester, VA). The pericarp was removed from the seed and photos were taken with a Leica MZ16 microscope (Leica Microsystems, Wetzlar, Germany) at each hour until radicle emergence (approximately 15 h) to trace radicle growth as protrusion through the endosperm and seed coat.

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23 Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30 ml of solution per 1 g of seed) of 30% -1.2 MPa PEG 8000 (30 g /100 ml water) (Fischer Scientific, Philadelphia, PA) for 24, 48 or 72 hours at 15C with constant light (fluorescent ~30 mol m -2 s -1 ) in an incubator (Precision Scientific, Winchester, VA). At each priming duration, 0.5 g samples were removed from the PEG priming solution and rinsed once with 25 ml of distilled water. Following the rinse, the pericarp was removed using forceps and a surgical blade. Microscope images were immediately recorded digitally. The remaining primed seeds were damp dried and stored for 7 days in uncovered 5.5 cm Petri dishes in a storage room at 16C, 50% RH. After 7 days, the seeds were removed from storage and imbibed on moistened #1 filter paper (2 ml distilled water) in a 5.5 cm Petri dish on ice for 3 hours. The pericarp was removed and images were recorded immediately. Seed Priming Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30 ml of solution per 1 g of seed) of 30% -1.2 MPa PEG 8000 (30 g/100 ml water) (Fischer Scientific, Philadelphia, PA) for 24, 48 or 72 hours at 15C with constant light in an incubator (Precision Scientific, Winchester, VA). After each priming time, seeds were removed and placed in a Buchner funnel and rinsed three times with distilled water, damp dried, and stored in uncovered 5.5 cm Petri dishes in a cold storage room at 16C and 50% RH for four to six days. Germination Experiments For each cultivar, seed lot, and priming time, 25 seeds each placed on one layer of 5.0 cm diameter Whatman #1 filter paper, moistened with 2 ml of distilled water in 5.5 cm Petri dishes, and incubated under constant light (fluorescent ~26 mol m -2 s -1 ) in

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24 incubators (Precision Scientific, Winchester, VA). Germination, defined as visible radicle protrusion through the pericarp, was recorded at three hour intervals beginning at 9 h, 12 h, 15 h, 18 h, 21 h, 24 h, 48 h and 72 hours after initial incubation. Experiment #1: Non-primed seeds were germinated in each incubator (chamber) at 20C and 36C in the light. Experiment #2: Non-primed seeds were incubated in each incubator (chamber) at 15C, 20C, 24C, 30C, 36C in the light. Experiment #3: Primed seeds were germinated in each incubator (chamber) 15C, 20C, 24C, 30C,36C in the light. Seeds were primed according to previously listed protocol for seed priming. Experiment #4: Primed seeds were germinated in each incubator (chamber) 15C, 20C, 24C, 30C, 36C in the dark similar to Experiment #3. Dark germination was monitored under a green safe light. Primed dark and light seed germination experiments were not conducted concurrently. Experiment #5: Primed seeds from the endo-beta-mannanase assay test were germinated after 14 days in storage, 16C and 50% RH. Seeds were primed according to previously listed protocol for seed priming. The seeds were germinated in each incubator (chamber) 15C, 20C, 24C, 30C,36C in the light. Total percent germination and mean hours to germination (MHG) were calculated as a measure of response to the treatment priming time (ptime). The MHG was calculated according to the formula Ti/Ni, where Ni is the number of newly germinated seeds at hour interval Ti (Maguire, 1962). A split-block experimental design was used with temperature as the main block and cultivar, lots and priming time as the split block. Analysis of variance (ANOVA) was conducted by the Statistical System (SAS) software (SAS, 2002). Mean separation for

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25 significant main effects or interactions were performed by Least Significant Difference (LSD p=0.05). Endo-beta-mannanase Enzyme Activity Assay A gel-diffusion assay protocol was used (Downie et al. 1994; Still et al. 1997) as described by Nascimento et al. (2001). The assay was used to detect endo-beta-mannanase enzyme activity during the priming treatments. Gel plates were prepared by dissolving 0.05% (w/v) galactomannan (locust bean gum, Sigma Chemical Co., St. Louis, MO) in incubation buffer (0.1 M citric acid, 0.2M Sodium phosphate, pH 5.0), stirring and heating for 30 minutes. Afterward, the solution was clarified by centrifugation at 15,000 g for 15 minutes at 4C. Phytagar (Gibco Lab., Grand Island, NY) at 0.7% (w/v) was added to the clarified solution and stirred while heating to the boiling point. Thirty ml of solution was dispensed into 150x25 mm disposable Petri dishes (Falcon, Franklin Lakes, NJ). After solidification, 32 wells per plate were made using a 2-mm disposable plastic pipette to remove excised gel by aspiration. Seed samples (1 g) were primed in 200 mm test tubes in an aerated solution (30ml of solution per 1g of seed) of 30% -1.2 MPa PEG 8000 (30g/100 ml water) (Fischer Scientific, Philadelphia, PA) for 0, 24, 48 or 72 hours at 15C with constant light in an incubator (chamber) (Precision Scientific, Winchester, VA). Seed samples (0.5 g) were removed from the priming solution and assayed immediately. For each cultivar, seed lot, and priming time, four replications of 18 seeds were used in each study, assays were conducted on six whole individual endosperms, six micropylar (radicle tip only) and six lateral (remaining endosperm) from the lettuce seeds primed 24 h, 48 h, or 72 h. The remaining 0.5 g of each period of time was rinsed three times in distilled water, damped dry and stored in uncovered 5.5 cm Petri dishes in a cold storage room at 16C and 50%

