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Physiological aspects of hydrilla Hydrilla verticillata (L.f.) Royle growth and reproduction

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Title:
Physiological aspects of hydrilla Hydrilla verticillata (L.f.) Royle growth and reproduction
Creator:
MacDonald, Gregory E., 1963-
Publication Date:
Language:
English
Physical Description:
xii, 113 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Aquatic plants ( jstor )
Carotenoids ( jstor )
Chlorophylls ( jstor )
Photoperiod ( jstor )
Plant growth ( jstor )
Plant tissues ( jstor )
Plants ( jstor )
Species ( jstor )
Trucks ( jstor )
Tubers ( jstor )
Agronomy thesis Ph.D
Dissertations, Academic -- Agronomy -- UF
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 100-112).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Gregory E. MacDonald.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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002030146 ( ALEPH )
33038030 ( OCLC )
AKL7778 ( NOTIS )

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Full Text









PHYSIOLOGICAL ASPECTS OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION















By

GREGORY E. MacDONALD


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

UNIVERSITY OF FLORIDA


1994




PHYSIOLOGICAL ASPECTS OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION
By
GREGORY E. MacDONALD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994


In memory of George E. MacDonald; your love, support, guidance and
understanding are the basis for all I have attained.


ACKNOWLEDGEMENTS
For their support and guidance during my tenure at the University of Florida
I wish to express my sincere appreciation to my committee members: Dr. Thomas
Bewick, Dr. George Bowes, Dr. William Haller, and Dr. Michael Kane. I would like
to extend my most sincere appreciation to my major advisor, Dr. Donn Shilling, for
his patience, understanding and guidance throughout my tenure at Florida. I would
also like to acknowledge the advice and support of Drs. Daniel Colvin, Ken
Langeland, and Bert McCarty.
In addition, I wish to acknowledge Christine Kelly, Bob Querns, Ron Kern,
Jim Gaffney, Jigna Thakore, Sean and Cindy Ragland, Laura Rankin, Margaret
Glenn, Jan Miller, Jamey Carter, Sandra McDonald, Brian Smith, Ken Smith,
Carlene Chase, Eric Bish, and Ron Doong. Without their valuable time and
assistance this research would have been extremely difficult, if not impossible.
For the two most important women in my life, I wish to express my deepest
gratitude to my wife Mickey and my mother Marilyn MacDonald. No words can
adequately describe the value of their support. I would also like to thank my
brother, Tim MacDonald, for his support and friendship.
During my tenure at the University of Florida I have had the opportunity to
build lasting friendships. The experiences I have shared will always be cherished.
in


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES x
ABSTRACT xi
CHAPTERS
I INTRODUCTION 1
II EVALUATION OF AN ENZYME LINKED
IMMUNOASSAY PROCEDURE FOR THE
ANALYSIS OF ABSCISIC ACID
IN HYDRILLA 20
Introduction 20
Materials and Methods 23
Elution Profiles and Column/ABA
Characterization 23
ELISA Internal Standardization
for ABA in Hydrilla 25
Results and Discussion 26
III THE INTERACTIVE EFFECT OF PHOTOPERIOD
AND FLURIDONE ON THE GROWTH,
REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata
(L.f.) Royle] 39
Introduction 39
Materials and Methods 43
Chlorophyll and Carotenoid Analyses 45
Anthocyanin Analysis 45
Abscisic Acid Analysis 46
IV


Statistical Analysis 46
Results and Discussion 47
IV THE INFLUENCE OF FLURIDONE, PHOTOPERIOD,
AND PLANT GROWTH REGULATORS ON THE
GROWTH AND REPRODUCTION OF MONOECIOUS
AND DIOECIOUS HYDRILLA (Hvdrilla verticillau:
(L.f.) Royle] 62
Introduction 62
Materials and Methods 65
Photoperiodic Response 67
Effect of Fluridone 67
Effect of Exogenous Abscisic Acid and
Gibberellic Acid 68
Statistical Analysis 68
Results and Discussion 69
V SUMMARY AND CONCLUSIONS 89
APPENDICES
A ELISA PLATE COATING
PROCEDURE 91
B ELISA SAMPLE QUANTIFICATION 92
C ELISA STOCK SOLUTIONS AND
STANDARDS 93
D ELISA BUFFERS AND SOLUTIONS 94
E ELISA STANDARD CURVE AND SAMPLE
ABA DETERMINATION 95
F ABSCISIC ACID COLUMN EXTRACTION
PROCEDURE 96
G THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS 97
v


H THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS 98
I THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF YOUNG HYDRILLA 99
LITERATURE CITED 100
BIOGRAPHICAL SKETCH 113
vi


LIST OF TABLES
TABLE PAGE
2.1 The effect of elution volume and methanol
concentration on abscisic acid C18 column retention
under neutral pH conditions 27
2.2 The effect of elution volume and methanol
concentration on abscisic acid Clg column retention
under low pH conditions 28
2.3 The effect of plant tissue and column extraction on
the recovery of abscisic acid. Abscisic acid was added
before or after column clean-up 30
3.1 The effect of photoperiod and fluridone on the dry
weight of mature hydrilla plants 48
3.2 The effect of photoperiod and fluridone on the dry
weight of young hydrilla plants 49
3.3 The effect of photoperiod and fluridone on the
subterranean turion production of
mature hydrilla plants 51
3.4 The effect of photoperiod and fluridone on the
subterranean turion production of young
hydrilla 52
3.5 The effect of photoperiod and fluridone on the
chlorophyll content of mature hydrilla plants 55
3.6 The effect of photoperiod and fluridone on the
carotenoid content of mature hydrilla plants 56
3.7 The effect of photoperiod and fluridone on the
anthocyanin content of mature hydrilla plants 57
vii


3.8 The effect of fluridone concentration on the
abscisic acid content of mature hydrilla apical stem
segments, average across photoperiodic regime 60
4.1 The effect of photoperiod on the growth of 8 week
old dioecious and monoecious hydrilla 70
4.2 The effect of photoperiod on the axillary turion
production of 8 week old dioecious and monoecious
hydrilla 71
4.3 The effect of photoperiod on the subterranean turion
production of 8 week old dioecious and monoecious
hydrilla 72
4.4 The effect of fluridone on the growth of dioecious
and monoecious hydrilla. Plants were grown under
long-day conditions for 12 weeks 74
4.5 The effect of fluridone on the axillary turion
production of monoecious hydrilla. Plants were
grown under long-day conditions for 12 weeks 76
4.6 Axillary turion production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 78
4.7 Axillary turion production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 79
4.8 Axillary turion weight per plant by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 80
4.9 Axillary turion weight per plant by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 81
4.10 Percent weight of axillary turions produced by 8 week old,
long-day grown dioecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications 83
viii


4.11 Percent weight of axillary turions produced by 8 week
old, long-day grown monoecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications 84
4.12 Shoot biomass production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous abscisic
and gibberellic acid applications 85
4.13 Shoot biomass production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 86
IX


LIST OF FIGURES
FIGURE PAGE
2.1 Internal abscisic acid standardization for column
cleanup procedure with hydrilla tissue 32
2.2 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.02 to 0.8 pmol 34
2.3 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.2 to 5.0 pmol 35
2.4 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.02 to 0.6 pmol 36
2.5 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.2 to 5.0 pmol 37
x


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHYSIOLOGICAL ASPECTS OF HYDRILLA
[Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION
By
Gregory E. MacDonald
August, 1994
Chairman: Dr. D.G. Shilling
Major Department: Agronomy
The goal of this research was to provide a better understanding of the
fundamental processes involved in hydrilla growth and reproduction. Abscisic acid
from hydrilla tissue was analyzed by enzyme-linked immunoassay. Although solid-
phase extraction provided good purification of abscisic acid present in hydrilla tissue,
no clean up of the extract was necessary prior to analysis. Crude, unpurified extracts
of hydrilla stem and turion tissue were analyzed by ELISA. Validation of abscisic
acid extraction and analysis procedure was verified by internal standardization.
Accurate quantification could be obtained from plant extracts containing 0.02 to 5.0
pmol of abscisic acid. Short days promoted subterranean turion formation but this
effect was reduced by long days and 5 and 10 ppb fluridone. Fluridone caused a
significant reduction in chlorophyll and carotenoid levels and the abscisic acid
xi


content of mature plant shoot tips. The concentration of abscisic acid in young
plants was higher under short days. These studies provide further evidence that
fluridone can be used as a fall herbicide treatment to reduce turion formation in
hydrilla. The growth of both biotypes was reduced under short-day conditions.
Monoecious hydrilla formed axillary and subterranean turions regardless of
photoperiod, while dioecious hydrilla formed only subterranean turions only under
short-day conditions. Fluridone reduced the growth of both biotypes and reduced
turion production in monoecious hydrilla. Exogenously applied abscisic acid at 0.1,
1.0, and 10 /iM induced axillary turion formation by dioecious hydrilla under long day
conditions and abscisic acid at 1.0 and 10 /M induced axillary turion formation by
monoecious hydrilla. This process could be reversed by gibberellic acid in both
biotypes. However, subterranean turion formation was not observed under any
treatment for the dioecious biotype, but was observed in control monoecious hydrilla
plants. Monoecious hydrilla could be induced to flower by 50 iM gibberellic acid
applications. This study indicates abscisic acid alone does not control axillary turion
formation in monoecious and dioecious hydrilla, but rather a balance between
abscisic acid and gibberellic acid may be the key to regulation of this phenological
process.
Xll


CHAPTER I
INTRODUCTION
Hydrilla [Hvdrilla verticillata (L.f.) Royle] is an exotic, submersed aquatic
vascular plant that causes major problems in the freshwater ecosystems of Florida
and the southeastern United States (Haller, 1976). Hydrilla interferes with
navigation, flood control and most recreational water activities, including fishing,
through a displacement of native vegetation (Langeland, 1990). Hydrilla was
discovered in 1959 in Florida, in a Miami canal. It was also reported in Crystal
River during the same period and was originally called Florida Elodea (Blackburn
et al.. 1969).
Hydrilla is a monotypic genus in the family Hydrocharitaceae, taxonomically
an ancient monocot family, with the center of origin thought to be tropical Asia
(Cooke and Luond, 1982). However, hydrilla is cosmopolitan, and is reported to be
broadly distributed throughout Germany, Lithuania, England, Poland, the upper Nile
of Africa, southeast Asia, Australia, Madagascar, India, China, Japan and the
southern United States (Lazor, 1978). A review of hydrilla by Mitra (1955) provided
the first detailed description of the species with historical background information.
Linnaeus filius first described hydrilla as Serpicula verticillata and classified it in the
Haloragaceae family. Hydrilla was later placed into the Hydrocharitaceae family
under the name Hydrilla ovalifolia but it was later changed to Hydrilla verticillata.
1


2
Hydrilla has been characterized by many scientists, as indicated by several describing
authors including Presl., Royle, and Casp. Today, Hydrilla verticillata (L.f.) Royle or
Casp. is the scientific name used to denote hydrilla. Langeland et al. (1992) showed
hydrilla to be endopolyploid, with monoecious hydrilla being triploid and dioecious
hydrilla being diploid; but, evidence from root tip karyotypes suggest sexual
compatibility across hydrilla populations and biotypes.
Anatomically, hydrilla is considered to be a primitive plant with reduced
vasculature. The xylem tissue is vestigial and phloem members are greatly reduced
(Yeo et al.. 1984). The leaves are comprised of two contiguous epidermal layers with
cuneate plastid inclusions, lacking in starch granules -- characteristic of most
monocots. The upper epidermis is much thicker than the lower layer (Pendland,
1979). Hydrilla leaves are borne in whorls of 4 or 5, with a distinctive spiny midrib
on the abaxial side of the leaf, distinguishing this species from elodea. Plants are
attached to the hydrosoil through fibrous roots and may resprout from root crowns
at the surface of the hydrosoil (Haller, 1976). Flowers are borne on stems near the
water surface (Cooke and Luond, 1982). Male and female flowers may be borne on
the same (monoecious) or separate (dioecious) plants. The control of floral
production is not well known but Pieterse et al. (1984), reported that low nitrogen
and phosphorus concentrations stimulated flower production in monoecious hydrilla.
Hydrilla regrows in the spring, as water temperature begins to increase, from
root crowns and/or subterranean turions in the hydrosoil (Haller, 1976). Shoot
growth is regulated by light quality with green light (prominent at deeper depths)


3
promoting stem elongation and red light (prominent at the surface) stimulating
branching (Van et al.. 1977). Hydrilla has also been shown to increase the level of
chlorophyll b at greater depths, enabling the plant to use longer wavelengths of light
for photosynthesis (Van et al.. 1977).
The development of a thick, entangled growth at the water surface (mat)
occurs when the shoots reach the water surface. This forms a dense canopy and
reduces light penetration in the first 0.3 m by 95% (Haller and Sutton, 1975). The
rapid growth of hydrilla has been reported to be 4.2 g dry wt'm2'day'1 (DeBusk et
al 1981). Hydrilla is extremely competitive, forming large, monotypic stands.
Studies on the photosynthetic characteristics revealed hydrilla to have a C02
compensation point of 44 fiL L"1 with light saturation occurring at 600 to 700
/mol'm s PPFD (Van et al.. 1976). Hydrilla can adapt to low light levels with a
light compensation point of 10 to 12 unol m'^s'1 PPFD (Bowes et al.. 1977).
Holaday and Bowes (1980) reported hydrilla could exist with low or normal C02
compensation points. Those plants with low compensation points had high activity
of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) and pyruvate, P dikinase (E.C.
2.7.9.1) enzyme levels, suggesting that hydrilla is capable of both C3 and C4
photosynthetic metabolism and the change was due to low C02 concentrations in the
water (Barko and Smart, 1981; Holaday et al.. 1983). Salvucci and Bowes (1983)
implicated carbonic anhydrase (E.C. 4.2.1.1) in the reduction of photorespiration in
hydrilla during periods of low C02 compensation points. Ascencio and Bowes (1983)
also reported a major increase in PEP carboxylase under these conditions.


4
Collectively, these characteristics allow hydrilla to utilize the free C02 from the water
sooner after sunrise than other aquatic plants due its low light compensation point.
Furthermore, hydrilla can maintain net photosynthesis under low C02 levels through
the activity of PEPcarboxylase. This increases hydrillas competiveness, decreasing
competition from other species and allows hydrilla to maintain the luxuriant growth
of the mat under low C02 and high 02 conditions. In addition, Kulshreshtha and
Gopal (1983) reported allelopathic properties of hydrilla on species of Ceratophyllum,
further increasing the competitive advantage of this species.
Several studies have evaluated the effect of mineral nutrition on hydrilla
growth. Sutton (1985) suggested the growth of hydrilla is controlled by nutrients in
the root zone, while Spencer (1986) showed hydrilla could grow equally well under
low or high organic matter sediment composition. Stewart (1984) showed phosphorus
in the rooting media was the limiting factor in growth for hydrilla. Basiouny and
Garrard (1984) demonstrated that calcium and phosphorus uptake in hydrilla was
passive while potassium, copper, iron and manganese uptake required metabolic
energy. Iron appeared to be particularly important for hydrilla growth with
tremendous iron uptake and an exceptionally high iron to manganese ratio required
for optimum growth (Basiouny et al.. 1977b). Basiouny et al. (1977a) showed iron
was absorbed by roots and leaves of hydrilla and translocated throughout the plant.
Hydrilla is extremely difficult to manage and control because spread can occur
through a variety of mechanisms including fragmentation and specialized dormant
buds called turions (Sculthorpe, 1967). Langeland and Sutton (1980) reported


5
regrowth from hydrilla fragments with a single node and Sutton et al. (1980) reported
one shoot tip will produce as much biomass as 16 tips. Seed production has been
reported for the monoecious biotype but does not occur in dioecious hydrilla in the
United States.
Turions can be formed in the axials of leaves, or at the ends of positively
geotropic rhizomes which extend into the hydrosoil (Haller, 1976; Yeo et al.. 1984).
Subterranean turions (tubers) can remain dormant for as long as 5 years (Van and
Stewart, 1990). Miller et al. (1976) reported that increased tuber size and density
were correlated to increased water depth. There appears to be an environmentally
enforced dormancy of tubers, preventing rapid depletion of the tuber bank (Van and
Stewart, 1990). Basiouny et al. (1978b) showed gibberellic acid, ethephon, thiourea
and storage at 5 C to increase turion sprouting, and hypothesized the existence of
two types of dormancy in hydrilla subterranean turions. Stewart (1969) found
gibberellic acid enhanced sprouting of subterranean turions while IAA and 2,4-D
enhanced axillary turion development. Subterranean turions are thought to maintain
hydrilla within a given area, possibly through periods of drought. Work by Basiouny
et al. (1978a) showed that tubers are better able to survive drought than axillary
turions with almost 17% germination rate for tubers dried for 64 hours.
Axillary turions are smaller than subterranean turions and are thought to
function in dispersal (Thullen, 1990). These structures are generally formed on
detached floating mats of hydrilla (Thullen, 1990; Miller et al.. 1993). Furthermore,
axillary turions could last only 1 year in the hydrosoil (Van and Stewart, 1990),


6
presumably due to their smaller size.
Turion formation in dioecious hydrilla has been shown to be a photoperiodic
response with a critical photoperiod of 13 hours for subterranean turion formation
(Van et al.. 1978a). Haller et al. (1976) reported subterranean turion production
occurred in north Florida between October and April. Klaine and Ward (1984)
demonstrated the turion formation response was phytochrome mediated, with red
light (650 nm) stimulation and far-red light (750 nm) repression of turion formation
in the dioecious biotype. Exogenous ABA applications induced turion formation
under non-inductive conditions. Van et al. (1978b) also showed exogenous ABA
promoted turion formation under 16 hour daylengths. Axillary turion formation
appears to respond similarly to subterranean turion formation with regard to
photoperiod. Miller et al. (1993) showed hydrilla axillary turion production was
similar to subterranean turion formation occurring in the fall and winter months
only.
Both the monoecious and dioecious hydrilla biotypes occur in the United
States. The dioecious female plant occurs throughout Florida, the southeast and
California, whereas monoecious hydrilla is found in Washington D.C., Virginia,
Maryland and North Carolina. Besides a difference in flowering, there seems to be
several distinct differences between the two biotypes in terms of vegetative growth
and turion production.
Monoecious hydrilla produces subterranean turions under both 10 and 16 hour
photoperiods, with a greater number of turions produced under short-day conditions.


