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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
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
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."
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.
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
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
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
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
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
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
Jennifer R. Bonina
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.
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
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.
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
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%)
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
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.
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
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.
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
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. Production locations and conditions for Bennett and Connick cultivars
Cultivar Production Location Harvest Date Avg. max Avg. min
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 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.
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
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
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%
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
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).
0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324
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
80 -- CAU
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72
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
Cultivar 200C 360C
Bennett 73 4
Connick 95 4
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 (%)
Temperature (T) **
Cultivar (C) NS
Lot (SL) 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
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)
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
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-
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)
Priming time (h)
Cultivar (C) NS
Lot (SL) *
Priming time (PT) **
*,**,NS Significant 5% (*), 1%
Significant Difference test p
(**) confidence levels,
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)
Micropylar Lateral Whole
Priming Time '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
LSDo.05 12.8 13.3 10.0
Cultivar Seed Lot
Bennett Australia 16
Connick Australia 29
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).
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.
- 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
Micropylar Lateral Whole
Priming Time Bennett Connick
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
Connick Australia ND
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.
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
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
Germination Temp C
Priming time (hr) Germ MHG Germ MHG
% 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
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
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
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.
ANALYSIS OF VARIANCE TABLES
Table A-1. Analysis of variance for germination (%) non-primed lettuce in light at 200C
20'C Source DF MSE(Tgerm)
Cultivar (C) 1 2025*
Lot (SL 1 2601*
C*SL 1 961
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
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
Priming Time (PT)
Priming Time (PT)
Priming Time (PT)
Table A-3. Continued.
30 C Source D
Priming Time (PT)
36 C Source DI
Cultivar (C) 1
Lot (SL) 1
Priming Time 3
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
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
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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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