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26 RH for 14 days prior to subsequent EBM assay testing. Endosperms were excised by pressing on the cotyledon end using forceps and the tip of a surgical blade. A portion of the excised endosperms remained intact (whole) while the others were separated into the micropylar tip and the lateral endosperm with forceps and a surgical blade. Each seed part (whole, micropylar tip or lateral) was placed into an individual microtiter plate (Nalge Nunc, Naperville, IL) well containing 20 L of sterile incubation buffer (0.1 M citric acid, 0.2 M sodium phosphate, pH5.0) and incubated in the dark for 2 hours at 25C. After incubation, 10 L of buffer from each well was transferred to the gel-diffusion plates and incubated for 24 h. A serial dilution of endo-beta-d-mannanase A. Niger enzyme was added as a standard to each plate, 0.5 ul EBM into 2 ml H 2 O=stock, #1 5 ul/1 ml H 2 O, #2 5 ul/10 ml H 2 O, #3 5 ul/50 ml H 2 O, #4 5 ul/100 ml H 2 O, #5 5 ul/500 ml H 2 O, #6 5 ul/1000 ml H 2 O (Megazyme International Ireland Ltd., Wicklow, Ireland). Petri dishes were covered with a lid and aluminum foil and wrapped in Parafilm (American National Can., Greenwich, CT). Gels were stained by adding 10 mL of Congo Red (Sigma Chemical Co., St. Louis, MO) in water (0.4% w/v) to each plate. Plates were shaken for 20 minutes at 60 rpm during staining. The Congo red solution was decanted and the gel was gently rinsed with distilled water for 1 min, then 10 mL of citrate-phosphate buffer pH 7.0 was added. After 3 min on the orbital shaker at 60 rpm, the buffer was decanted. Plates were scanned within 5-10 min using a Scan Jet 3c/T (Hewlett Packard, Palto Alto,CA). The diameter of the cleared areas on the plates indicated endo-beta-mannanase activity. Only seed parts with observed activity were used for analysis using WinRhizo tm (Regent Instruments Inc., Quebec, Canada) software.

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27 Percentage of seeds assayed with endo-beta-mannanase activity per seed part and calculated; (number of seeds with activity / total number of seeds assayed) / x 100. A randomized complete block experimental design was used with treatments replicated four times. Analysis of variance (ANOVA) was conducted for the percentage data by seed part with the Statistical Analysis System (SAS) software (SAS,2002). Significant main effects means were reported by least significant difference (LSD), 5% level. Results and Discussion A significant difference in seed size was observed between the seed lots (~4 mm Australia and ~3 mm California) which may have occurred due to the higher maturation temperatures (~32C) in California during production compared to Australia (~26C) (Figure 3-1). Lettuce seed size and weight can be affected by seed maturation temperature. Sung et al. (1998) observed that when lettuce seeds matured at temperatures above 30C, seeds were smaller in size. Thus, larger seeds were produced under 26C than 32C but it also may be due to plant growth conditions such as irrigation, fertilization and sun light. The seed moisture content (SMC) of quiescent (dry) seeds varied by seed lot, 7.1% SMC for BAU, 7.0% SMC for CAU, 6.7% SMC for BCA and 6.5% SMC for CCA. This may have been due to a difference in seed size (Figure 3-2). Water uptake during imbibition followed a classical triphasic pattern in solutions of either water or PEG (Bewley and Black 1994). In water, rapid water uptake occurred during the first hour (~60% fresh weight) of imbibition (Figure 3-2). After 6 hours, SMC increased to ~80% and a plateau occurred between 6 h and 12 h with a final rapid uptake of water and radical emergence (Figure 3-3). Radicles were visible through the

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28 endosperm after 14 h (Figure 3-4) and through the pericarp between 14 h and 15 h (Figure 3-5). Between 16 hours and 20 hours, all cultivars attained approximately 115% increase in fresh weight as compared to original dry weight. In PEG, water uptake occurred rapidly in the first hour (~40%), followed by an increase in uptake between 2 h and 6 h (~58) (Figure 3-6). A plateau occurred after Hour 8 (Figure 3-6). Seeds primed in PEG remained at 60-62% moisture without visible radicles when removed from solution at 24 h, 48 h or 72 h (Figure 3-7). High levels of osmoticum (PEG) inhibited radicle emergence while allowing for metabolic reactions to occur during the lag phase of water uptake (Heydecker and Gibbins 1978; Karssen et al. 1989). Consequently, total percent germination and germination rate will increase when seeds are re-imbibed at planting, especially at high temperatures. Initial germination tests were conducted on non-primed seeds to determine germination rate and optimal germination temperature (Table 3-2). Therefore, cultivar and lot means were separated under these temperatures. At 20C, germination was 95% for Bennett and 73% for Connick cultivars. The California lot had lower germination at 20C, 71% as compared to the Australia lot which indicated extreme thermosensitivity, though 20C is an optimal temperature for lettuce (Khan, 1980/81). After eight months of storage at 10C and 50% RH, germination of all lots was nearly 100% for temperatures up to 25C whereas at 30C germination was above 80% (Table 3-3). After-ripening during storage may have led to improved germination at (30C). Thermodormancy persisted at the highest temperature 36C and germination of all lots was less than 10%. These results are typical for thermosensitive cultivars.