7
The growth of monoecious hydrilla is generally prostrate, near the hydrosoil with
many horizontal stems and higher shoot densities than dioecious hydrilla. In
comparative studies, Steward and Van (1987) found that the monoecious biotype
tolerated cooler temperatures and was able to sprout at lower temperatures. Ames
et al. (1986) also reported greater growth of monoecious hydrilla under cooler
temperatures compared to dioecious.
Subterranean turion production has been shown to be greatest under short
days for monoecious hydrilla with more turions produced than the dioecious form,
but there was a decline in biomass accumulation with short days. Monoecious
hydrilla also appears to able to reproduce much more rapidly than dioecious, forming
new subterranean turions in 4 weeks as compared to 8 weeks for dioecious hydrilla
(Van, 1989). Spencer et al. (1987) reported subterranean turions to be nearly 2 to
5 times as large as axillary turions, with monoecious subterranean turions smaller
than those of dioecious hydrilla. In addition, Ames et al. (1987) showed percent
sprouting was higher in monoecious than dioecious hydrilla. These studies indicate
monoecious hydrilla possesses an annual growth habit, along with rapid and prolific
turion production, adapting this biotype to northern areas which have cooler
temperatures and short growing seasons.
Earlier work suggested monoecious hydrilla was photoperiodic in response to
turion formation. Spencer and Anderson (1987) showed that monoecious hydrilla did
not produce subterranean turions under daylengths greater than 14 hours and
production increased as daylength decreased. They also reported photo-interruption


8
of monoecious hydrilla inhibited turion formation, similar to long-day grown plants
(Anderson and Spencer, 1986). However, monoecious hydrilla appears only to be
delayed in the formation of turion under long-day conditions, producing turions after
8 weeks under long-day conditions (Van, 1989).
Management of hydrilla is difficult due to rapid growth rate and prolific turion
formation. Cultural management schemes, such as drawdowns to deplete tuber
populations have had limited success (Haller et al 1976) and biocontrol agents, such
as grass carp, are unpredictable forms of control. Martyn (1985) used grass carp to
control 9000 acres of hydrilla in a 20,000 acre lake in Texas. All hydrilla was
removed after 2 years but over 270,000 fish were necessary. Insect biocontrol has
been studied, with several introductions but these have met with only limited success.
The herbicides diquat, endothall and copper sulfate, alone or in combination
provide good initial control but regrowth of the hydrilla quickly occurs. Fluridone
(l-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(li/)-pyridinone) has been shown
to be very effective for hydrilla control, but contact time and concentration are
critical issues affecting the efficacy of a given fluridone treatment (Van and Stewart,
1985; Haller et al.. 1990; Fox et ah. 1991).
Fluridone affects plant tissue by decreasing pigment levels (Maas and Dunlap,
1989). This is due to a decrease in carotenoid levels followed by a subsequent
decrease in chlorophyll content due to excess photooxidative stress (Devlin et al..
1978). Bartels and Watson (1978) demonstrated the mechanism of action of
fluridone to be carotenoid inhibition and this effect could be overcome by adding 5-


9
aminolevulinic acid (Fletcher et al.. 1984). The actual site is an inhibition of the
conversion of phytoene to phytofluene in the terpenoid biosynthetic pathway (Mayer
et al.. 1989). Fluridone was reported to be a non-competitive enzymatic inhibitor,
with reversible binding. Fluridone causes similar effects on hydrilla tissue, decreasing
the chlorophyll and carotenoid content, but interestingly, increasing anthocyanin
content (Doong et al.. 1993).
Fluridone was originally marketed for weed control in cotton ('Gossvpium
hirsutium L.). Translocation studies showed fluridone to be translocated
apoplastically following root absorption. Tolerant species, such as cotton, show
limited translocation and root uptake (Berard et al.. 1978). Banks and Merkle (1978)
reported good control with fluridone of many annual weeds in cotton but also
reported some soil persistence.
Fluridone is an off-white crystalline solid, with a melting point of 151-154 C
and a water solubility of 12 ppm (McCowen et al.. 1979). It is classified as a
pyridinone herbicide with a molecular weight of 329.3 (McCowen et al.. 1979).
Mossier et al. (1991) reported microbial breakdown for fluridone was very slow and
variable. Aqueous degradation rates from lake collected microbial consortia did not
correlate to previous fluridone use patterns in the aquatic environment. Fluridone
has a fairly high affinity to clay or organic matter (Mossier et al.. 1993). Therefore,
slow-release formulations could only be effectively used in aquatic ecosystems with
a sandy bottom substrate or in situations where the fluridone pellet does not contact
the hydrosoil.


10
Ultraviolet light breakdown is the major degradation mechanism in the natural
environment. Photolytic degradation of fluridone is highest at 297 to 325 nm with
a half-life of 26 hours although some degradation can occur at 325 to 355 where the
photolytic half-life was 840 hours (Mossier et al.. 1989).
Fluridone has also been shown to decrease the levels of ABA within plant
tissue (Jones and Davies, 1991). Abscisic acid is derived from carotenoid precursors
so the negative effect of fluridone on carotenoid levels also translates to lower ABA
content. Abscisic acid is thought to be involved in turion formation in hydrilla, and
fluridone could be used to regulate this process. Turion formation in hydrilla can be
inhibited by fluridone under short-day conditions at concentrations greater than 5
ppb (MacDonald et al.. 1993), but the effect on ABA and subsequent inhibition of
turion formation was not determined.
Abscisic acid (ABA) is a plant hormone involved in a multitude of
physiological responses including stomatal opening (Cornish and Zeevaart, 1985), bud
and seed dormancy (Quatrano, 1987; Barros and Neill, 1986), freeze tolerance
(Johnson-Flanagan et al.. 1991; Reaney and Gusta, 1987), and tuber and turion
formation (Smart and Trewavas, 1984). ABA is formed through the carotenoid
biosynthetic pathway by the conversion of 9-cry-neoxanthin to xanthin (Xan).
Xanthin is subsequently converted to ABA aldehyde and finally to cry- ABA (Gamble
and Mullet, 1986; Zeevaart et al.. 1989; Li and Walton, 1990; Parry and Horgan,
1991). Light can cause isomerization to trans-ABA but only cry-ABA is thought to
be the biologically active form. ABA biosynthesis is regulated by the production of


11
xanthin, not the conversion of xanthin to ABA (Parry and Horgan, 1991).
ABA has been implicated in the seed dormancy of many species (Hole et al..
1989; Singh and Browning, 1991; Le Page-Degivry and Garello, 1992), but the exact
mechanism not well understood. Morris et al. (1991) indicated that ABA induced
dormancy in wheat seeds at the gene level while Ozga and Dennis (1991) showed
changes in ABA in apple seeds were not related to dormancy. In a review, Quatrano
(1987) reported that ABA has been shown to modulate gene expression in cultured
embryos of certain crops. During seed development the increase in endogenous
ABA promotes maturation of the embryo and represses seedling growth. This is
accomplished by modulating gene sets, positively affecting the maturation set and
negatively affecting the germination set. As the seed begins to lose water, the
amount of ABA and the embryo sensitivity to ABA decreases and the dehydration
of the seed, not ABA, prevents germination.
Subbaiah and Powell (1992) reported that the chilling requirement of apple
seed is not related to ABA changes during stratification, and chilling, not ABA, was
required for germination. Also the decline in ABA was largely due to leaching of
the seed coat and nuclear membrane while ABA content of embryo remained
constant. They postulated that ABA leaching from the seed allows certain promotive
forces to develop, but these can only occur after the chilling requirement was met.
Earlier work also supports this contention. Schopfer et al. (1979) showed
ABA could reversibly arrest embryo development by inhibiting water uptake, not by
control of DNA, RNA or protein synthesis. Release from dormancy is not due to


12
lower ABA levels, but rather to a loss of sensitivity to ABA. Oishi and Bewley
(1990) found the decline in ABA will permit germination of maize kernels, but only
drying will allow the aleurone layer to become sensitive to gibberellic acid.
Gibberellic acid induces a-amylase activity, causing the remobilization of food
reserves for seedling growth.
Mutations are often used to study the role of ABA. Koornnef et al.. 1989
used three ABA mutants of Arabidopsis thaliana, termed abi loci, and found all
mutations reduced dormancy. However, one mutation did not effect the water
relations of leaf tissue. They hypothesized that the mutations affect different tissue-
specific receptors. Research by McCarty (unpublished) indicated that there was an
ABA receptor molecule complex on a promoter sequence of RNA. When ABA
binds to the promotor, RNA transcription begins and a specific enzyme(s) or event
is initiated.
There have been several indications that ABA plays an important role in plant
growth, especially under water-stressed conditions. Saab et al. (1990) showed that
ABA maintains primary root growth and inhibits shoot growth of maize seedlings
under low water potentials. In later work, Saab et al. (1992) suggested a differential
response to endogenous ABA by root and mesocotyl growing zones of maize. At low
water potentials, ABA had the greatest effects near the root tip and this effect
decreased from the tip. Mesocotyl tissue is more responsive to ABA in those cells
away from the meristematic region. Porter (1981) suggested that endogenous levels
of ABA in different plant organs could be a factor in the directional control of


13
assimilate transport in plants. Hoffmann-Benning and Kende (1992) reported that
the growth of rice was regulated by a ratio of gibberellic acid and abscisic acid, as
a growth promoter and inhibitor, respectively.
Interesting work on the effects of ABA on aquatic macrophytes has also been
reported. Kane and Albert (1987) demonstrated that ABA, possibly as an indicator
of water-stress, caused changes in aerial leaf morphology and vasculature in Hippuris
vulgaris. ABA also caused aerial leaf development in MyriophyHum and Proserpinaca,
and suggested possible ABA/ethylene interactions (Kane and Albert, 1989). Goliber
and Feldman (1989) measured the ABA content of the aerial leaves of Hippuris
vulgaris and showed much higher levels. They suggested the effect of osmotic stress
is the cue in aerial leaf development.
There is considerable changes in the compartmentation of ABA during light
and dark periods due to pH changes, with a 2-fold increase of ABA in the apoplast
in the dark. ABA can irreversibly conjugate to glucose forming ABA-glucoside.
This form is inactive and is restricted to the vacuole, and may be a sequestration
mechanism by which ABA is removed from the active pool. ABA has also been
shown to be a major factor in the control of stomatal opening through regulation of
guard cell turgor pressure (Zeevaart and Creelman, 1988). Guard cells under stress
appear to release ABA, initiating closure. Water-stressed roots can also produce
ABA that is translocated to the leaves and effects stomatal closure (Zeevaart and
Creelman, 1988).


14
Hartung (1983) reported the site of action of ABA in guard cells to be on the
outer surface of the plasmalemma. Raschke (1987) reported ABA prevented
stomatal opening by blocking H+ extrusion and K+ influx, and initiated rapid closure
by K+ release. Recent work suggested that ABA acts on a G-protein at the
membrane surface, activating phospholipase C, which catalyzes the hydrolysis of
phosphotidalinositol-pyrophosate to inositol-3-phosphate (IP3) and diglyceralhyde.
IP3 then causes intracellular Ca2+ release, blocking K+ channels and lowering
stomatal opening. There is also some evidence that internal ABA may have some
control over this process as well.
Abscisic acid is also thought to be involved in the process of turion and tuber
formation in hydrilla and other species. Tubers are among the most common form
of vegetative reproductive structures and are produced by several species including
potato (Solanum tuberosuml and jerusalem artichoke (Helianthus tuberosum!.
Tubers are swollen underground stems possessing multiple nodes and greatly reduced
leaf characteristics (Cutter, 1978). In potato, the morphology of tuber formation has
been well documented. Tubers are formed from stem tissue, generally at the ends
of basal stolons (Peterson, £t ah, 1985), and are initiated in the youngest elongating
intemode (Peterson, et ah, 1985). The pith cells in this area enlarge, followed by
rapid cell division of the perimedullary parenchyma and cortical cells. Cell division
occurs in early tuber development and final tuber size is achieved through cell
enlargement (Reeve et_ ah, 1973).


15
Turions are similar to tubers but are morphologically less complex, derived
from leaf tissue, with little differentiation of the stem tissue. Turion are often found
in freshwater aquatic species, such as green milfoil (Myriophyllum verticillatum L.)
(Weber and Nooden, 1976) and giant duckweed (Spirodella polvrhizal (Smart and
Trewavas, 1983). Unlike tubers, turions sprout from a single node at the tip of the
structure, although turions are comprised of several densely packed nodes (Yeo et
ah, 1984). The outer portion of the turion is made up of leaf tissue, which swells and
envelopes the terminal apex, giving the structure a scale-like appearance. These
leaves accumulate carbohydrates, with an abscission layer forming at the base of the
lowermost leaves. The turion detaches from the mother plant and remains dormant
until conditions favorable for growth arise.
Turion formation by dioecious hydrilla is promoted by short-days (Haller £t
ah, 1976). This response has been shown to be phytochrome mediated (Klaine and
Ward, 1984). Phytochrome has been implicated in several plant processes, including
flowering, seed germination and tuber formation (Quail, 1991). However, recent
work provided evidence that phytochrome may regulate more fundamental plant
processes (Lissemore et ah, 1987), (Smith and Whitelam, 1990), (Kendrick and
Nagatani, 1991), (Seeley et ah, 1992) and (Edgerton and Jones, 1992). Phytochrome
appears to be involved in the activation of K+ channels (Lew et ah, 1992) and has
been suggested to be a protein kinase (McMichael and Lagarias, 1990) (Doshi et ah,
1992). Recent research also indicated that at least two types of phytochrome [Type
I (Phytochrome A) or Type II (Phytochrome B] can be present within plant tissues


16
(Abe et ah, 1985) (Shimazaki and Pratt, 1985). Phytochrome A has been shown to
regulate stem elongation (Boylan and Quail, 1991), chloroplastic gene expression
(Sharrock et ah, 1988) and anthocyanin biosynthesis (Adamse et ah, 1988).
Phytochrome B has been linked to photoperiodic responses (Vince-Prue and
Takimoto, 1987; Takimoto and Saji, 1984) with respect to floral induction.
Changes in hormone levels occur soon after photoperiodic induction of tuber
formation. In potato, changes in ABA, gibberellin, and cytokinin levels have been
reported. Gibberellins are high Under long-day photoperiodic conditions and decline
under short days (Krauss, 1985), while the opposite effect is observed with abscisic
acid. Several researchers have reversed the effect of photoperiod with exogenous
applications of these hormones (i.e. preventing tuber formation under short-day
lengths with gibberellic acid while inducing tuberization with abscisic acid under long-
day lengths). However, a critical ratio or balance between these two hormones may
be needed for tuberization to occur (Menzel, 1980) (Vreugdenhill and Struik, 1989).
Cytokinins accumulate in response to tuberization but do not induce this process
(Krauss, 1985). In addition, increased nitrogen levels in the leaves have been shown
to delay or inhibit tuber formation in potato (Krauss, 1978), while increased
photosynthate concentration promotes this response (Wenzler et ah, 1989).
Exogenously applied abscisic acid also induced turion formation in hydrilla
(Van et ah, 1978), (Klaine and Ward, 1984). Klaine and Ward (1984) also reported
that turion formation in hydrilla was reduced by exogenous gibberellic acid, similar
to that observed for potato. However, cytokinin had no effect. Turion formation in