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29 1 mm 16x CAU BCA CCA BAU Figure 3-1. Size differences between Bennett and Connick lettuce se ed cultivars from left to right: Bennett produced in Australia (BAU), Connick produced in Australia (C AU), Bennett produced in California (BCA), and Connick produced in California (CCA).

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30 Time(hr) 0123456789101112131415161718192021222324%SMC 020406080100120140 BAU BCA CAU CCA Radicle Emergence Figure 3-2. Seed moisture content (SMC) of lettuce seeds imbibed in water. Vertical bars indicate standard errors

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31 Endosperm 100m 115x Radicle Figure 3-3. Radicle protrusion through the micropylar tip of the endosperm of non-primed lettuce after 15h of imbibition in water at 20C in light.

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32 100um 115x Endosperm Radicle Figure 3-4. Radicle protrusion through the endosperm (view of whole seed) of non-primed lettuce (pericarp removed) after 14h of imbibition in water at 20C in light

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33 100um 115x Endosperm Radicle Figure 3-5. Radicle protrusion through the endosperm and pericarp (view of whole seed) of non-primed lettuce (pericarp removed) after 15h of imbibition in water at 20C in light

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34 Time(hr) 04812162024283236404448525660646872%SMC 020406080100120140 BAU BCA CAU CCA Figure 3-6. Seed moisture content of lettuce seeds in polyethylene glycol. Vertical bars indicate standard error.

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35 100 m 115x 100 m 115x Endosperm Radicle tip A B 100 m 115x 100 m 115x C D Figure 3-7. Micropylar tip of lettuce seed after priming in PEG (no visible radicles). A) 0h. B) 24h. C) 48h. D) 72h. The pericarp and seed coat were removed, the endosperm is visible.

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36 Table 3-2. Germination (%) of non-primed lettuce in the light at 20C and 36C (Experiment #1) Germination (%) Cultivar 20C 36C Bennett 73 4 Connick 95 4 Significance NS Seed Lot Australia 97 6 California 71 2 Significance ** *,**,NS Significant at 5%(*) level, 1% (**) level respectively and non-significant (NS) Table 3-3. Germination (%) of non-primed Bennett and Connick lettuce seed after 8 months storage at 10C and 45% RH (Experiment 2) Temp (C) Germination (%) 15 100 20 98 24 98 30 84 36 8 LSD 0.05 5.8 ANOVA Temperature (T) ** Cultivar (C) NS Lot (SL) NS C*T NS L*T NS C*L NS C*SL*T NS **,NS Significant at 1% (**) level and non-significant (NS). Mean separation within temperature by Least Significant Difference (LSD) test p = 0.05

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37 The various seed lots were primed for 0 h, 24, 48 or 72 hours in order to determine optimal hydrothermal priming time for the lettuc e cultivars. There were no differences in germination between cultivar or seed lot. Only the main effects of priming time was significant at 30C and 36C (Table 3-4). Germ ination increased in all primed seeds at 30C and 36C in light and dark compared to non-primed seeds. At temperatures below 24C, all non-primed and primed seeds ge rminated over 90% in both light and dark (Table 3-4). In light, germination at 30C was 91% in non-primed seeds whereas priming for 24h or longer germination increased to 99%. At 36C, germination of primed seeds was over 90% while non-primed seeds germinated at 40% or less regardless of light or dark. Thus, priming overcame thermosensitivity of th e seeds at high temperature (36C). Germination of primed seeds in the dark at 30C was over 92% whereas nonprimed seed germination was 18% (Table 3-4). Primed seeds germinated over 90% at 36C regardless of priming time, whereas non-primed seed germinated 1%. Germination in dark at 36C resulted in reduced germin ation of seeds primed for 72 h compared to those primed for 24 h or 48 h. Low germina tion of seeds imbibed in the dark at high temperatures (30C) was reported for other lettuce cultivars (Sung et al 1998; Nascimento et al. 2000). Other studies have also observed an interaction between high temperature and light and suggested it is due to a phenomenon that occurs during mediation by the phytochrome system (Taylorson and Hendricks 1972; Fielding et al. 1992). However, priming improved total percen t germination, regardless of temperature, duration of priming time, or germinating in the dark.