17
giant duckweed (Perry and Byrne, 1969) can also be induced by exogenous ABA
applications, suggesting that the role of abscisic acid in regulating vegetative
reproduction is similar for a variety of species.
The role of ABA and gibberellic acid in tuber formation is well documented
but specific changes related to initiation of this process is unclear. Hannapel et ah
(1985) showed exogenous gibberellin prevented the accumulation of patatin and two
other potato tuber proteins and correlated this with the inhibition of tuber formation.
Abscisic acid had an inhibitory effect on overall protein synthesis in Spirodela.
polvrhiza turion formation (Smart and Trewavas, 1984), and the authors suggested
that there were several novel proteins specific to induced tissue.
{
Quantification of abscisic acid has traditionally been accomplished through the
use of gas chromatography-mass spectrometry (Dumbroff et al.. 1983; Reymond £t
ah, 1987). However, the use of enzyme-linked immunoassays (ELISA) for ABA
analysis has increased over the years. The advantage of ELISA is that limited
sample purification and increased number of samples can be analyzed within a given
time frame.
i Several researchers have developed ABA antibodies (Weiler, 1980; Mertens
et ah. 1983; Ross et ah. 1987; Quarrie et ah. 1988). Most monoclonal antibody
immunoassays were able to use crude aqueous extracts without interference,
providing accurate quantification comparable to GC-MS analysis (Leroux et ah. 1985;
Quarrie et ah. 1988; Soejima et ah. 1990; Tahara et ah. 1991). Some
radioimmunoassays can detect free and conjugated ABA; this is dependant on Cj


18
or C4 ABA coupling to proteins for total or free ABA, respectively (Weiler, 1980).
However, ABA analysis with ELISA from plant tissues varies with plant
species and tissue type. Some extracts may require purification while other extracts
do not. Therefore, validation through internal standardization is recommended for
ABA analysis from a previously unvalidated tissue type.
Hydrilla is a perennial species, with regrowth from overwintering turions often
being the main source for re-establishment. Turions sprout in the spring, forming a
large, monotypic stand. The hydrilla then reproduces in the fall, replenishing the
turion supply within the hydrosoil. Therefore, turion depletion in the hydrosoil would
be the most effective means for long-term hydrilla control. However, herbicide
treatments during fall conditions (turion formation) would result in applications to
established hydrilla plants. However, fluridone has been shown to be very effective
for the control of hydrilla, and can reduce turion formation at concentrations greater
than 5 ppb. Fluridone will cause decreased ABA levels within plant tissues and this
may be the reason for the reduction in turion production.
The ability of hydrilla to reproduce by turions is the greatest restraint to the
control of this species. Therefore, a better understanding of the fundamental
processes involved in turion initiation and development would greatly assist in efforts
to effectively manage this species. To accomplish this goal the objectives of this
research were to 1) develop an ELISA based ABA analysis procedure for quantifying
ABA from hydrilla; 2) test the hypothesis that fluridone reduces turion formation
from a reduction in ABA content, not a lethal impact on plant growth; and 3)


19
compare the response (growth and turion development) of monoecious and dioecious
hydrilla biotypes cultured under controlled conditions to long and short-day
conditions, fluridone and exogenous plant growth regulators.


CHAPTER II
EVALUATION OF AN ENZYME LINKED IMMUNOASSAY PROCEDURE
FOR THE ANALYSIS OF ABSCISIC ACID IN HYDRILLA
Introduction
Abscisic acid (ABA) has been implicated in many plant developmental
processes, including control of gene expression, plasma membrane ion flux, and seed
dormancy (Neill et al. 1986; Barratt et al.. 1989). In addition, ABA plays a vital role
in plant water relations, through the regulation of stomata and growth under water
stressed conditions (Saab et al.. 1990; Saab et al.. 1992). ABA has also been
implicated in turion formation and heterophylly of aquatic macrophytes (Smart and
Trewavas, 1984; Kane and Albert, 1987).
Abscisic acid is a 15-carbon compound with a molecular weight of 264.3. It
exists in two forms: cis and trans, which isomerize in light. Cis-ABA is the
biologically active form, therefore quantification of ABA should take this fact into
account. The acidic nature of abscisic acid (pKa of 4.8) has been utilized in analysis
protocols (Jones and Davies, 1991).
Abscisic acid is derived from the terpenoid biosynthetic pathway, but the exact
synthesis route has only recently been elucidated. Earlier work suggested ABA was
formed through two separate pathways: a direct and indirect pathway (Walton,
1980). The direct pathway was suspected to involve a C15 precursor derived from
farnesyl-pyrophosphate, which may itself be xanthoxin (Milborrow, 1983). Xanthoxin
20


21
was discovered in the late 1960s (Taylor and Burden, 1973) as a precursor to ABA
and led to the indirect pathway hypothesis, as this compound is derived from the
breakdown of carotenoids. Therefore, ABA was also proposed to be derived from
carotenoid breakdown. However, elucidation of the exact biosynthetic pathway has
been extremely difficult due to the large number of potential ABA precursors versus
the minute amount of ABA (Cowan and Railton, 1986; Milborrow, 1983).
Subsequent research has provided the most definitive evidence that ABA is
produced via the indirect pathway of carotenoid breakdown. Biosynthetic inhibitors,
such as fluridone, have been shown to block carotenoid synthesis and also lower
ABA levels (Moore and Smith, 1984; Gamble and Mullet, 1986; Oishi and Bewley,
1990). In addition, carotenoid deficient mutants have lower levels of ABA (Wang
et al.. 1984; Neill et al 1986). Since these studies, most researchers have concluded
the synthesis of ABA involves epoxidation and isomerization of specific carotenoid
molecules, the formation of xanthoxin, and subsequent ABA formation (Zeevaart et
ah, 1989; Li and Walton, 1990; Parry and Horgan, 1991).
Abscisic acid can be extracted from plant tissue through a variety of methods.
Goliber and Feldman (1989) used 80% methanol for extraction from Hippuris
vulgaris while Gamble and Mullet (1986) found adequate extraction with 100%
acetone. Loveys and vanDijk (1988) found boiling water sufficient for extracting
ABA from grape (Vitis spp.l leaves and Tahara et al. (1991) found extraction with
tris-buffered saline to provide excellent extraction of ABA from wheat (Triticum
aestivum L. em. Thell.) leaves.


22
Traditional methods of ABA quantification include high performance liquid
chromatography (HPLC) and/or gas-liquid chromatography (GC). These methods
provide excellent quantification but require a large amount of purification (Dumbroff
et al.. 1983; Leroux et al.. 1985; Soejima et al.. 1990). Recently, enzyme-linked
immunoassays (ELISA) have been developed for ABA detection, greatly reducing the
rigorous purification required prior to analysis (Raikhel et al.. 1987; Tahara et al..
1991). In addition, the number of samples that can be processed at a single time is
much greater with ELISA.
Hydrilla [Hydrilla verticillata (L.f.) Royle] is a submersed aquatic plant that has
become a weed problem throughout much of southeastern United States and
California (Langeland, 1990). Hydrilla is a highly aggressive species, forming large,
monotypic stands that interfere with flood control, navigation and many recreational
water activities including boating and fishing (Haller, 1976; Langeland, 1990).
Hydrilla persists within a given area primarily through the production of specialized
vegetative reproductive structures called turions. Turion formation in hydrilla and
several other aquatic species is thought to be directly affected by ABA but the exact
role of ABA is not known (Van et al.. 1978; Klaine and Ward, 1984). Therefore,
a better understanding of the affect of ABA on turion formation in hydrilla could
provide more effective management strategies for this noxious species.
Several researchers have found the enzyme-linked immunoassay (ELISA)
procedure specific enough to quantify ABA in crude extracts without purification
(Tahara et al, 1991). However, each individual plant species must be tested and


23
validated if crude extraction is to be used (Zeevaart and Creelman, 1988). In
addition, plant tissue types may also vary in the content of interfering compounds,
particularly between chlorophyllous and achlorophyllous tissue. Therefore, the major
impediment to the universal applicability of ELISA is the unpredictable presence of
compounds that cross-react with the ABA-specific antibody. A procedure known as
internal standardization must be conducted on tissue prior to analysis for ABA using
ELISA. This technique allows a determination of whether purification of the sample
is necessary before ELISA quantification.
Although ABA has been implemented in hydrilla turion development, ABA
quantification from hydrilla tissue by ELISA has not been widely used. Therefore,
studies were conducted to develop a purification procedure for detecting ABA from
hydrilla and to determine if such a procedure was warranted for quantification by
ELISA.
Materials and Methods
Elution profiles and column/ABA characterization. Reverse-phase cartridges,
particularly C18, have been widely used for abscisic acid purification (Hubick and
Reid, 1980). Therefore, initial studies were performed to characterize the
relationship between eluant volume, methanokwater ratio and pH on the retention
of ABA to this type of column. Columns1 were prepared prior to use by washing
1 PrepSep; Fisher Scientific, Pittsburgh, PA 15219.


24
once with methanol and twice with deionized water. All samples were passed
through the column under vacuum (90 kPa) and the resulting eluant was collected
in 20 ml glass vials. During elution these vials were placed within a vacuum
manifold .
In the first experiment, 100 tL of 1 mM ABA was added to the top of the
column and eluted with varying concentrations of methanohwater (20:80, 50:50,
70:30, 80:20, 100:0) in 10 mL aliquots up to 50 mL. The elution pH was
approximately 7, with the majority of the ABA molecules in the protonated form.
The ABA content was then determined spectrophotometrically at 250 nm. Recovery
was expressed as a percent of ABA added.
i
A similar experiment was conducted with varying concentrations of
methanohwater (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 70:30, 80:20, and 100:0) in
10 ml aliquots up to 50 mL. In addition, the methanohwater elutions were acidified
with 1% formic acid to obtain a pH of less than 3.0. This resulted in most the ABA
in an unprotonated, neutral form. Recovery was expressed as a percent of ABA
added.
The preceding elution studies allowed the development of a purification and
concentrating procedure that was used in the following two experiments (see results
and discussion). An experiment was conducted to determine if the presence of
compound(s) in hydrilla extract would interfere with accurate quantification of ABA
2 PrepTorr; Fisher Scientific, Pittsburgh, PA 15219.


25
using UV spectroscopy. Tissue extract contained 20 mg-fresh weight hydrilla shoot
tips which were homogenized in 10 to 15 mL 100% methanol and the extract filtered
under vacuum. This extract contained three levels of plant tissue (0, 12.5, and 25 mg-
fresh weight/sample). Abscisic acid was added to the plant extracts at concentrations
of 0, 8, 13, or 18 nM prior to column extraction. ABA was detected
spectrophotometrically at 250 nm.
A study was also conducted to determine if components in hydrilla extract
would influence the recovery of ABA purified using column chromatography. This
study involved fortifying hydrilla extract with different concentrations of ABA before
or after purification. Tissue extract was prepared using the aforementioned methods
and three concentrations (150, 250, or 350 nM) of ABA were used for fortification.
Abscisic acid recovered was detected spectrophotometrically at 250 nm.
ELISA internal standardization for ABA in hydrilla. An internal
standardization experiment was conducted to determine if purification of hydrilla
tissue was necessary prior to ABA quantification with ELISA. Antibody and
antibody tracer specific for ABA were obtained directly from the manufacturer3.
Procedures for the preparation of ELISA plates, sample preparation and ABA
concentration determination are outlined in Appendices A-E.
Internal standardization was performed on crude extracts of hydrilla stem and
turion tissue utilizing ELISA. Due to the large range of values to be tested,
3 Phytodetek, Idetek, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089.


26
standardization of each tissue type was performed in two experiments. In the first
experiment, 100 iL of 0, 0.02, 0.04, 0.06, 0.08, 0.2, 0.4, or 0.6 pmol ABA was added
to dilutions of hydrilla stem tissue (1:2 and 1:4) and turion tissue (1:4 and 1:8).
Dilutions were based on 20 mg-dry weight/sample as a 1:1 dilution and ABA was
added prior to quantification by ELISA. Samples were extracted according to the
procedures outlined in the previous section. The methanolic extract was then dried
under a stream of nitrogen gas and reconstituted in tris-buffered saline prior to
quantification by ELISA.
In the second experiment, 100 /L of 0.0, 0.2, 0.5, 1.0, 2.0, or 5.0 pmol ABA
was added to dilutions of hydrilla stem tissue (1:1 and 1:3) and turion tissue (1:1 and
1:4). Dilutions were based on 12 or 8 mg-dry weight/sample as the 1:1 dilution) for
stem or turion tissue, respectively. ABA was added prior to quantification by ELISA.
Tissue extracts for both experiments were prepared in a manner similar to plant
material used in the column extraction procedure.
Results and Discussion
Abscisic acid under neutral pH conditions showed little affinity for the C18
column when the mobile phase contained greater than 30% methanol. The majority
of ABA was recovered with only 10 mL of 30% methanol (Table 2.1). Much higher
concentrations of methanol were necessary to elute ABA from the C18 column when
the mobile phase was acidic (Table 2.2). Under low pH conditions ABA retention
increased at similar methanol concentrations to the previous experiment. However,
complete retention was observed at concentrations below 20% methanol.


27
Table 2.1. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under neutral pH conditions.
Cumulative
Elution
Volume
0
10
20
Methanol Concentration (%)
30 40 50 70 80
100
mL
% ABA eluted
10
8+31
504
77+2
872
94 + 1
94+3
895
763
80+4
20
33+3
21 2
182
2+2
0
0
--
5+3
5+3
30
15+2
111
21
2+2
0
0
0
0
0
40
7 + 1
62
21
0
0
0
0
0
0
50
41
0
0
0
0
0
0
0
0
1 Means (four replications) followed by standard errors.


28
Table 2.2. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under low pH conditions.
Cumulative
Elution
Volume
0
10
20
Methanol Concentration (%)
30 40 50 70 80
100
mL
% ABA eluted
10
0
0
0
21!
14+6 572
903
992
923
20
0
0
0
0
593 44+3
15 3

11 4
30
0
0
0
84
13+4 0
0
0
0
40
0
0
0
28 4
0 0
0
0
0
50
0
0
0
22 3
0 0
0
0
0
1 Means (four replications) followed by standard errors.


29
Concentrations greater than 50% removed ABA from the column after 20 mL of
elution volume. Extraction of ABA using Ci8 solid-phase columns is highly
dependent on pH. Low pH conditions allows greater retention of the ABA to the
column while neutral conditions do not. These results agree with the work of Parry
and Horgan, 1991 and other researchers.
The purpose of these studies was to develop a two-stage procedure for the
purification and concentration of ABA for analysis by ELISA. In the first
purification step, it was imperative that the appropriate solvent strength (%
methanol) be achieved to allow the retention on the column of as many extract
components as possible while allowing the elution of ABA. Because the volume of
solvent increased in this step, it was then necessary to concentrate the purified ABA
extract. This was accomplished by determining the appropriate solvent strength of
the mobile phase that would allow the retention of ABA by the column. This
technique is known as solid phase extraction and has many advantages over liquid
to liquid extraction, which results in large quantities of waste solvent. In addition,
solid phase extraction is cheap, rapid and allows for the simultaneous preparation of
numerous samples. The results provided information necessary to devise a clean-up
procedure that could be utilized for ABA analysis. This procedure provided over
92% recovery for all concentrations when ABA was extracted in the absence of plant
tissue (data not shown). Details of this procedure can be found in Appendix F.
When this procedure was used with the addition of plant tissue, it resulted in
greater than 89% recovery for all concentrations of ABA added (Table 2.3). Visual


30
Table 2.3. The effect of plant tissue and column extraction on the recovery of
abscisic acid. Abscisic acid was added before or after column purification.
ABA
Added
Plant + ABA
Before Column
Plant + ABA
After Column
Recovery
(nM)
0
0.276+0.401
- absorbance
0.276 0.40
----%
150
0.5800.20
0.632 0.34
92
200
0.703 0.09
0.752 0.45
93
250
0.7860.18
0.880 0.42
89
1 Means (three replications) followed by standard errors.