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38 Table 3-4. Germination (%) of primed and non-primed lettuce seed in dark and light at several temperatures (Experiment 3 and 4) Temperature (C) Light Dark Priming time (h) 15 20 24 30 36 15 20 24 30 36 0 98 93 99 91 35 98 95 95 18 1 24 91 96 98 99 91 96 99 97 94 96 48 96 99 100 99 98 100 100 100 99 97 72 98 100 100 99 96 99 100 99 92 88 LSD 0.05 4.0 8.3 8.8 8.7 ANOVA Cultivar (C) NS NS NS NS NS NS NS NS NS NS Lot (SL) NS NS NS NS NS NS NS NS NS NS Ptime (PT) NS NS NS ** ** NS NS NS ** ** PT*SL NS NS NS NS NS NS NS NS NS NS C*SL NS NS NS NS NS NS NS NS NS NS C*PT NS NS NS NS NS NS NS NS NS NS C*SL*PT NS NS NS NS NS NS NS NS NS NS **,NS Significant at 1% (**) level and non-significant (NS) Mean separation within temperature by Least Significant Difference test p= 0.05.

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39 Priming decreased mean hour to germination (MHG), and improved germination rate at each temperature in light or dark when compared to non-primed seeds (Table 3-5). The increases in uniformity, synchrony and rate confirm findings reported in other studies of seed priming (Argerich and Bradford 1989; Brocklehurst et al. 1984). In the light, seeds primed for 48 or 72 h germinated most rapidly, 50% faster as compared to the non-primed seeds at 30 to 36C. The three priming treatments had similar germination rates at the lower temperatures (15C to 24C), but germinated approximately 30% faster as compared to the non-primed seeds. In the dark, all priming treatments had similar MHG and germination rate was ~65% faster as compared to control with the exception of the 24 h treatment at 15C which was slower, 51%. Therefore, the 24 h priming treatment led to slower germination and priming was not as complete as 48 h or 72 h. In the dark, germination was slower at 36C (~50%) as compared to the control. MHG in the dark at 72 h of priming was less than 48 h, thus, priming may have gone too far. EBM activity was observed in a low number of seeds or non-detectable levels in the control or seeds primed for 24 hours (Figure 3-8). Studies conducted by Nascimento et al. (1998) also noted that EBM activity was not detected in dry (not imbibed) non-primed lettuce seeds but suggested that EBM synthesis occurred during priming hence EBM was observed. After priming for 48 h or 72 h, 30% of the micropylar and whole seeds exhibited EBM activity regardless of location, seed lot, or cultivar (Figure 3-8). The percentage of seeds exhibiting EBM after 72 h were slightly higher as compared to the 48 h prime duration, however the values were not significantly different (Table 3-6). In the lateral seed part there was a fluctuation in activity at both 48 h and 72 h prime durations. After 48 h of priming EBM activity was in 48% of the seeds in the Australia

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40 seed lot and 2% in the California seed lot, respectively. At 72 h EBM activity was observed in higher percentages (54%) in the Australia seed lot as compared to 36% in the California seed lot. It is also important to note that in the Connick California seed lot, a lower percentage of seeds exhibited EBM activity in the micropylar region (Table 3-6). The difference in observed EBM in the lateral endosperm and the micropylar region of the Connick California seed lot may have occurred for several reasons. When the micropylar and lateral endosperm were separated during sampling, the tissues were disrupted. This may have caused lower levels of EBM to be detected in the lateral region at 24 h and 48 h, as EBM may have leached out. Nascimento (1998) observed a correlation between higher EBM activity in primed seeds incubated at 20C and faster germination rate compared to non-primed seeds. It was suggested that the elevated amount of EBM in the primed seeds caused endosperm cell wall weakening leading to more rapid germination. MHG was 35% slower at 20C in the California seed lot after 48 h of priming than the Australia seed lot when the primed seeds were germinated (Table 3-8). This may be why lower EBM was observed in the Connick California lot. Thus, lower levels of EBM were observed when germination rate was slower. EBM was also detected in re-dried seeds (Table 3-7) as well as during priming (Table 3-6). Radicles were also not visible in any of the seeds during priming (Figure 3-9) and after three hours of imbibition of re-dried primed stored seeds (14 days). EBM activity was observed in a low number of seeds or non-detectable levels in the control or seeds primed for 24 hours (Figure 3-10). EBM was observed in the micropylar tip in all the seed lots, except in the Connick California seed lot (Table 7). This may be due to disruption during sampling. At 48h prime duration, EBM increased to 20% or higher in

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41 all seed parts (Figure 3-10). The difference in observed EBM in the lateral endosperm was similar to that of seeds removed directly from the priming solution (Table 3-6). At 48h prime duration Bennett had a higher pe rcentage of seeds that exhibited EBM activity than Connick, 61 and 22%. Af ter 72h prime duration, Connick was higher with 39% and 22% seeds with activity, respectiv ely. This may also have been due to the separation of the micropylar and lateral endos perm leading to tissue disruption causing EBM to leach out. Also, it is likely that th e EBM enzyme may be inactive in portions of the endosperm cell walls, thus EBM could not be detected. Which may be the case in the lateral region of the endosperm as EBM m obilizes storage reserv es from the lateral endosperm immediately prior to radicle emergence. Similar amounts of seeds exhibited EBM activity (<25%) in whole seeds after 48 h or 72 h of priming. Since there was no significa nt increase in EBM in the total percent of the seeds exhibiting EBM activity it is proposed that 48 h pr ime duration is the optimal hydrothermal priming time. Therefore, if 25% of seeds in the priming solution exhibit EBM activity optimal hydrothermal time has been reached. Also as noted, the 24 h priming treatment led to slower germination and 72 h of priming led to reduced total percent germination (Table 3-4 and Table 3-5). Thus, priming for 24 h was not as complete as 48 h and priming for 72 h may have gone too far. Therefore, it is proposed that the optimal hydrothermal priming time is 48 h as it led to the highest total percent germin ation and increase in uniformity and rate for all lots in both light conditions.

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42 Table 3-5. Mean hours to germination (MHG) of primed and non-primed lettuce seed in dark and light at several temperatures (Experiments 3 and 4) Temperature (C) Light Dark Priming time (h) 15 20 24 30 36 15 20 24 30 36 0 40 23 24 24 28 43 33 34 44 23 24 28 16 16 11 12 21 13 13 13 11 48 17 11 10 10 10 15 11 11 12 11 72 16 11 10 10 10 17 12 11 14 12 LSD 0.05 6.4 3.6 3.8 3.0 4.9 3.9 3.8 4.5 7.1 2.6 ANOVA Cultivar (C) NS NS NS NS NS NS NS NS NS NS Lot (SL) NS NS NS NS NS NS NS NS NS Priming time (PT) ** ** ** ** ** ** ** ** ** ** PT*SL NS NS NS NS NS NS NS NS NS NS C*L NS NS NS NS NS NS NS NS NS NS C*PT NS NS NS NS NS NS NS NS NS NS C*SL*PT NS NS NS NS NS NS NS NS NS NS *,**,NS Significant 5% (*), 1% (**) confidence levels, and non-significant (NS) Mean separation within temperature by Least Significant Difference test p =0.05.

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43 Table 3-6. Total percent of seeds with endo-beta-mannanase activity (EBM) in the endosperm during seed priming. (seeds were assayed at 0h time and after termination of each hydrothermal priming duration) Seed Part Micropylar Lateral Whole Lot Priming Time (h) AU CA Percent of Seeds Exhibiting EBM Activity 0 ND ND ND ND 24 2 8 ND 2 48 30 48 2 25 72 41 54 36 31 LSD 0.05 12.8 13.3 10.0 Cultivar Seed Lot Bennett Australia 16 California 22 Connick Australia 29 California 5 LSD 0.05 9.5 ANOVA Cultivar (C) NS NS NS Lot (SL) NS ** Priming time (PT) ** ** ** C*SL NS NS C*PT NS NS NS SL*PT NS NS C*SL*PT NS NS NS *,**,NS Significant 5% (*), 1% (**) confidence levels, and non-significant (NS). Endo-beta-mannanase enzyme activity was not detected (ND).

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44 01020304050600244872Priming Time (hr)Percent of seeds with EBM activity micropylar lateral whole Figure 3-8. Percent of seeds exhibiting endo-beta-mannanase activity (by seed part) during priming. Means pooled for cultivar, lot and rep. n= 16. Micropylar LSD=12.8, Lateral LSD=13.3, Whole LSD=10.0.

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45 100 m 115x 100 m 115x Endosperm A Radicle tip B 100 m 115x 100 m 115x C D Figure 3-9. Micropylar tip of lettuce seed after priming in PEG, stored and re-imbibed for 3hrs on ice (no visible radicles). A) 0h. B) 24h. C) 48h. D) 72h. The pericarp and seed coat were removed, the endosperm is visible.

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46 Table 3-7. Total percent of seeds with endo-beta-mannanase activity (EBM) in the endosperm after termination of each hydrothermal priming duration-seeds were assayed at 0h time and after dry back and 14 days of storage Seed Part Micropylar Lateral Whole Cultivar Priming Time (h) Bennett Connick Percent of Seeds Exhibiting EBM Activity 0 ND 1 ND ND ND 24 4 3 3 6 48 22 61 22 36 72 14 22 39 38 LSD 0.05 10.0 17.0 16.0 Cultivar Seed lot Bennett Australia 15 California 11 Connick Australia ND California 14 LSD 0.05 9.4 ANOVA Cultivar (C) NS NS NS Lot (SL) NS NS Priming time (PT) ** ** ** C*SL NS NS C*PT NS NS SL*PT NS NS NS C*SL*PT NS NS NS *,**,NS Significant 5% (*), 1% (**) confidence levels, and non-significant (NS). Endo-beta-mannanase enzyme activity was not detected (ND) Mean separation within priming treatment by Least Significant Difference test p=0.05 Mean separation for cultivar x priming time interaction by Least Significant Difference test p=0.05.