31
observations indicated that the column purification procedure provided good
separation of chlorophyll and carotenoids. However, anthocyanins were extracted
with the ABA, as indicated by a pinkish color during the second column extraction.
Furthermore, the presence of interfering compounds in hydrilla tissue extract
was not detected using this procedure (Figure 2.1). An internal standardization
procedure allows for the detection of an interfering compound. If the addition of
ABA to various extract dilutions does not cause a change in the rate of absorbance
then interfering components are not present. The absence of such a compound
would be indicated by parallel lines as the rate at which ABA detected as a function
of ABA added was the same regardless of the extract dilution. Although different
amounts of ABA were detected, dilutions as a function of ABA added was the same.
The column clean-up procedure developed reported herein provided good
recovery of ABA and separation of possible interfering compounds. Although
additional purification could be necessary with chromatographic methods of
quantification, this procedure provided an excellent initial purification step.
Furthermore, this procedure provided adequate purification of hydrilla tissue for
quantification by ELISA, if purification was needed.
Internal standardization of ELISA allowed for the detection of an interfering
compound (s) by dilution of the interfering compound (s) with the addition of higher
levels of ABA. The absence of interference would be indicated by parallel lines of
the two levels of plant tissue and/or the lack of a significant interaction (P > 0.05).
The results from hydrilla stem tissue indicated the absence of interfering compounds


ABA ADDED (nM)
Figure 2.1. Internal abscisic acid standardization for column cleanup
procedure with hydrilla tissue.


33
(Figures 2.2 and 2.3). Good separation of the two dilutions was observed in the first
experiment at 0.6 and 0.8 pM ABA. However, the ability to detect differences in
dilution was limited to a range of 0.2 to 0.5 pM in the second experiment. Hydrilla
turion tissue also appeared to be free of interfering compounds (Figures 2.4 and 2.5).
Although the lines do not appear to be absolutely parallel, statistically there was no
interaction (P >0.05) between ABA added and ABA detected for the different
extract dilutions. This implies that there was a consistent change in the amount ABA
detected regardless of the amount of ABA added (i.e. the lines representing
independent and dependent variables for the different dilutions were parallel
although differing in magnitude).
Furthermore, the presence of interfering compounds would result in response
convergence at a higher added ABA, because the interfering compound (s) would
react with the antibody, over-estimating ABA present. However, these data do not
fit this scenario. In addition, preliminary experiments were conducted by adding
ABA prior to purification and the same relationship between ABA added and ABA
detected was observed (data not shown). Based on these results analysis of ABA can
be accurately determined from hydrilla tissue by analysis of crude extraction.
However, prudence should be exercised during analysis to ensure proper dilution of
the respective sample; only those readings within the linear range of the standard
curve are valid. The internal standardization reported herein provided an accurate
determination within the range of 0.02 to 5.0 pmol per sample, allowing a broad
range of sample ABA concentrations to exist. This also provides a large degree of


ABA ADDED
Figure 2.2 Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.02 to 0.8 pmol.


Figure 2.3. Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.2 to 5.0 pmol.


ELISA with hydrilla turion tissue. Range of values was 0.02 to 0.6 pmol.


o
LU
>
cc
LU
CD
G
O
<
GQ
<
Q iiii I i i i i l i i i i I i i i i l
0 1 2 3 4 5
ABA ADDED
"igure 2.5. Internal standardization of abscisic acid quantification using
ELISA with hydrilla turion tissue. Range of values was 0.2 to 5.0 pmol.


38
flexibility when undertaking ABA analysis from hydrilla tissue. Using this
methodology, 8.0 pmol per sample for crown tissue (20 mg dry weight) and 12 pmol
per sample for turion tissue (8 mg dry weight) were detected from these hydrilla
tissue types. From these data, the appropriate amount of tissue needed for ABA
extraction would be 10 to 20 mg dry weight.
However, this procedure should be used to correlate large differences in ABA
levels between tissue types, biotypes or seasonal hormone fluctuations. The minute
quantities of ABA found endogenously can be depleted by a multitude of factors
before actual analysis, therefore relative (large) differences only should be used when
determining the effect of ABA.


CHAPTER III
THE INTERACTIVE EFFECT OF PHOTOPERIOD AND FLURIDONE
ON THE GROWTH, REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
Introduction
Hydrilla is an exotic, submersed vascular plant that has become one of the
most troublesome aquatic weeds in the state of Florida. Hydrilla was first observed
in a Miami canal and in Crystal River around 1960 and is thought to have been
introduced from Asia by the aquarium plant industry (Blackburn et al. 1969; Haller,
1976). Since introduction, hydrilla has spread throughout Florida, the southeastern
United States, and California (Langeland, 1990). Infestations have also been
reported in areas of Maryland, Virginia and Washington D.C., but this is thought to
be a separate introduction involving a different hydrilla biotype (Stewart et al.. 1984).
Hydrilla is a major problem in many freshwater ecosystems, forming a dense,
entangled mat of shoots at the water surface (Haller and Sutton, 1975). This
extensive growth interferes with most recreational water activities, navigation, and
flood control. In addition, hydrilla effectively outcompetes most native vegetation,
changing the natural balance of the aquatic ecosystem. Changes in the native flora
and fauna occur that, ultimately impact the freshwater fishing industry of Florida.
Hydrilla regrows in the spring as the water temperature begins to rise (Haller,
1976). During the initial phases of growth, hydrilla shoots quickly reach the water
39


40
surface and begin to branch, forming a dense canopy (mat)(Van et al.. 1977). This
mat effectively absorbs nearly 95% of the available sunlight (PPFD), limiting light
penetration to understory vegetation and reducing competition. In addition, hydrilla
can adapt to extremely low light levels. Bowes et al. (1977) reported the light
compensation point of hydrilla to be 12 to 20 ^mol m^ sec'1. Further work by
Salvucci and Bowes (1983) showed that a change in photosynthetic metabolism, from
C3 to C4 like photosynthesis occurred under certain conditions. This ability,
coupled with the low light compensation point, allows hydrilla to outcompete other
species and maintain the monotypic mat.
Hydrilla is difficult to control because spread can occur through a variety of
mechanisms including stem fragmentation and turion development. In hydrilla,
turions are produced at the tips of positively geotropic rhizomes, which extend from
the root crown into the hydrosoil (Sculthorpe, 1967). These structures, termed
subterranean turions or tubers, become detached from the mother plant and can
remain dormant for 1 to 5 years (Van and Stewart, 1990). Turions may also be
formed in leaf axils and are termed axillary turions (Yeo et al.. 1984). Axillary
turions are generally smaller than subterranean turions and are usually formed on
detached, floating plants (Haller, 1976; Miller et al.. 1993). Due to the smaller size,
axillary turions are thought to function in dispersal, whereas subterranean turions act
primarily in the persistence of hydrilla in a given area (Spencer et al.. 1987; Thullen,
1990).


41
Hydrilla forms turions during the late summer and early fall in North Florida
(Haller, 1976; Miller et al.. 1993). Van et al. (1978) reported that hydrilla responded
to photoperiod, with turions produced only under short-day conditions. Studies by
Klaine and Ward (1984) elucidated the role of phytochrome in controlling this
response. The level of ABA is thought to rise in response to short-day conditions,
preceding turion formation. ABA applied exogenously to hydrilla under long-day
conditions promotes turion production, but the differentiation between subterranean
and axillary turion formation has not been studied (Van et al.. 1978; Klaine and
Ward, 1984).
Management of hydrilla is difficult and results are variable (Schmidtz et al..
1987), with control efforts focusing primarily on herbicide use for logistical reasons.
Endothall, (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid), diquat, (6,7-
dihydrodipyridol[l,2-a:2,r-c]pyrazinediium ion), and copper sulfate alone or in
combination (s) provide good initial control but hydrilla quickly regrows. A slow
acting herbicide, fluridone, (l-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(li/)-
pyridinone), was introduced for aquatic weed control during the 1980s and has
shown great promise for the control of hydrilla (McCowan et al.. 1979). However,
proper exposure periods and concentrations are necessary for effective control
(Netherland et al.. 1993).
Fluridone inhibits carotenoid biosynthesis, causing photooxidation of
unprotected chlorophyll molecules (Bartels and Watson, 1978; Devlin et al.. 1978;
Maas and Dunlap, 1989). Fluridone has also been shown to inhibit the production


42
of ABA, which is thought to form from the carotenoid biosynthetic pathway (Li and
Walton, 1990; Parry and Horgan, 1991). Because of this, fluridone is used in
physiological experiments to study the effect of ABA on plant growth and
development (Hole et al.. 1989; Oishi and Bewley, 1990; Saab et al.. 1990).
Short-term control of hydrilla is often realized within a given area but long
term eradication is rarely achieved. Fluridone provides excellent control of hydrilla
and is generally applied in the spring as the hydrilla begins to regrow. Good initial
control is observed but regrowth from dormant turions occurs during the mid to late
summer months. This regrowth provides substantial biomass for reproduction in the
fall and turion levels in the hydrosoil are replenished. Thus the cycle of spring
treatment but regrowth and subsequent turion formation in the fall never results in
effective, long-term management.
One management strategy would be to treat hydrilla during the fall before or
during turion formation. However, hydrilla may not be sensitive to herbicides at this
time. Herbicides are most active on young, rapidly growing plants. Hydrilla growing
in the fall would be near maturity and should require a higher dose to achieve
control. Fluridone has been shown to reduce turion formation at concentrations _>
5 ppb (MacDonald et al.. 1993). However, the actual mechanism (i.e., reduction in
ABA levels and subsequent turion inhibition or turion inhibition as a result of plant
death), is not known. MacDonald et al. (1993) postulated that lower ABA levels
result from sublethal fluridone concentrations and indicated a potential growth
regulator phenomenon.


43
Clearly, the key in long-term hydrilla management is turion depletion in the
hydrosoil. Two strategies have been suggested concerning this issue; inhibition of
turion production or elimination of turion dormancy allowing precocious germination.
The objective of this study focused on the former strategy. Hydrilla was grown under
long (vegetative growth) and short-day (reproductive growth) conditions and
evaluated for changes in growth, turion formation and biochemical parameters in
response to varying rates of fluridone.
Materials and Methods
Hydrilla was initially planted on July 1, 1993 from apical stem segments and
grown under natural conditions in 900 L concrete vaults at the Center for Aquatic
Plants in Gainesville, FL. Stock hydrilla tissue was obtained from the Waccissa River
in north Florida; a pre-determined "fluridone-free" water system. Treatment plants
were grown in 10 x 10 cm plastic pots filled with organic potting soil4 and covered
with a 2 cm deep sand "cap" to prevent floating. Two 10 cm long apical hydrilla
sprigs were planted per pot and 6 pots were placed in 24 x 30 cm plastic dishpans.
On August 16, 1993 the plants were transferred to 540 L fiberglass vaults under short
(natural) or long (artificially extended) day greenhouse conditions. Environmental
conditions of the long-day greenhouse were 16 hr light/8 hr dark photoperiod, day
temperature of 30 5 C, night temperature of 25 5 C, with a mean quantum
4 Ace Hardware Corp., Oak Brook, IL 60521.


44
irradiance at noon of 1200 anol m'^sec'1 (PPFD). Photoperiod was extended for
4 hours after sunset with incandescent bulbs [100 /mol m^ sec'1 (PPFD)]. The
short-day greenhouse was maintained at similar conditions under natural (short-day)
photoperiodic conditions.
A second population of young hydrilla plants was established on August 18,
1993 in a manner similar to the older plants and placed under the same conditions.
The 8 week old population represented summer hydrilla growth while the younger
plants represented hydrilla regrowing from overwintering turions. Throughout the
remainder of this chapter, mature and young will be used to denote the 8 week old
and newly established hydrilla plants, respectively.
Each greenhouse (photoperiodic condition) contained 8 vaults, 4 comprising
mature hydrilla plants, 4 containing younger hydrilla. Each vault contained a total
of 10 dishpans. Fluridone concentrations of 0, 1, 5, and 10 ppb were established
within each population and photoperiodic regime beginning Sept. 2, 1993. Periodic
flushing and insecticide treatment were necessary to maintain optimum growth
conditions. After flushing fluridone was re-applied to each vault to achieve the
desired concentration.
Hydrilla (one dishpan) was harvested from each vault on Sept. 2, 1993 and
\
harvests continued on a weekly basis for 8 weeks. Each dishpan contained 6
v.
replications (1 replication = 1 pot). Parameters measured from both plant age
groups included fresh weight and subterranean turion number. Mature plants were
subsequently divided into apical stem, crown, and subterranean turion sections.


45
Subsamples were a composite from all replications. Immediately following
measurements and/or subsampling, young plants (entire plant) and mature plant
subsamples were frozen in liquid nitrogen and stored at -20 C. Samples were
lyophilized at -SO'C and dry weights recorded. The tissue ground was then to 0.5 mm
fineness and stored at 20 C prior to biochemical analyses.
Chlorophyll/carotenoid analyses. Approximately 10 mg dry apical stem tissue
was placed in a 20 ml glass scintillation vial and filled with 10 ml of
chloroforrmmethanol (2:1 v/v). Vials were placed on a rotary shaker (150 rpm) for
12 hours at 4 C. The homogenate was filtered and the crude extract dried under a
stream of nitrogen gas at room temperature under dim light (< 5 /xmolm'^sec'1).
The residue was resuspended in 80% acetone and absorbances at 470, 646, and 663
nm were measured spectrophotometrically. Total chlorophyll concentration was
calculated using the formula: 17.32 x 7.18 x A663. Total carotenoid content
was determined using the formula: 1000 x A470 3.27 x Chla 104 x Chlb)/229
(Lichtenthaler and Wellburn, 1983). Chlorophyll a (Chla) and chlorophyll b (Chlb)
concentrations (fig/mL) were calculated from the following formulas: 12.21 x A^ -
2.81 x Ah4b and 20.13 x A^ 5.03 x A^, for Chi a and b, respectively. Three
replications of the composite apical stem section subsample from mature plants only
(described above) were analyzed for chlorophyll and carotenoid content.
Anthocvanin analysis. Approximately 10 mg dry apical stem tissue was placed
into a 20 ml scintillation vial filled with 5 ml of 1% acidic methanol (v/v). Tissue
was extracted for 12 hours on a rotary shaker (150 rpm) at 4 C. The resulting


46
mixture was allowed to settle and absorbance of the supernate was measured
spectrophotometrically at 530 and 657 nm. Chlorophyll has some absorbance at 530
nm in acidic methanol, therefore corrections were made using the formula: A530 -
0.25 x Ag57, which assumes an extinction coefficient of 34,000 M^cm'1 (Jonsson et
al, 1984; Mancinelli, 1990). Anthocyanin content was determined from apical stem
section subsamples from mature plants only (described above) with 3 replications per
subsample.
Abscisic acid analysis. Tissue (10 to 20 mg dry weight) was extracted with
100% methanol for 12 hours on a rotary shaker (150 rpm) at 4 C. Methanol was
evaporated at -50 C and the residue resuspended in 0.5 to 1.0 ml of tris-buffered
saline and re-extracted as described previously. Following extraction, the
homogenate was centrifuged at 9000 rpm for 4 minutes. Analysis for ABA was
performed on the resulting supernate using an enzyme-linked immunoassay specific
for ABA. Details of the immunoassay procedure are presented in Appendices A-E.
A single sample from composite apical stem segments and crown subsamples of
mature were analyzed for ABA content. A single sample from 3 replicates of
younger plants 0, 2,4, 6, and 7 weeks after treatment (WAT) were analyzed for ABA
content.
Statistical analysis. Data were initially analyzed by analysis of variance to test
for treatment effects (WAT, photoperiod, and fluridone concentration) and
interactions. The effect of photoperiod was separated with Fishers protected LSD
test at the 0.05 level of significance. The effect of fluridone concentration was


47
compared to the untreated control using Dunnetts "T" test at the 0.05 level of
significance. Means are presented with standard errors.
Results and Discussion
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod and fluridone concentration on the dry weight of mature and
young hydrilla plants. The dry weight of untreated plants increased over time in both
photoperiodic regimes for both mature and young hydrilla populations (Tables 3.1
and 3.2). Young plants showed a greater increase in biomass than the mature
hydrilla over the duration of the study. Seven WAT, the biomass of young plants
increased by 83 and 90% under long and short-day conditions, respectively.
However, mature plants only showed an increase of 39 and 25% for long and short
day photoperiods, respectively. The dry weight of mature plants was reduced by
fluridone concentrations of 5 and 10 ppb by more than 63%, regardless of
photoperiod at 7 WAT. Young hydrilla plant biomass was reduced by fluridone
concentrations of 5 and 10 ppb greater than 95% under short-day conditions and
greater than 68% under long days. Fluridone at 1 ppb under long-day conditions
caused 50 and 42% reduction in dry weight of mature plants compared to the
untreated control after 6 and 7 weeks of treatment, respectively. Short-day grown
mature hydrilla, however, did not show significant biomass reduction at 1 ppb
fluridone, while younger plants were significantly reduced at 5 and 7 WAT under
short day conditions.