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47 01020304050600244872Priming Time (hr)Percent of seeds with EBM activity micropylar lateral whole Figure 3-10. Percent of seed exhibiting endo-beta-mannanase activity (by seed part) after dry back and re-imbibition. Means pooled for cultivar, lot and rep. n= 16. Micropylar LSD=10.0, Lateral LSD=17.0, Whole LSD=16.0

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48 The ability of seed lots to germinate at high temperatures after priming, dry-back and subsequent storage correlated with the occurrence of percent EBM activity detected in a seed lot. Primed seeds were germinated after 14 days in storage (from EBM assay). At 20C, non-primed seeds germinated 92%, whereas primed seeds germinated 99% (Table 3-8). Germination at 36C in primed seeds was 100% while non-primed seed germination was 8%. Priming overcame thermosensitivity after 24 hours of priming duration with no significant improvement to total percent germination when seeds were primed for longer durations. The MHG of non-primed seeds was approximately 26 hours at 20C and decreased to 12 hours after 72 h prime duration, thereby improving germination rate of lettuce. The 72 h prime duration led to the most rapid MHG, however total percent germination did not significantly increase as compared to he 48 h prime duration. Thus, the 48 h prime duration was considered the optimal hydrothermal priming time as there would be no further advantages of priming another 24 h to 72 h priming time. Variation in MHG occurred in the seed lots at 20C. The Australia lot germinated more rapidly than the California lot after 48 h prime duration, yet after 72 h prime duration, MHG were similar between seed lots (Table 3-8). The mean hour to germination was similar in non-primed and primed seeds at 36C, but total percent germination was significantly lower in non-primed seeds. It is clear that although a short 24 h prime duration significantly improved both germination rate and total germination in light and dark at 36C as compared to the control, the total percent germination and consistency of germination (MHG) were improved in seeds primed for 48 h. The 72 h prime duration did not significantly

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49 increase total percent germination or germination rate as compared to 48 h prime duration in the light. In the dark, germination rate decreased at 36C. This indicated that a 72 h prime duration may not be needed, therefore priming for 48 h may be sufficient. Endo-beta-mannanase activity varied within the lots and seed parts in seeds removed directly from priming solution and those primed and stored. Variation in endo-beta-mannanase activity in the whole endosperm, have been observed in tomato and datura (Dirk et al. 1995; Bewley 1997a; Still and Bradford 1997). Variations in EBM depend on the cultivar and the threshold amount needed for germination has not been determined (Dirk et al.1995). Therefore, EBM activity may be observed in varying amounts with in the endosperm but as reported by Nascimento et al. (2001), low amounts of EBM seem to be adequate for germination to occur, even at high temperature. EBM activity was observed in low number of seeds or non-detectable levels in the control or seeds primed for 24 hours. However, in seeds primed for 48 h or 72 h, EBM was detected in up to 30% of all seeds regardless of location, seed lot, or cultivar. Therefore, if similar amounts of EBM activity occurred between 48 h or 72 h prime duration, and if germination rate and total percent germination is optimal at 48 h prime duration, then potentially the optimal hydrothermal priming time has been determined. The EBM activity observed in the whole endosperm at 48 h prime duration for all lots was <25 % while total percent germination and rate were optimal. Thus, it is proposed that 25% of seeds in the priming solution should exhibit EBM activity, in order to achieve optimal hydrothermal time.

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50 Table 3-8. Germination (%) and Mean Hour to Germination (MHG) of primed lettuce seeds (from EBM study) after 14 days of storage in the light at 20C and 36C (Experiment 5) Germination Temp C 20 36 Priming time (hr) Germ MHG Germ MHG Lot % AU CA % h 0 92 28 25 8 17 24 99 20 21 100 17 48 99 13 20 100 17 72 100 12 12 100 17 LSD 0.05 2.5 11.1 2.8 ANOVA Cultivar (C) NS NS NS NS Lot (SL) NS NS NS NS Priming time (PT) ** ** ** NS PT*SL NS NS NS C*SL NS NS NS NS C*PT NS NS NS NS C*SL*PT NS NS NS NS **,NS Significant at 1% (**) level and non-significant (NS) Mean separation within priming time by Least Significant Difference test p= 0.05.

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51 Though the average percentage of seeds exhibiting EBM activity was relatively low, (30%) that does not mean that the EBM enzyme was not present. The EBM assay selected for this study was chosen because it is rapid, simple, and inexpensive. However, it is likely that more activity would be observed if a more precise test were developed. It has been suggested that there may be more than one isoform of EBM which this test may not presently detect. Also, EBM may be inactive in parts of the endosperm, particularly the lateral region as the enzyme may be cell wall bound prior to mobilization of storage reserves. Currently, a more precise and specific test is not available and if there was one it would be likely that the test would require more time, labor and would not be as cost efficient. The objective of this investigation was to determine if this cost-efficient EBM assay test could be used as an indicator of optimal priming time in lettuce. EBM activity was associated with hydrothermal priming time in all lettuce seed lots tested. Using this assay, seeds can periodically be removed directly from the priming solution and assayed for EBM activity. Rather than priming seeds and using germination tests to determine optimal hydrothermal time, the assay will serve as a more rapid indicator of proper hydrothermal timing. Results of the EBM assay test can be obtained rapidly, within 12 hours. Whole endosperms have been determined to be an accurate indicator of total percent EBM activity per seed. The extraction technique is rapid and simple since the seeds do not need to be separated by seed part for assay. The EBM assay could be conducted by one person. Under research laboratory conditions, the test costs approximately $11.56 US per 1000 seeds. By defining optimal hydrothermal priming through the EBM assay, the lettuce protocol might possibly be useful for other species