48
Table 3.1. The effect of photoperiod and fluridone on the dry weight of mature
hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
4.6
3.9
11.9
g/plant
9.6
3.4
7.4
7.5
1
4.4
4.6
8.0
5.3
3.6
3.7*3
3.2*
5
4.3
4.3
4.0*
3.5*
1.7
2.2
1.1*
10
4.2
3.9
5.6*
bo
*
4.1
2.8*
2.8*
Short Day
0
5.3
4.7
5.5
4.8
4.6
6.9
7.1
1
4.3
4.1
5.1
3.5
4.1
6.0
4.3
5
3.9
3.5
3.8
2.3*
1.7*
1.9*
1.4*
10
4.9
3.3
4.5
1.6*
1.9*
2.3*
1.7*
1 Average hydrilla plant dry weight at the initiation of the study was 4.25 g.
LSD0 os = 2.3 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by are significantly different from
the control (Dunnettst test 0 os)-


49
Table 3.2. The effect of photoperiod and fluridone on the dry weight of young
hydrilla plants. '
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4 5 6 7
Long Day
0
0.17
0.17
g/plant
0.40 0.36 0.58 0.75 0.66
1
0.20
0.26
0.37
0.44 0.57 0.51*30.71
5
0.20
0.25
0.21*
0.19* 0.10* 0.07* 0.03*
10
0.30*
0.44*
0.20*
0.17* 0.23* 0.12* 0.21*
Short Day
0
0.28
0.43
0.55
0.60 0.90 1.38 2.97
1
0.25
0.34
0.41
0.57 0.49* 0.86 0.81*
5
0.33
0.43
0.42
0.42* 0.29* 0.27* 0.31*
10
0.28
0.23*
0.18*
0.14* 0.07* 0.07* 0.05*
1 Average hydrilla plant dry weight at the initiation of the study was 0.18 g.
1 LSD0 os = 0 .17 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by are significantly different from
the control (Dunnetts Y test 0 05).


50
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod, and fluridone concentration on the subterranean turion
production of mature and younger hydrilla plants. Turions were not produced during
the first 4 weeks of treatment for either mature or young plants (data not shown).
Mature hydrilla grown under short-day conditions produced a greater number of
turions at 0 and 1 ppb fluridone 5 WAT or longer than under long-day conditions
(Table 3.3). Subterranean turion production was reduced by more than 80% in
mature hydrilla by fluridone at 5 and 10 ppb at 4 to 7 WAT under both
photoperiods. Fluridone at 1 ppb also reduced turion production of mature plants
at 4 to 7 WAT under long and short day conditions, with the exception of 5 and 7
WAT under short days.
Subterranean turion production by younger hydrilla plants was reduced under
long day conditions and 5 and 10 ppb fluridone by nearly 100% (Table 3.4).
Fluridone at 1 ppb reduced turion production in the younger plant 6 and 7 WAT
under short and long photoperiods, respectively.
Young and mature hydrilla plants responded similarly to photoperiod and
fluridone in terms of growth and reproduction. Fluridone is very effective for
hydrilla control and rates of 5 and 10 ppb reduced dry weight and nearly eliminated
turion production when hydrilla was exposed for greater than 5 weeks, similar to
previous research (MacDonald et al 1993). At 1 ppb fluridone, hydrilla growth was
arrested in the mature plant population, while young plants at this treatment
continued to accumulate biomass. Both mature and young plants gained biomass


51
Table 3.3. The effect of photoperiod and fluridone on the subterranean turion
production of mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment
period1 (ppb)
4
5
6
7
Long Day
0
3.5
turions/plant
2.2 2.0
4.0
1
0.3*2
0.3*
0*
0*
5
0.2*
0*
0*
0.2*
10
0.2*
0*
0.2*
*

Short Day
0
4.3
6.3
8.0
11.7
1
3.0
5.8
3.0*
9.0
5
0.2*
0.2*
0.3*
2.3*
10
0.2*
0*
0*
0.8*
1 LSD0 os = 1.7 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by are significantly different from
the control (Dunnetts Y test q.os)-


52
Table 3.4. The effect of photoperiod and fluridone on the subterranean turion
production of young hydrilla.
Photo- Fluridone
Weeks After Treatment
period1 (ppb)
4
5
6
7
Long Day
0
0
turions/plant
0 1.7
0.7
1
0
0.7
0*
0.7
5
0
0
0*
0
10
0
0
0*
0
Short Day
0
0.8
4.0
6.2
12.0
1
3.3
4.2
5.0
4.0*
5
0.2
0*
0*
0*
10
0*
0*
0*
0*
1 LSD0 os = 1.0 to separate the effect of photoperiod within week and fluridone
concentration.
9
Values within a week and photoperiod followed by are significantly different from
the control (Dunnetts t test q.qs)-


53
in untreated conditions but the younger, more rapidly growing plants should have
been more susceptible than mature plants to fluridone treatment.
A possible explanation for this apparent contradiction may be related to light
intensity. Mature hydrilla plants have the majority of photosynthetic tissue near the
water surface, exposed to a greater amount of light and potential photooxidative
stress versus younger plant tissue emerging through the water column. In addition,
the removal of apical dominance would cause branching at the water surface for the
mature plant (Van et al.. 1977). The new tissue produced by the mature hydrilla
plants would then be more susceptible to the affect of fluridone due to the higher
light intensity at the surface. This would result in a reduction of growth. However,
the loss of apical dominance in young hydrilla resulting from fluridone treatment
would cause branching near the hydrosoil, where light intensity and potential
photooxidative stress is much lower. Hydrilla can survive under very low light
intensities (Bowes et al.. 1977) and this could explain the ability of the younger plants
to increase in dry matter over the study period. Even though carotenoid levels would
be lower as a result of fluridone treatment, so would potential photooxidative stress.
Another explanation could be the rapid growth of the younger plants as compared
to the mature hydrilla plants. The rapid growth could overwhelm the lethal affects
of fluridone, with continued growth before fluridone began to take affect.
Subterranean turion production appeared to be directly related to growth in
terms of dry matter for both young and mature plants, where reduction in growth
also resulted in a reduction of turion number. There was turion formation under


54
long-day conditions by the mature hydrilla plants. However, the number of turions
produced did not increase over time as in the short-day grown plants, which indicated
a breach in long-day extension, or pre-induction before transfer to greenhouse
conditions.
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod and fluridone concentration for total chlorophyll, carotenoid
and anthocyanin content. Chlorophyll and carotenoid content of the mature hydrilla
plants was decreased at 5 and 10 ppb fluridone after 2 weeks of treatment in both
long and short-day photoperiodic conditions (Tables 3.5 and 3.6). Chlorophyll and
carotenoid content under long-day conditions was also decreased by 1 ppb fluridone,
but the effect under short days was variable. There was also a general decline in
these pigment levels as a function of weeks after treatment, with greater decreases
observed at higher levels of fluridone. Conversely, anthocyanin content increased
over time and short-day conditions (Table 3.7). The effect of fluridone on
anthocyanin content was highly variable and anthocyanin content appeared to
decrease anthocyanin content at higher fluridone rates.
Fluridone decreased carotenoid levels at concentrations of 5 and 10 ppb after
1 week of exposure as expected, as the mechanism of action of this compound has
been demonstrated to be an inhibition of carotenoid biosynthesis (Bartels and
Watson, 1978). Chlorophyll content also decreased in a similar fashion. Earlier
work by Doong et al.. 1993 demonstrated a lag in the chlorophyll content of hydrilla
at 5 ppb fluridone following carotenoid decrease but this was not evident from the


55
Table 3.5. The effect of photoperiod and fluridone on the chlorophyll content of
mature hydrilla plants.
Weeks After Treatment
l
Photo- Fluridone
pb)
1
2
3
4
5
6
7
0
673
847
679
695
543
450
489
1
981 *3
529*
322*
398*
170*
364*

5
498*
310*
91*
94*
75*
198*
219
10
299*
167*
134*
25*

62*
207
0
616
613
527
465
438
269
486
1
552
603
320*
489
278*
254
292
5
534*
357*
207*
225*
46*
52*
128
10
200*
85*
63*
278*
32*
29*
28*
Long Day
Short Day
1 Average hydrilla chlorophyll content at the initiation of the study was 904 ig/g
fresh wt.
9
LSDo.05 = 58 to separate the effect of photoperiod within week and fluridone
concentration.
a ,
Values within a week and photoperiod followed by are significantly different from
the control (Dunnetts Y test Q 05).


56
Table 3.6. The effect of photoperiod and fluridone on the carotenoid content of
mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
68
74
-- /xg/g-fresh weight -
86 92 87
65
92
1
91*3
56*
44*
54*
24*
72
70
5
49*
36*
15*
15*
12*
61
39*
10
26*
21*
23*
3*

11*
42*
Short Day
0
67
67
74
84
77
48
106
1
52*
63
44*
76
55*
46
47*
5
54*
50*
25*
44*
9*
12*
26*
10
19*
10*
10*

7*
9*
6*
1 Average hydrilla carotenoid content at the initiation of the study was 89 fig/g fresh
wt.
LSD0 05 = 10 to separate the effect of photoperiod within week and fluridone
concentration.
'I
Values within a week and photoperiod followed by are significantly different from
the control (Dunnetts Y test o.os)-


57
Table 3.7. The effect of photoperiod and fluridone on the anthocyanin content of
mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
58
76
- ig/g-fresh weight -
69 76 123
76
113
1
70*3
69
75
128*
68
66
156*
5
52
41*
62
101*
94*
80

10
54
53*
86*
141*

30*

Short Day 0
63
75
73
143
117
*
329
419
1
56
55*
85
131
206*
346
300
5
70
61*
143*
110*
95
81*
216
10
51
*
o
U1
83
109*
94
99*

1 Average hydrilla anthocyanin content at the initiation of the study was 63 fxg/g
fresh wt.
9
LSDo.05 = 39 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by are significantly different from
the control (Dunnettst test q.qs)-


58
present study. A possible reason for this effect could be that high light intensity
caused a greater degree of photooxidative stress and thus, more rapid chlorophyll
breakdown. Chlorophyll content of untreated plants declined throughout the
duration of the study in both photoperiodic regimes. Berg (1977) and Kar and
Choudhuri (1987) reported a loss of chlorophyll content in hydrilla during late
summer and hypothesized that a senescence had occurred. However, a decline in
chlorophyll was also observed under long-day conditions, when senescence should not
have been a factor.
The effect of fluridone on anthocyanin content was highly variable and did not
cause an increase in the level of this pigment, as demonstrated by earlier studies
(Doong et al.. 1993). However, the level at which Doong et al.. reported elevated
anthocyanin content was 50 ppb fluridone, far above the levels tested in this study.
Interestingly, anthocyanin levels increased dramatically under short-day conditions,
with higher fluridone concentrations suppressing the level of this pigment. Berg
(1977) reported an increase in anthocyanin content as hydrilla began to decline
during late summer and this was probably the case for the short-day grown hydrilla
plants. The decline in anthocyanin content from fluridone treatment probably
resulted from tissue deterioration.
There was no significant differences in the ABA content of crown tissue from
mature plants (P > 0.05 see Appendix G). The average ABA content of these
structures was 25 and 204 pmol/g fresh weight for crown and turion tissue types,
respectively. There was a significant interaction between weeks after treatment and


59
fluridone concentration for apical stem tissue but not a significant (P >0.05) effect
of photoperiod (Appendix H). Abscisic acid content increased in control and 1 ppb
fluridone treated plants 1 and 2 weeks after study initiation, respectively, while
fluridone at 5 and 10 ppb decreased ABA content (Table 3.8).
ABA content in younger plant tissue was only affected by photoperiod (P <
0.05), not by fluridone concentration and there was not an interaction (Appendix I).
Abscisic acid content in young plant tissue was highest under short-day conditions
when averaged over time and fluridone: 0.53 + 0.07 and 0.71 0.05 pmol/g-fresh
weight, long and short days, respectively.
The results of the ABA analyses performed in this study are highly variable
but follow similar findings of other researchers (Van et al.. 1978; Klaine and Ward,
1984; Anderson et al.. 1990 [unpublished]). ABA content was shown to rise soon
after treatment, but the increase was transient. In addition, fluridone reduced ABA
content and turion production in mature plants at 5 and 10 ppb, but this could also
be the indirect consequence of plant death. ABA was higher under short-day length
conditions, which favor turion formation. However, direct evidence linking
turionformation and increased ABA content was not established. Although ABA is
probably involved in some aspect of turion formation and/or development, the exact
mechanism is still unknown.
The results from this study provided further evidence that hydrilla can be
effectively managed by late summer to early fall applications of fluridone, which
prevent turion formation. Mature hydrilla tissue was shown to be as susceptible to


60
Table 3.8. The effect of fluridone concentration on the abscisic acid content of
mature hydrilla apical stem segments, averaged across photoperiodic regime.
Weeks After
Treatment1
0
Fluridone (ppb)
1 5
10
pmol/g-fresh
weight
1
18
36
13
13
2
39
25
13
15
3

18
11
5
4
19
13
11
11
5

11
7

6
26
19
12
3
7
15
16
10
15
1 Average abscisic acid content at the initiation of the study was 15 pmol/g fresh wt.
9
LSDg.05 = 13 to separate the effect of fluridone concentration within week.


61
fluridone as younger tissue and fluridone concentrations of 5 ppb or higher will
dramatically reduce subterranean turion production. This contradicts the earlier
hypothesis of MacDonald et al. (1993) who proposed that sublethal concentrations
of fluridone could block turion formation. However, the present studies support the
argument that lowering ABA concentration sufficiently to block reproduction would
require a lethal reduction in carotenoids. Therefore, the reduction in turion
formation is probably a coincidental result of plant death. Prudence should be
exercised when applying these results to real-world situations, as factors such as
exposure time and critical concentrations have yet to be evaluated.


CHAPTER IV
THE INFLUENCE OF FLURIDONE, PHOTOPERIOD AND
PLANT GROWTH REGULATORS ON THE GROWTH AND
REPRODUCTION OF MONOECIOUS AND DIOECIOUS
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
Introduction
Hydrilla is a submersed aquatic vascular plant that has become a major
aquatic weed problem in the southeastern U.S. (Haller, 1976; Langeland, 1990). This
species is particularly important in Florida, where it was introduced in the late 1950s
by the aquarium plant industry (Blackburn et ah, 1969). Since introduction hydrilla
has spread throughout the southeastern United States and California.
There are both monoecious and dioecious biotypes of hydrilla which both can
be found in the United States. Until recently, each biotype was believed to grow in
distinct regions, but monoecious and dioecious have been reported in a common lake
in North Carolina (Coley et al.. 1993). Dioecious hydrilla exists primarily in those
areas mentioned above, and consists of only the female plant. Monoecious hydrilla
was introduced during the late 1970s and can now be found in areas of Virginia,
Maryland, Washington D.C., and North Carolina (Stewart et al.. 1984; Coley et al..
1993).
Hydrilla causes problems in many freshwater ecosystems, displacing most
native vegetation. This highly aggressive species exists in large, monotypic stands,
which form a dense mat at the water surface (Haller and Sutton, 1975; Bowes et al..
62


63
1977). Hydrillas extensive growth interferes with navigation and flood control, and
severely limits most water recreation activities. Furthermore, hydrilla growth will
alter the flora and fauna of a given area, impacting freshwater fish populations.
Hydrilla possesses several physiological and morphological adaptations
including a low light compensation point, C4-like photosynthetic metabolism, and
specialized vegetative reproduction [turions] (Bowes et al.. 1977; Van et al.. 1976;
Salvucci and Bowes, 1983). Turions are specialized vegetative structures that
function in persistence and dispersal of many aquatic macrophytes (Sculthorpe, 1967).
In hydrilla, turions may be formed at the tips of rhizomes that penetrate into the
hydrosoil (subterranean turions), or in leaf axils (axillary turions)[Yeo et al.. 1984],
Subterranean turions are slightly larger and function to insure persistence of hydrilla
within a given area, while the smaller axillary turions are thought to function in
dispersal (Haller, 1976; Spencer et al.. 1987; Thullen, 1990).
Hydrilla turion production varies between biotypes, from both a physiological
and morphological standpoint. Dioecious hydrilla forms turions only under short-day
(< 12 hours light) conditions, while monoecious hydrilla appears to be day-neutral
(Klaine and Ward, 1984; Van et ah. 1978; Anderson and Gee, 1986). Monoecious
hydrilla will produce a greater number of smaller turions than dioecious hydrilla
(Spencer and Anderson, 1987; Van, 1989). This size characteristic is true for both
axillary and subterranean turions. Turion formation in the dioecious biotype is
believed to be regulated by phytochrome (Klaine and Ward, 1984; Van et al.. 1978).
This response has been associated with increased levels of abscisic acid (ABA) and


64
exogenous applications of this hormone have resulted in turion formation under non-
photoinductive conditions (Klaine and Ward, 1984; Van et al.. 1978). However,
delineation between axillary and subterranean turion formation was not addressed.
Furthermore, the effect of ABA on monoecious hydrilla was not studied.
ABA has been associated with a number of plant processes including water
relations, growth and development, and seed dormancy (Neill et al.. 1986; Zeevaart,
J.A.D. et al.. 1988; Saab et al.. 1990). ABA has also been shown to cause
heterophylly in some aquatic plants (Kane and Albert, 1987; Goliber and Feldman,
1989).
Turion formation is similar to tuber formation in terrestrial species, such as
potato (Solanum tuberosum! and Jerusalem artichoke (Helianthus tuberosum!.
Tuber formation in potato involves changes in abscisic and gibberellic acid content,
with an increase in ABA during tuberization and a subsequent decline in gibberellic
acid levels (Krauss, 1985). Similar to turion formation in hydrilla, ABA will promote
tuber formation under non-photoinductive conditions while gibberellic acid will
inhibit tuberization under inductive conditions. However, most researchers feel there
exists a critical ratio between these two hormones, not simply an on-off
phenomenon (Menzel, 1980; Vreugdenhill and Struik, 1989). This interactive effect
has not been evaluated on either biotype of hydrilla.
Plant growth regulator experiments are usually performed under laboratory
conditions to limit variability. Axenic culture is sometimes employed to study various
plant processes under highly controlled conditions. Axenic culture has proven to be


65
a valuable technique for studying the physiology of aquatic plants (Kane and Albert,
1987). In addition, axenic culture permits plant growth under heterotrophic
conditions. This allows for the potential use of a compound that influences
photosynthesis and other autotrophic processes. Fluridone is often used to regulate
ABA biosynthesis, but is lethal under autotrophic conditions.
Monoecious and dioecious hydrilla biotypes differ in the process of turion
formation but little is known regarding the processes involved. Therefore, the
objectives of these studies were to evaluate the growth and reproductive development
of monoecious and dioecious hydrilla in axenic culture to 1) long and short-day
photoperiod, 2) fluridone under long-day conditions, and 3) exogenous abscisic acid
and gibberellic acid applications.
Materials and Methods
Axenic plant stock material was obtained from Dr. Michael E. Kane,
Environmental Horticulture Dept., University of Florida. From this material, stock
cultures of both biotypes were established from 3 to 5 cm long apical stem segments.
Stock cultures were maintained in aluminum foil capped 0.94 L glass vessels filled
with 150 cm quartz silica sand (as a rooting medium) + 500 ml of liquid growth
medium. Basal medium consisted of half-strength Murashige and Skoog mineral
salts1 and 0.75% (w/v) sucrose, adjusted to pH 6.5 with 1.0 N KOH prior to
1 Murashige and Skoog Basal Salt Mixture (1962); Sigma Chemical Co.,
St. Louis, MO 63178.


66
autoclaving. Growth medium was autoclaved at 22 psi and 250 F for 20 minutes.
Deionized water was added to the vessels containing sand prior to autoclaving to
prevent breakage. After autoclaving, medium was added to the vessels and plant
material transferred. All transfers were performed under sterile conditions. Stock
cultures were maintained on a monthly basis and sterility was checked by indexing,
according to the procedures of Kane (unpublished) and Knauss (1976). Stock
cultures were maintained in an environmentally controlled growth chamber set at the
following conditions: 11 + 1 hr light/6 + 6 hr dark photoperiod (lhr light
interruption at the middle of the dark cycle to maintain long-day conditions); 150
9 1
/xmol'm sec (PPFD); 30 5 C. The light source consisted of fluorescent and
i
incandescent bulbs.
Experimental plant material was grown in 38 x 300 mm glass tubes2 filled with
50 cm3 quartz-silica sand and 175 ml growth medium. Plant material was obtained
from stock plants. Establishment of hydrilla was accomplished by transferring a 3 to
5 cm long (0.005 g dry wt) apical stem section into each tube and forcing the basal
end into the sand 1 to 2 cm deep to enhance rooting. Each tube was considered a
replication. Experimental cultures were maintained in an environmentally controlled
growth chamber set at the following conditions: 9 + 1 h light/7 + 7 h dark
photoperiod (1 h light interruption at the middle of the dark cycle to maintain long-
day conditions); 600 /mol'm^ sec'1 (PPFD); 30 5 C mean light temperature and
2 Blico Glass, Inc., Vineland, N.J. 08360.


67
25 5 C mean dark temperature. Short-day grown plants were cultured in a
chamber maintained at: 10 h light/14 h dark photoperiod -- 200 /zmol m'^sec'1
(PPFD) and 25 5 C mean light temperature and 20 + 5 C mean dark
temperature. Lighting in both chambers consisted of a mixture of incandescent and
fluorescent bulbs.
Photoperiodic response. Monoecious and dioecious hydrilla biotypes were
grown under long or short-day conditions for 8 weeks with a minimum of 6
replications. The experiment was repeated once. After 8 weeks, the plants were
harvested and subterranean and axillary turion number was determined.
Subterranean turion development was determined by the formation a rhizome
penetrating the sand substrate. Axillary turion formation was distinguished by a
swelling of shoot tissue, compact leaf development and the lack of prominent
internodes. The plants were then dried at 60 C for 48 h and dry weights recorded.
Effect of fluridone. A preliminary experiment was conducted to determine
if hydrilla could be grown heterotrophically using axenic techniques with
supplemental sucrose. Monoecious and dioecious hydrilla biotypes were grown under
long-day conditions for 12 weeks. The experiment was repeated once with 4
replications. Fluridone was prepared as a concentrated stock in water from technical
grade material3 and filter-sterilized (0.22 un)4 prior to addition to the tubes.
Fluridone was added at concentrations of 0, 0.5, 1.0, 5.0, or 10 ppb at the time of
3 DowElanco, Inc., Indianapolis, IN 46268.
4 MSI, Fisher Scientific, Pittsburgh, PA. 15219.


68
establishment. After 12 weeks, the plants were harvested and the number of axillary
turions produced was determined. The plants were then dried at 60 C for 48 h and
dry weights recorded.
Effect of exogenous abscisic acid and gibberellic acid. Monoecious and
dioecious hydrilla biotypes were grown under long-day conditions for a total of 8
weeks. Biotypes were run as separate experiments and each experiment conducted
twice with a minimum of three replications. After 4 weeks of growth the growth
medium was exchanged and hormone treatments were applied. Abscisic acid (99%
mixed isomers)5 and gibberellic acid (90% GA3)5 were prepared as concentrated
stocks in water and filtered-sterilized prior to treatment. Small amounts of ethanol
was used to facilitate dilution and the appropriate ethanol concentration was added
to the control plants. Abscisic acid was applied at 0, 0.1, 1.0, or 10 M and
gibberellic acid at 0, 5, 50, or 500 iM to establish a matrix of 16 total treatments.
Four weeks after treatment, the plants were harvested and the number of axillary
turions, subterranean turions, and flowers produced were counted. Axillary turions
were subsampled and the plants dried at 60 C for 48 h and dry weights recorded. No
attempt was made to distinguish between male and female flowers of the monoecious
biotype.
Statistical analysis. Data were initially analyzed by analysis of variance to test
for treatment effects (biotype, photoperiod, fluridone concentration, ABA or GA
content) and interactions. Treatment means were separated with Fishers
5 Sigma Chemical Co., St. Louis, MO 63178.


69
protected LSD test or Dunnetts T test at the 0.05 level of significance. Means are
presented with standard errors.
Results and Discussion
Although plant material was obtained from axenic stocks, a certain degree of
contamination, bacteria and/or fungal, occurred in each of the experiments. Because
of the relatively short duration of the experiments, this contamination did not cause
plant death. There was no observed trend for any treatment to be more
contaminated than the others. Therefore, it is not the authors intent to claim axenic
culture was used or maintained, and conclusions drawn from these studies are not
intended to reflect that premise.
There was a significant (P < 0.05) interaction between biotype and
photoperiod for subterranean and axillary turion production and dry weight. Both
biotypes produced greater biomass under long-day conditions (Table 4.1). Dioecious
hydrilla grown under short days, produced less than 70% of the biomass produced
by long-day grown plants. There was a 20% difference for monoecious hydrilla.
Axillary turion production was not observed for dioecious hydrilla, while monoecious
hydrilla produced these structures under both photoperiods (Table 4.2). Dioecious
hydrilla produced subterranean turions only under short-day conditions, while the
monoecious hydrilla produced subterranean turions under both photoperiods (Table
4.3).
In this study, photoperiod had a tremendous effect on plant growth and
subterranean turion formation for dioecious hydrilla. Dioecious hydrilla has been


70
Table 4.1. The. effect of photoperiod on the growth of 8 week old dioecious and
monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
g dry/plant
Long-Day
0.92 0.091
1.21 0.05
Short-Day
0.28 0.04
0.95 0.05
1 Means followed by standard errors.


71
Table 4.2. The effect of photoperiod on the axillary turion production of 8 week old
dioecious and monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
turions/plant -
Long-Day
0
0.88+0.401
Short-Day
0
2.71 1.41
1 Means followed by standard errors.


72
Table 4.3. The effect of photoperiod on the subterranean turion production of 8
week old dioecious and monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
turions/plant -
Long-Day
0
0.75 0.491
Short-Day
1.29 0.42
1.00 0.53
1 Means followed by standard errors.


73
shown to undergo annual decline (Berg, 1977), which is characterized by a loss of
biomass prior to the onset of winter. In these experiments short days may have
caused this phenomenon in dioecious hydrilla, thus limiting biomass production.
Several species of plants cease vegetative growth during the onset of reproduction
(Gardener et al.. 1985). However, this does not seem to be the case with
monoecious hydrilla. Another possible explanation could be the lower light intensity
in the short-day chamber. Monoecious hydrilla also displayed significantly less
biomass in this environment, but to a much lesser extent than dioecious hydrilla.
Dioecious hydrilla has been reported to be a short-day plant in terms of turion
formation, with a critical photoperiod of 11 to 12 hours (Van et al.. 1978; Miller et
ah, 1993). This characteristic response was also observed in these studies. However,
axillary turion development was not observed under either day length. Haller (1976)
and Miller et al. (1993) reported axillary turion formation occurred on floating
hydrilla plants. Plants used in these experiments were intentionally rooted which
possibly precluded axillary turion formation. Monoecious hydrilla produced axillary
and subterranean turions under both photoperiods. This contrasts with earlier
research, which showed no turion formation under 14 to 16 hour daylengths or photo
interruption of long nights (Anderson and Spencer, 1986; Spencer and Anderson,
1987).
Monoecious and dioecious hydrilla biotypes were adversely affected by
increasing concentrations of fluridone (Table 4.4). Ten ppb fluridone caused 91 and
82 % reduction in shoot biomass of dioecious and monoecious hydrilla, respectively.


Table 4.4. The effect of fluridone on the growth of dioecious and monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.
Fluridone
Dioecious
Monoecious
(PPb)
g dry/plant
0
0.56 + 0.181
0.63 0.26
0.5
0.450.16
0.71 0.08
1.0
1.03 0.18
0.600.08
5.0
0.260.10
0.320.10
10
0.050.01*2
0.110.04*
LSDq.05
0.383
1 Means followed by standard errors.
Values within a given biotype followed by are significantly different from the
control (Dunnetts Y test0 0s)-
a
To separate the effect of photoperiod within a given level of fluridone.


75
One ppb fluridone caused a 46 % increase in dioecious shoot biomass. Fluridone
decreased turion production at higher concentrations (5 and 10 ppb), while
stimulating turion production at 0.5 ppb in monoecious hydrilla (Table 4.5). This
observation is similar to results reported from earlier field experiments with
dioecious hydrilla (MacDonald et ah. 1993). This indicates that these biotypes
behave similarly to fluridone in response to turion formation.
Fluridone is a very effective herbicide, inhibiting carotenoid biosynthesis and
causing subsequent photooxidation of unprotected chlorophyll molecules (Bartels and
Watson, 1978; Devlin et al.. 1978). In this study, plant tissue became whitish in color,
indicating pigment loss. Monoecious hydrilla appeared to recover as indicated by
green coloration, but still showed a decrease in biomass over the course of the study.
The lethal effect of fluridone has been demonstrated to be an inhibition of
photosynthesis and plant death due to starvation. However, plants grown with
sucrose amendments should have been able to survive fluridone treatment. If
hydrilla could have been grown heterotrophically then fluridone could have been
used to regulate ABA formation without the lethal effects from reduced
photosynthesis. This could have allowed a determination of whether fluridone acts
as a growth regulator at low concentrations or a herbicide at higher concentrations.
Hydrilla could not be grown heterotrophically and therefore separation of the growth
regulator and herbicidal effects of fluridone could not be ascertained.
In this experiment, fluridone decreased the growth of both monoecious and
dioecious hydrilla biotypes, with monoecious being affected to a lesser degree than


76
Table 4.5. The effect of fluridone on the axillary turion production of monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.
Fluridone
Turions/Plant
(PPb)
0
2.00.71
0.5
5.0+1.I*2
1.0
2.71.7
5.0
0.3 0.3
10
0*
1 Means followed by standard errors.
2 Values followed by are significantly different from the control (Dunnetts t
testo>05).


77
dioecious hydrilla. Although the goal of fluridone treatment in this experiment was
to lower ABA levels thus precluding turion formation, it is possible that lowered
ABA may be causing the reduction in growth. Studies with ABA-deficient mutants
have demonstrated lower growth rates, epinasty and wiltiness associated with these
types of mutations (Koornneef et al 1982).
Fluridone at 5 and 10 ppb caused a deep purple coloration in dioecious
hydrilla. Fluridone at high rates has been shown to increase the anthocyanin content
of hydrilla but only in mature tissue (Doong et al.. 1993). This phenomenon was
thought to be in response to oxidative stress due to the loss of carotenoids.
Anthocyanin production is also associated with high levels of carbohydrate (Creasy,
1968), present in mature tissue, and which was also present in this study.
There was not a significant (P > 0.05) effect of experiment for exogenous
applications of abscisic and gibberellic acid, therefore experiments for each biotype
were pooled. In dioecious hydrilla, exogenous applications of ABA alone induced
axillary turion formation at all concentrations (Table 4.6). The greatest number of
turions were observed following treatments of 0.1 and 1.0 /M ABA. The effect from
ABA was reversed by applications of GA, but not completely. ABA also stimulated
axillary turion production in monoecious hydrilla, but only at concentrations of 1.0
and 10 xM (Table 4.7). Similarly, GA at 50 and 500 M completely reversed this
effect at 1.0 /xM ABA.
Total subterranean turion weight per plant reflected the same trend observed
from turion number per plant (Tables 4.8 and 4.9). The size of dioecious turions
were always larger than monoecious hydrilla regardless of treatment (0.005 and 0.002


78
Table 4,6. Axillary turion production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (xM)1
0
Gibberellic Acid (/xM)1
5 50
500
-- turions/plant
0
0
0 0
0
0.1
15.65.12
6.432.8 5.0+2.6
0
1.0
27.0+6.2
0.9+0.9 17.79.4
4.63.2
10
11.4 4.0
7.64.3 5.4+2.6
4.41.3
1 LSD0 os = 10.6 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.


Table 4,7. Axillary turion production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (1M)1
0
Gibberellic Acid (iM)1
5 50
500
- turions/plant
0
0
0
0
0
0.1
0
0
0
0
1.0
42.614.72
9.3+6.1
0
0
10
52.3 17.1
35.6 + 10.6
22.4 + 10.2
4.1+3.1
1 LSD0 05 = 4.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.