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52 including tomato, pepper, carrot and celery. By using this model developed for lettuce, the seed industry can improve the efficiency and consistency of results of the priming process for different vegetable seed species, cultivars, and seed lots. Cost analysis of the endo-beta-mannanase assay test was determined for 1000 primed seeds (10 seeds per 100 seed lots). The analysis was based on chemical and material expenses for a research lab based on bulk pricing (Table 3-9). The gel plates cost $165.00 per 100 plates (35 will be used for this study). However, this test does not require sterile conditions so the plates can be washed and re-used up to ten times thus, the price per plate is $.165 ($5.77). The cost of the test in 2005 is estimated to be $11.56US and does not include the cost of lab equipment. The labor required to conduct this assay was determined for each step of the EBM test. The skill level to conduct this test would require a high school education. To extract 1000 whole endosperms it would take 8 hours (30 seconds per seed), to incubate the endosperms in the buffer it would take two hours, to transfer the buffer to the gel plates it would take 2 hours and to scan and stain 35 plates it would take two hours. The analysis of the test was done by hand using scanned copies of the gel plates and it is estimated that this method would two hours. However, a computer program could be developed to scan and analyze the plates to determine the percentage of seeds with EBM (diameter of the cleared areas), thus improving the efficiency of the analysis. In summary, one of the primary drawbacks of priming is that the optimal duration of priming time must be determined for each cultivar and seed lot due to variations in seed age, vigor and thermosensitivity. This can take additional time and labor and add subsequent cost. The objective of this research was to determine if an endo-beta

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53 mannanase (EBM) activity assay could be used as an indicator of optimal priming time thereby providing an efficient priming procedure for improving seed quality rather than the conventional priming technique with subsequent germination trials. Two cultivars of lettuce, Bennett and Connick, and two seed lots (Australia and California) were primed for 0 h, 24 h, 48h and 72 h. EBM activity was determined. The EBM assay is a rapid and simple test that can be completed in 12 hours. Seeds were re-dried, and then germinated at 36C to determine the effectiveness of the priming process. Germination in all lots was improved to over 90% when the seeds were primed for 48 h or 72 h. Essentially no EBM activity was detected in non-primed seeds or seeds primed for 24 h. By testing for EBM in the whole seeds, it was suggested that priming for 48 h would be optimal for all seed lots used in this study and there is no advantage to priming for a period beyond (48 h).

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54 Table 3-9. Cost analysis for Endo-beta-mannanase assay test for 1000 seeds (100 lots) Material Amount Total Disposable Petri Plates 35 $5.77 Citric acid monohydrate 25g $0.61 Sodium phosphate 32g $1.52 Galactomannan 4g $0.36 Phytagar 7g $1.05 EBM Standard 1U $0.27 Congo red 1.58g $0.44 Parafilm 560 sq ft $1.01 Aluminum foil 36 sq ft $0.54 Total Cost $11.56 or $0.011/seed

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APPENDIX A ENDO-BETA-MANNANASE ASSAY Figure A-1. Assay of Endo-beta-mannanase in individual lettuce endosperm seed parts using a gel diffusion. The four wells at the top represent enzyme dilutions. (1 to r) #5, #4, #2, #1 dilutions of Endo-beta-D-mannanase A. niger. Whole Lateral Micropylar Standards 55

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APPENDIX B ANALYSIS OF VARIANCE TABLES Table A-1. Analysis of variance for germination (%) non-primed lettuce in light at 20C and 36C 20C Source DF MSE(Tgerm) Cultivar (C) 1 2025* Lot (SL 1 2601* C*SL 1 961 Error b 12 264 36C Source DF MSE (Tgerm) Cultivar (C) 1 1 Lot (SL) 1 81* C*SL 1 9 Error b 12 10 Table A-2. Analysis of variance for germination of non-primed Bennett and Connick lettuce seed after 8 months storage at 10C and 45% RH Source DF MSE (Tgerm) Temperature (T) 4 24363* Error a 15 97 Cultivar (C) 1 28 Lot (SL) 1 132 C*T 4 79 C*SL 1 39 SL*T 4 58 C*SL*T 4 88 Error b 45 96 56

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57 Table A-3. Analysis of variance for mean hours to germination (MHG) and total germination of primed and non-primed lettuce seed in dark and light at a range of temperatures Light Dark 15C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm) Rep 3 287.1 95.6 246.5* 108.3 Cultivar (C) 1 2.6 506.2 10.5 9.0 Lot (SL) 1 523.3* 30.2 33.0 4.0 Priming Time (PT) 3 2122.1** 178.2 2712.3** 45.6 PT*SL 3 41.5 78.9 25.2 60.6 C*SL 1 123.8 210.2 36.0 64.0 C*PT 3 31.0 66.9 52.6 36.3 C*SL*PT 3 78.3 208.2 25.2 20.6 Error 45 117.3 209.7 44.5 42.7 Light Dark 20C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm) Rep 3 402.9** 108.9 73.2 76.9 Cultivar (C) 1 58.1 12.2 87.9 90.2 Lot (SL) 1 153.1 156.2 1.3 72.2 Priming Time (PT) 3 529.3** 157.6 1807** 74.9 PT*SL 3 43.4 93.0 17.6 97.6 C*SL 1 21.3 6.2 26.3 90.2 C*PT 3 5.1 106.2 76.2 92.9 C*SL*PT 3 35.3 22.9 78.1 90.3 Error 45 38.6 90.3 41.0 93.2 Light Dark 24C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm) Rep 3 423.8** 26.9* 67.1 102.9 Cultivar (C) 1 3.5 20.3 60.0 132.3 Lot (SL) 1 23.7 0.3 22.6 42.3 Priming Time (PT) 3 692.5** 12.9 2131.7** 101.6 PT*SL 3 45.5 5.6 2.0 170.3 C*SL 1 21.4 20.2 5.0 0.3 C*PT 3 8.3 3.0 32.9 54.9 C*SL*PT 3 21.0 11.0 29.7 134.9 Error 45 39.3 8.42 59.6 122.4