80
Table 4.8. Axillary turion weight per plant by 8 week old, long day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic Gibberellic Acid (iM)1
Acid (mM)1 0 5 50 500
mg dry/plant
0
0
0
0
0
0.1
74312
33 15
2012
0
1.0
128 45
33
65 40
53
10
52 22
523
31 15
15 8
1 LSD0 os = 0.06 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.


81
Table 4.9. Axillary turion weight per plant by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic
Acid (xM)1
0
0.1
1.0
10
Gibberellic Acid (/xM)1
5 50 500
g dry/plant
0
0
0
0 0 0
0.231+0.0762 0.020+0.013 0
0
0
0.146+0.052 0.057+0.025 0.042+0.03 0.006 + 0.005
1 LSD0 05 0 .07 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.


82
g'1 dry wt turion for dioecious and monoecious hydrilla, respectively). The
percentage of total plant weight represented by turions also reflected the same
response as described for turion number (Tables 4.10 and 4.11). nterestingly,
monoecious hydrilla appears to partition a greater percentage of biomass for
reproduction than dioecious hydrilla, producing a greater number of smaller turions.
The effect of ABA and GA on the shoot dry weight of both hydrilla biotypes
was different than the effects on turion formation (Tables 4.12 and 4.13). Both ABA
and gibberellic acid inhibited shoot growth of dioecious hydrilla with GA being the
more inhibitory compound. At the highest concentration of both compounds, ABA
ameliorated the activity of GA. Monoecious hydrilla was inhibited equally by ABA
and GA in terms of shoot growth. However, these two compounds appeared to act
additively to inhibit shoot growth. Although a reduction in shoot growth was
observed on a dry weight basis, the plants treated with GA elongated to the top of
the tube. It is probable that the reduction in dry weight was caused by decreased
leaf production by the plants.
Dioecious hydrilla did not produce subterranean turions or flowers (data not
shown). Monoecious hydrilla produced an average of 5 subterranean turions per
tube only in the absence of hormone applications (untreated control). In addition,
monoecious hydrilla produced an average of 44 flowers per tube the addition of
gibberellic acid at 50 ¡xM. Floral production has been reported to be influenced by
nitrogen and phosphorus (Pietrse et al.. 1984). Flowering has been associated with
short day conditions in dioecious hydrilla, suggesting turion formation and flower


83
Table 4.10. Percent weight of axillary turions produced by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic
Acid (M)1 0
0
0
0.1
12.06.22
1.0
22.5 8.1
10
0.65 0.10
Gibberellic Acid (/xM)1
5 50 500
% of total dry weight
0 0 0
0.8 0.09 0.72 0.08 0
0.41 0.07 0.580.14 0.420.12
0.620.13 0.44 0.10 0.50+0.12
1 LSD0 os = 8.95 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.


84
Table 4.11. Percent weight of axillary turions produced by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic Gibberellic Acid (/MJ1
Acid (/M)1 0 5 50 500
% of total dry weight
0
0
0
0
0
0.1
0
0
0
0
1.0
31.2+9.32
2.5 1.7
0
0
10
29.09.6
13.3+3.9
8.74.8
1.2+0.8
1 LSDq q5 = 9.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.


Table 4.12. Shoot biomass production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (/xM)1
0
Gibberellic Acid (xM)1
5 50
500
g dry/plant
0
0.80+0.042
0.73 0.15
0.660.10
0.35 0.14
0.1
0.580.11
0.80+0.09
0.72 0.08
0.50 0.04
1.0
0.69+0.13
0.41 0.07
0.580.14
0.42 0.12
10
0.650.10
0.620.13
0.44 0.11
0.50 0.12
1 LSD0 05 0 .08 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.


86
Table 4.13. Shoot biomass production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic Gibberellic Acid (1M)1
Acid (/iM)1 0 5 50 500
g dry/plant
0
0.680.092
0.53 0.12
0.72 0.07
0.49 0.07
0.1
0.42 0.03
0.58 0.07
0.540.10
0.34 0.06
1.0
0.58 0.11
0.470.12
0.42 0.11
0.31 0.05
10
0.46 0.05
0.37 0.05
0.33 0.06
0.32 0.06
1 LSD0 os = 0.23 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.


87
production are linked to a common physiological process. However, floral
stimulation by exogenous gibberellic acid, implied a different, separate mechanism
was involved in this process.
Several researchers have demonstrated the effect of exogenous ABA
applications on turion formation in dioecious hydrilla but little work has been done
with the monoecious biotype (Klaine and Ward, 1984; Van et al 1978). Dioecious
hydrilla appeared to be more sensitive to ABA applications than monoecious hydrilla,
as evidenced by turion formation at 0.1 /M and reduced sensitivity to GA
applications. ABA has been shown to induce tuber formation in potato and this
effect can be reversed by application of GA (Vreugdenhill and Struik, 1989). The
increased sensitivity of the dioecious biotype to exogenously applied ABA could be
related to photoperiodic responses, whereby applications of ABA could induce a
cascade of reactions that lead to turion formation. However, this should have
resulted in subterranean turion formation, not in the production of axillary turions.
Interestingly, monoecious hydrilla produced subterranean turions without
treatment, similar to those plants in the photoperiod experiment. If ABA was
directly responsible for the formation of these structures, then exogenous applications
should have induced this process. This suggested an indirect role of ABA in turion
development, with the formation of the rhizome being the initial step and not related
to changes in ABA. ABA could be produced after rhizome differentiation to
maintain dormancy and induce swelling of the rhizome tip. In a normally developing
hydrilla plant, the amount of ABA produced during subterranean turion development


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81,9(56,7< 2) )/25,'$


UNIVERSITY OF FLORIDA
3 1262 08554 5605


PHYSIOLOGICAL ASPECTS OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION
By
GREGORY E. MacDONALD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994

In memory of George E. MacDonald; your love, support, guidance and
understanding are the basis for all I have attained.

ACKNOWLEDGEMENTS
For their support and guidance during my tenure at the University of Florida
I wish to express my sincere appreciation to my committee members: Dr. Thomas
Bewick, Dr. George Bowes, Dr. William Haller, and Dr. Michael Kane. I would like
to extend my most sincere appreciation to my major advisor, Dr. Donn Shilling, for
his patience, understanding and guidance throughout my tenure at Florida. I would
also like to acknowledge the advice and support of Drs. Daniel Colvin, Ken
Langeland, and Bert McCarty.
In addition, I wish to acknowledge Christine Kelly, Bob Querns, Ron Kern,
Jim Gaffney, Jigna Thakore, Sean and Cindy Ragland, Laura Rankin, Margaret
Glenn, Jan Miller, Jamey Carter, Sandra McDonald, Brian Smith, Ken Smith,
Carlene Chase, Eric Bish, and Ron Doong. Without their valuable time and
assistance this research would have been extremely difficult, if not impossible.
For the two most important women in my life, I wish to express my deepest
gratitude to my wife Mickey and my mother Marilyn MacDonald. No words can
adequately describe the value of their support. I would also like to thank my
brother, Tim MacDonald, for his support and friendship.
During my tenure at the University of Florida I have had the opportunity to
build lasting friendships. The experiences I have shared will always be cherished.
in

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES x
ABSTRACT xi
CHAPTERS
I INTRODUCTION 1
II EVALUATION OF AN ENZYME LINKED
IMMUNOASSAY PROCEDURE FOR THE
ANALYSIS OF ABSCISIC ACID
IN HYDRILLA 20
Introduction 20
Materials and Methods 23
Elution Profiles and Column/ABA
Characterization 23
ELISA Internal Standardization
for ABA in Hydrilla 25
Results and Discussion 26
III THE INTERACTIVE EFFECT OF PHOTOPERIOD
AND FLURIDONE ON THE GROWTH,
REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata
(L.f.) Royle] 39
Introduction 39
Materials and Methods 43
Chlorophyll and Carotenoid Analyses 45
Anthocyanin Analysis 45
Abscisic Acid Analysis 46
IV

Statistical Analysis 46
Results and Discussion 47
IV THE INFLUENCE OF FLURIDONE, PHOTOPERIOD,
AND PLANT GROWTH REGULATORS ON THE
GROWTH AND REPRODUCTION OF MONOECIOUS
AND DIOECIOUS HYDRILLA (Hvdrilla verticillau:
(L.f.) Royle] 62
Introduction 62
Materials and Methods 65
Photoperiodic Response 67
Effect of Fluridone 67
Effect of Exogenous Abscisic Acid and
Gibberellic Acid 68
Statistical Analysis 68
Results and Discussion 69
V SUMMARY AND CONCLUSIONS 89
APPENDICES
A ELISA PLATE COATING
PROCEDURE 91
B ELISA SAMPLE QUANTIFICATION 92
C ELISA STOCK SOLUTIONS AND
STANDARDS 93
D ELISA BUFFERS AND SOLUTIONS 94
E ELISA STANDARD CURVE AND SAMPLE
ABA DETERMINATION 95
F ABSCISIC ACID COLUMN EXTRACTION
PROCEDURE 96
G THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS 97
v

H THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS 98
I THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF YOUNG HYDRILLA 99
LITERATURE CITED 100
BIOGRAPHICAL SKETCH 113
vi

LIST OF TABLES
TABLE PAGE
2.1 The effect of elution volume and methanol
concentration on abscisic acid C18 column retention
under neutral pH conditions 27
2.2 The effect of elution volume and methanol
concentration on abscisic acid C18 column retention
under low pH conditions 28
2.3 The effect of plant tissue and column extraction on
the recovery of abscisic acid. Abscisic acid was added
before or after column clean-up 30
3.1 The effect of photoperiod and fluridone on the dry
weight of mature hydrilla plants 48
3.2 The effect of photoperiod and fluridone on the dry
weight of young hydrilla plants 49
3.3 The effect of photoperiod and fluridone on the
subterranean turion production of
mature hydrilla plants 51
3.4 The effect of photoperiod and fluridone on the
subterranean turion production of young
hydrilla 52
3.5 The effect of photoperiod and fluridone on the
chlorophyll content of mature hydrilla plants 55
3.6 The effect of photoperiod and fluridone on the
carotenoid content of mature hydrilla plants 56
3.7 The effect of photoperiod and fluridone on the
anthocyanin content of mature hydrilla plants 57
vii

3.8 The effect of fluridone concentration on the
abscisic acid content of mature hydrilla apical stem
segments, average across photoperiodic regime 60
4.1 The effect of photoperiod on the growth of 8 week
old dioecious and monoecious hydrilla 70
4.2 The effect of photoperiod on the axillary turion
production of 8 week old dioecious and monoecious
hydrilla 71
4.3 The effect of photoperiod on the subterranean turion
production of 8 week old dioecious and monoecious
hydrilla 72
4.4 The effect of fluridone on the growth of dioecious
and monoecious hydrilla. Plants were grown under
long-day conditions for 12 weeks 74
4.5 The effect of fluridone on the axillary turion
production of monoecious hydrilla. Plants were
grown under long-day conditions for 12 weeks 76
4.6 Axillary turion production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 78
4.7 Axillary turion production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 79
4.8 Axillary turion weight per plant by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 80
4.9 Axillary turion weight per plant by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 81
4.10 Percent weight of axillary turions produced by 8 week old,
long-day grown dioecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications 83
viii

4.11 Percent weight of axillary turions produced by 8 week
old, long-day grown monoecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications 84
4.12 Shoot biomass production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous abscisic
and gibberellic acid applications 85
4.13 Shoot biomass production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications 86
IX

LIST OF FIGURES
FIGURE PAGE
2.1 Internal abscisic acid standardization for column
cleanup procedure with hydrilla tissue 32
2.2 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.02 to 0.8 pmol 34
2.3 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.2 to 5.0 pmol 35
2.4 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.02 to 0.6 pmol 36
2.5 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.2 to 5.0 pmol 37
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PHYSIOLOGICAL ASPECTS OF HYDRILLA
[Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION
By
Gregory E. MacDonald
August, 1994
Chairman: Dr. D.G. Shilling
Major Department: Agronomy
The goal of this research was to provide a better understanding of the
fundamental processes involved in hydrilla growth and reproduction. Abscisic acid
from hydrilla tissue was analyzed by enzyme-linked immunoassay. Although solid-
phase extraction provided good purification of abscisic acid present in hydrilla tissue,
no clean up of the extract was necessary prior to analysis. Crude, unpurified extracts
of hydrilla stem and turion tissue were analyzed by ELISA. Validation of abscisic
acid extraction and analysis procedure was verified by internal standardization.
Accurate quantification could be obtained from plant extracts containing 0.02 to 5.0
pmol of abscisic acid. Short days promoted subterranean turion formation but this
effect was reduced by long days and 5 and 10 ppb fluridone. Fluridone caused a
significant reduction in chlorophyll and carotenoid levels and the abscisic acid
xi

content of mature plant shoot tips. The concentration of abscisic acid in young
plants was higher under short days. These studies provide further evidence that
fluridone can be used as a fall herbicide treatment to reduce turion formation in
hydrilla. The growth of both biotypes was reduced under short-day conditions.
Monoecious hydrilla formed axillary and subterranean turions regardless of
photoperiod, while dioecious hydrilla formed only subterranean turions only under
short-day conditions. Fluridone reduced the growth of both biotypes and reduced
turion production in monoecious hydrilla. Exogenously applied abscisic acid at 0.1,
1.0, and 10 /iM induced axillary turion formation by dioecious hydrilla under long day
conditions and abscisic acid at 1.0 and 10 /¿M induced axillary turion formation by
monoecious hydrilla. This process could be reversed by gibberellic acid in both
biotypes. However, subterranean turion formation was not observed under any
treatment for the dioecious biotype, but was observed in control monoecious hydrilla
plants. Monoecious hydrilla could be induced to flower by 50 ¿iM gibberellic acid
applications. This study indicates abscisic acid alone does not control axillary turion
formation in monoecious and dioecious hydrilla, but rather a balance between
abscisic acid and gibberellic acid may be the key to regulation of this phenological
process.
Xll

CHAPTER I
INTRODUCTION
Hydrilla [Hvdrilla verticillata (L.f.) Royle] is an exotic, submersed aquatic
vascular plant that causes major problems in the freshwater ecosystems of Florida
and the southeastern United States (Haller, 1976). Hydrilla interferes with
navigation, flood control and most recreational water activities, including fishing,
through a displacement of native vegetation (Langeland, 1990). Hydrilla was
discovered in 1959 in Florida, in a Miami canal. It was also reported in Crystal
River during the same period and was originally called Florida Elodea (Blackburn
et al.. 1969).
Hydrilla is a monotypic genus in the family Hydrocharitaceae, taxonomically
an ancient monocot family, with the center of origin thought to be tropical Asia
(Cooke and Luond, 1982). However, hydrilla is cosmopolitan, and is reported to be
broadly distributed throughout Germany, Lithuania, England, Poland, the upper Nile
of Africa, southeast Asia, Australia, Madagascar, India, China, Japan and the
southern United States (Lazor, 1978). A review of hydrilla by Mitra (1955) provided
the first detailed description of the species with historical background information.
Linnaeus filius first described hydrilla as Serpicula verticillata and classified it in the
Haloragaceae family. Hydrilla was later placed into the Hydrocharitaceae family
under the name Hydrilla ovalifolia but it was later changed to Hydrilla verticillata.
1

2
Hydrilla has been characterized by many scientists, as indicated by several describing
authors including Presl., Royle, and Casp. Today, Hydrilla verticillata (L.f.) Royle or
Casp. is the scientific name used to denote hydrilla. Langeland et al. (1992) showed
hydrilla to be endopolyploid, with monoecious hydrilla being triploid and dioecious
hydrilla being diploid; but, evidence from root tip karyotypes suggest sexual
compatibility across hydrilla populations and biotypes.
Anatomically, hydrilla is considered to be a primitive plant with reduced
vasculature. The xylem tissue is vestigial and phloem members are greatly reduced
(Yeo et al.. 1984). The leaves are comprised of two contiguous epidermal layers with
cuneate plastid inclusions, lacking in starch granules -- characteristic of most
monocots. The upper epidermis is much thicker than the lower layer (Pendland,
1979). Hydrilla leaves are borne in whorls of 4 or 5, with a distinctive spiny midrib
on the abaxial side of the leaf, distinguishing this species from elodea. Plants are
attached to the hydrosoil through fibrous roots and may resprout from root crowns
at the surface of the hydrosoil (Haller, 1976). Flowers are borne on stems near the
water surface (Cooke and Luond, 1982). Male and female flowers may be borne on
the same (monoecious) or separate (dioecious) plants. The control of floral
production is not well known but Pieterse et al. (1984), reported that low nitrogen
and phosphorus concentrations stimulated flower production in monoecious hydrilla.
Hydrilla regrows in the spring, as water temperature begins to increase, from
root crowns and/or subterranean turions in the hydrosoil (Haller, 1976). Shoot
growth is regulated by light quality with green light (prominent at deeper depths)

3
promoting stem elongation and red light (prominent at the surface) stimulating
branching (Van et al.. 1977). Hydrilla has also been shown to increase the level of
chlorophyll b at greater depths, enabling the plant to use longer wavelengths of light
for photosynthesis (Van et al.. 1977).
The development of a thick, entangled growth at the water surface (mat)
occurs when the shoots reach the water surface. This forms a dense canopy and
reduces light penetration in the first 0.3 m by 95% (Haller and Sutton, 1975). The
rapid growth of hydrilla has been reported to be 4.2 g dry wt'm2'day'1 (DeBusk et
al 1981). Hydrilla is extremely competitive, forming large, monotypic stands.
Studies on the photosynthetic characteristics revealed hydrilla to have a C02
compensation point of 44 ¿xLL*1 with light saturation occurring at 600 to 700
/ímol'm s PPFD (Van et al.. 1976). Hydrilla can adapt to low light levels with a
light compensation point of 10 to 12 /¿moPm'^'s"1 PPFD (Bowes et al.. 1977).
Holaday and Bowes (1980) reported hydrilla could exist with low or normal C02
compensation points. Those plants with low compensation points had high activity
of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) and pyruvate, P¿ dikinase (E.C.
2.7.9.1) enzyme levels, suggesting that hydrilla is capable of both C3 and C4
photosynthetic metabolism and the change was due to low C02 concentrations in the
water (Barko and Smart, 1981; Holaday et al.. 1983). Salvucci and Bowes (1983)
implicated carbonic anhydrase (E.C. 4.2.1.1) in the reduction of photorespiration in
hydrilla during periods of low C02 compensation points. Ascencio and Bowes (1983)
also reported a major increase in PEP carboxylase under these conditions.