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58 Table A-3. Continued. Light Dark 30C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm) Rep 3 129.0** 34.0 142.9 532.9 Cultivar (C) 1 12.2 36.0 115.6 600.2 Lot (SL) 1 110.2* 121.0 56.2 6.3 Priming Time (PT) 3 740.8** 278.0** 4012.2** 23706.9** PT*SL 3 78.8* 13.6 31.8 50.9 C*SL 1 42.2 1.0 81.0 132.5 C*PT 3 39.0 55.3 18.6 60.9 C*SL*PT 3 56.8 11.0 69.1 38.3 Error 45 25.4 45.7 144.7 220.3 Light Dark 36C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm Rep 3 91.9 267.0 12.7 256.6 Cultivar (C) 1 256.0 484.0 0.4 12.2 Lot (SL) 1 33.0 144.0 21.4 56.2 Priming Time (PT) 3 1189.9** 14436.3** 550.5** 34221.6** PT*SL 1 49.0 1.0 0.8 6.2 C*SL 1 26.8 56.6 3.1 128.9 C*PT 6 9.4 83.1 3.0 138.6 C*SL*PT 45 66.5 193.4 12.6 216.0 Table A-4. Analysis of variance for total percent of seeds with endo-beta-mannanase activity in the endosperm during seed priming Source DF MSE(micro) MSE(lateral) MSE(whole) Rep 3 340.6 511.0 85.8 Cultivar (C) 1 68.0 435.8 729.0 Lot (SL) 1 1387.6 5643.8** 1560.2* Priming Time (PT) 3 6615.2** 6542.0** 3845.2** PT*L 3 1225.0 1591.0* 685.0 C*SL 1 3422.3* 276.4 351.6 C*PT 3 287.0 530.7 269.0 C*SL*PT 3 845.4 466.2 190.7 Error 45 463.7 247.4 303.2

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59 Table A-5. Analysis of variance for total percent of seeds with endo-beta-mannanase activity in the endosperm priming and 14 days of storage Source DF MSE(micro) MSE(lateral) MSE(whole) Rep 2 384.9 220.2 156.3 Cultivar (C) 1 475.0 374.0 50.0 Lot (SL) 1 285.2 200.0 2537.5* Priming Time (PT) 3 1193.7** 5072.0** 4704.6** PT*SL 3 404.4 207.4 1300.9 C*SL 1 981.0* 833.3 50.0 C*PT 3 163.3 1674.0* 670.7 C*SL*PT 3 266.9 334.6 20.7 Error 30 191.3 310.4 538.3 Table A-6. Analysis of variance for germination of primed lettuce seeds (from EBM study) after 14 days of storage in Light at 20C and 36C 20C 36C Source DF MSE(MHG) MSE(Tgerm) MSE(MHG) MSE(Tgerm) Rep 3 32.1 1.4 59.6 6.6 Cultivar (C) 1 3.0 0.4 2.6 66.0 Lot (SL) 1 20.2 9.8 21.3 9.8 Priming Time (PT) 3 587.6** 190.0** 60.8 34148.3** PT*SL 3 78.8* 3.5 37.8 5.6 C*SL 1 1.0 31.7 252.0 19.1 C*PT 3 14.2 10.8 63.4 80.6* C*SL*PT 3 29.5 25.3 101.0 12.8 Error 45 17.9 18.0 92.8 23.5

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BIOGRAPHICAL SKETCH Jennifer Rita Bonina was born on October 6, 1979 (one of triplets, along with Jessica and Jolene) in Gloucester, Massachusetts to Jill and Andrew Bonina. She was raised in Gloucester, MA, until she enrolled in the Biology Department at Gordon College, Wenham, MA. She worked as a teaching assistant and tutor for the department. She is a member of the American Society of Plant Biology and attended several meetings as an undergraduate and won The American Society of Plant Biology Educational Exhibit Award for the best educational booth. She graduated in May 2002 with a Bachelor of Science degree in biology. She was accepted into the graduate program at the University of Florida in Fall 2002 in the Horticultural Sciences Department. She was a teaching assistant in the department, and the founding president of the Horticultural Sciences Graduate Student Club. She is also a member of Gamma Sigma Delta Honor Society. 68