4
Collectively, these characteristics allow hydrilla to utilize the free C02 from the water
sooner after sunrise than other aquatic plants due its low light compensation point.
Furthermore, hydrilla can maintain net photosynthesis under low C02 levels through
the activity of PEPcarboxylase. This increases hydrilla’s competiveness, decreasing
competition from other species and allows hydrilla to maintain the luxuriant growth
of the mat under low C02 and high 02 conditions. In addition, Kulshreshtha and
Gopal (1983) reported allelopathic properties of hydrilla on species of Ceratophyllum,
further increasing the competitive advantage of this species.
Several studies have evaluated the effect of mineral nutrition on hydrilla
growth. Sutton (1985) suggested the growth of hydrilla is controlled by nutrients in
the root zone, while Spencer (1986) showed hydrilla could grow equally well under
low or high organic matter sediment composition. Stewart (1984) showed phosphorus
in the rooting media was the limiting factor in growth for hydrilla. Basiouny and
Garrard (1984) demonstrated that calcium and phosphorus uptake in hydrilla was
passive while potassium, copper, iron and manganese uptake required metabolic
energy. Iron appeared to be particularly important for hydrilla growth with
tremendous iron uptake and an exceptionally high iron to manganese ratio required
for optimum growth (Basiouny et al.. 1977b). Basiouny et al. (1977a) showed iron
was absorbed by roots and leaves of hydrilla and translocated throughout the plant.
Hydrilla is extremely difficult to manage and control because spread can occur
through a variety of mechanisms including fragmentation and specialized dormant
buds called turions (Sculthorpe, 1967). Langeland and Sutton (1980) reported

5
regrowth from hydrilla fragments with a single node and Sutton et al. (1980) reported
one shoot tip will produce as much biomass as 16 tips. Seed production has been
reported for the monoecious biotype but does not occur in dioecious hydrilla in the
United States.
Turions can be formed in the axials of leaves, or at the ends of positively
geotropic rhizomes which extend into the hydrosoil (Haller, 1976; Yeo et al.. 1984).
Subterranean turions (tubers) can remain dormant for as long as 5 years (Van and
Stewart, 1990). Miller et al. (1976) reported that increased tuber size and density
were correlated to increased water depth. There appears to be an environmentally
enforced dormancy of tubers, preventing rapid depletion of the tuber bank (Van and
Stewart, 1990). Basiouny et al. (1978b) showed gibberellic acid, ethephon, thiourea
and storage at 5 C to increase turion sprouting, and hypothesized the existence of
two types of dormancy in hydrilla subterranean turions. Stewart (1969) found
gibberellic acid enhanced sprouting of subterranean turions while IAA and 2,4-D
enhanced axillary turion development. Subterranean turions are thought to maintain
hydrilla within a given area, possibly through periods of drought. Work by Basiouny
et al. (1978a) showed that tubers are better able to survive drought than axillary
turions with almost 17% germination rate for tubers dried for 64 hours.
Axillary turions are smaller than subterranean turions and are thought to
function in dispersal (Thullen, 1990). These structures are generally formed on
detached floating mats of hydrilla (Thullen, 1990; Miller et al.. 1993). Furthermore,
axillary turions could last only 1 year in the hydrosoil (Van and Stewart, 1990),

6
presumably due to their smaller size.
Turion formation in dioecious hydrilla has been shown to be a photoperiodic
response with a critical photoperiod of 13 hours for subterranean turion formation
(Van et al.. 1978a). Haller et al. (1976) reported subterranean turion production
occurred in north Florida between October and April. Klaine and Ward (1984)
demonstrated the turion formation response was phytochrome mediated, with red
light (650 nm) stimulation and far-red light (750 nm) repression of turion formation
in the dioecious biotype. Exogenous ABA applications induced turion formation
under non-inductive conditions. Van et al. (1978b) also showed exogenous ABA
promoted turion formation under 16 hour daylengths. Axillary turion formation
appears to respond similarly to subterranean turion formation with regard to
photoperiod. Miller et al. (1993) showed hydrilla axillary turion production was
similar to subterranean turion formation - occurring in the fall and winter months
only.
Both the monoecious and dioecious hydrilla biotypes occur in the United
States. The dioecious female plant occurs throughout Florida, the southeast and
California, whereas monoecious hydrilla is found in Washington D.C., Virginia,
Maryland and North Carolina. Besides a difference in flowering, there seems to be
several distinct differences between the two biotypes in terms of vegetative growth
and turion production.
Monoecious hydrilla produces subterranean turions under both 10 and 16 hour
photoperiods, with a greater number of turions produced under short-day conditions.

7
The growth of monoecious hydrilla is generally prostrate, near the hydrosoil with
many horizontal stems and higher shoot densities than dioecious hydrilla. In
comparative studies, Steward and Van (1987) found that the monoecious biotype
tolerated cooler temperatures and was able to sprout at lower temperatures. Ames
et al. (1986) also reported greater growth of monoecious hydrilla under cooler
temperatures compared to dioecious.
Subterranean turion production has been shown to be greatest under short
days for monoecious hydrilla with more turions produced than the dioecious form,
but there was a decline in biomass accumulation with short days. Monoecious
hydrilla also appears to able to reproduce much more rapidly than dioecious, forming
new subterranean turions in 4 weeks as compared to 8 weeks for dioecious hydrilla
(Van, 1989). Spencer et al. (1987) reported subterranean turions to be nearly 2 to
5 times as large as axillary turions, with monoecious subterranean turions smaller
than those of dioecious hydrilla. In addition, Ames et al. (1987) showed percent
sprouting was higher in monoecious than dioecious hydrilla. These studies indicate
monoecious hydrilla possesses an annual growth habit, along with rapid and prolific
turion production, adapting this biotype to northern areas which have cooler
temperatures and short growing seasons.
Earlier work suggested monoecious hydrilla was photoperiodic in response to
turion formation. Spencer and Anderson (1987) showed that monoecious hydrilla did
not produce subterranean turions under daylengths greater than 14 hours and
production increased as daylength decreased. They also reported photo-interruption

8
of monoecious hydrilla inhibited turion formation, similar to long-day grown plants
(Anderson and Spencer, 1986). However, monoecious hydrilla appears only to be
delayed in the formation of turion under long-day conditions, producing turions after
8 weeks under long-day conditions (Van, 1989).
Management of hydrilla is difficult due to rapid growth rate and prolific turion
formation. Cultural management schemes, such as drawdowns to deplete tuber
populations have had limited success (Haller et al„ 1976) and biocontrol agents, such
as grass carp, are unpredictable forms of control. Martyn (1985) used grass carp to
control 9000 acres of hydrilla in a 20,000 acre lake in Texas. All hydrilla was
removed after 2 years but over 270,000 fish were necessary. Insect biocontrol has
been studied, with several introductions but these have met with only limited success.
The herbicides diquat, endothall and copper sulfate, alone or in combination
provide good initial control but regrowth of the hydrilla quickly occurs. Fluridone
(l-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(li/)-pyridinone) has been shown
to be very effective for hydrilla control, but contact time and concentration are
critical issues affecting the efficacy of a given fluridone treatment (Van and Stewart,
1985; Haller et al.. 1990; Fox et ah. 1991).
Fluridone affects plant tissue by decreasing pigment levels (Maas and Dunlap,
1989). This is due to a decrease in carotenoid levels followed by a subsequent
decrease in chlorophyll content due to excess photooxidative stress (Devlin et al..
1978). Bartels and Watson (1978) demonstrated the mechanism of action of
fluridone to be carotenoid inhibition and this effect could be overcome by adding 5-

9
aminolevulinic acid (Fletcher et al.. 1984). The actual site is an inhibition of the
conversion of phytoene to phytofluene in the terpenoid biosynthetic pathway (Mayer
et al.. 1989). Fluridone was reported to be a non-competitive enzymatic inhibitor,
with reversible binding. Fluridone causes similar effects on hydrilla tissue, decreasing
the chlorophyll and carotenoid content, but interestingly, increasing anthocyanin
content (Doong et al.. 1993).
Fluridone was originally marketed for weed control in cotton ('Gossvpium
hirsutium L.). Translocation studies showed fluridone to be translocated
apoplastically following root absorption. Tolerant species, such as cotton, show
limited translocation and root uptake (Berard et al.. 1978). Banks and Merkle (1978)
reported good control with fluridone of many annual weeds in cotton but also
reported some soil persistence.
Fluridone is an off-white crystalline solid, with a melting point of 151-154 C
and a water solubility of 12 ppm (McCowen et al.. 1979). It is classified as a
pyridinone herbicide with a molecular weight of 329.3 (McCowen et al.. 1979).
Mossier et al. (1991) reported microbial breakdown for fluridone was very slow and
variable. Aqueous degradation rates from lake collected microbial consortia did not
correlate to previous fluridone use patterns in the aquatic environment. Fluridone
has a fairly high affinity to clay or organic matter (Mossier et al.. 1993). Therefore,
slow-release formulations could only be effectively used in aquatic ecosystems with
a sandy bottom substrate or in situations where the fluridone pellet does not contact
the hydrosoil.

10
Ultraviolet light breakdown is the major degradation mechanism in the natural
environment. Photolytic degradation of fluridone is highest at 297 to 325 nm with
a half-life of 26 hours although some degradation can occur at 325 to 355 where the
photolytic half-life was 840 hours (Mossier et al.. 1989).
Fluridone has also been shown to decrease the levels of ABA within plant
tissue (Jones and Davies, 1991). Abscisic acid is derived from carotenoid precursors
so the negative effect of fluridone on carotenoid levels also translates to lower ABA
content. Abscisic acid is thought to be involved in turion formation in hydrilla, and
fluridone could be used to regulate this process. Turion formation in hydrilla can be
inhibited by fluridone under short-day conditions at concentrations greater than 5
ppb (MacDonald et al.. 1993), but the effect on ABA and subsequent inhibition of
turion formation was not determined.
Abscisic acid (ABA) is a plant hormone involved in a multitude of
physiological responses including stomatal opening (Cornish and Zeevaart, 1985), bud
and seed dormancy (Quatrano, 1987; Barros and Neill, 1986), freeze tolerance
(Johnson-Flanagan et al.. 1991; Reaney and Gusta, 1987), and tuber and turion
formation (Smart and Trewavas, 1984). ABA is formed through the carotenoid
biosynthetic pathway by the conversion of 9’-cú-neoxanthin to xanthin (Xan).
Xanthin is subsequently converted to ABA aldehyde and finally to cis- ABA (Gamble
and Mullet, 1986; Zeevaart et al.. 1989; Li and Walton, 1990; Parry and Horgan,
1991). Light can cause isomerization to trans-ABA but only cis-ABA is thought to
be the biologically active form. ABA biosynthesis is regulated by the production of

11
xanthin, not the conversion of xanthin to ABA (Parry and Horgan, 1991).
ABA has been implicated in the seed dormancy of many species (Hole et al..
1989; Singh and Browning, 1991; Le Page-Degivry and Garello, 1992), but the exact
mechanism not well understood. Morris et al. (1991) indicated that ABA induced
dormancy in wheat seeds at the gene level while Ozga and Dennis (1991) showed
changes in ABA in apple seeds were not related to dormancy. In a review, Quatrano
(1987) reported that ABA has been shown to modulate gene expression in cultured
embryos of certain crops. During seed development the increase in endogenous
ABA promotes maturation of the embryo and represses seedling growth. This is
accomplished by modulating gene sets, positively affecting the maturation set and
negatively affecting the germination set. As the seed begins to lose water, the
amount of ABA and the embryo sensitivity to ABA decreases and the dehydration
of the seed, not ABA, prevents germination.
Subbaiah and Powell (1992) reported that the chilling requirement of apple
seed is not related to ABA changes during stratification, and chilling, not ABA, was
required for germination. Also the decline in ABA was largely due to leaching of
the seed coat and nuclear membrane while ABA content of embryo remained
constant. They postulated that ABA leaching from the seed allows certain promotive
forces to develop, but these can only occur after the chilling requirement was met.
Earlier work also supports this contention. Schopfer et al. (1979) showed
ABA could reversibly arrest embryo development by inhibiting water uptake, not by
control of DNA, RNA or protein synthesis. Release from dormancy is not due to

12
lower ABA levels, but rather to a loss of sensitivity to ABA. Oishi and Bewley
(1990) found the decline in ABA will permit germination of maize kernels, but only
drying will allow the aleurone layer to become sensitive to gibberellic acid.
Gibberellic acid induces a-amylase activity, causing the remobilization of food
reserves for seedling growth.
Mutations are often used to study the role of ABA. Koornnef et al.. 1989
used three ABA mutants of Arabidopsis thaliana, termed abi loci, and found all
mutations reduced dormancy. However, one mutation did not effect the water
relations of leaf tissue. They hypothesized that the mutations affect different tissue-
specific receptors. Research by McCarty (unpublished) indicated that there was an
ABA receptor molecule complex on a promoter sequence of RNA. When ABA
binds to the promotor, RNA transcription begins and a specific enzyme(s) or event
is initiated.
There have been several indications that ABA plays an important role in plant
growth, especially under water-stressed conditions. Saab et al. (1990) showed that
ABA maintains primary root growth and inhibits shoot growth of maize seedlings
under low water potentials. In later work, Saab et al. (1992) suggested a differential
response to endogenous ABA by root and mesocotyl growing zones of maize. At low
water potentials, ABA had the greatest effects near the root tip and this effect
decreased from the tip. Mesocotyl tissue is more responsive to ABA in those cells
away from the meristematic region. Porter (1981) suggested that endogenous levels
of ABA in different plant organs could be a factor in the directional control of

13
assimilate transport in plants. Hoffmann-Benning and Kende (1992) reported that
the growth of rice was regulated by a ratio of gibberellic acid and abscisic acid, as
a growth promoter and inhibitor, respectively.
Interesting work on the effects of ABA on aquatic macrophytes has also been
reported. Kane and Albert (1987) demonstrated that ABA, possibly as an indicator
of water-stress, caused changes in aerial leaf morphology and vasculature in Hippuris
vulgaris. ABA also caused aerial leaf development in MyriophyHum and Proserpinaca,
and suggested possible ABA/ethylene interactions (Kane and Albert, 1989). Goliber
and Feldman (1989) measured the ABA content of the aerial leaves of Hippuris
vulgaris and showed much higher levels. They suggested the effect of osmotic stress
is the cue in aerial leaf development.
There is considerable changes in the compartmentation of ABA during light
and dark periods due to pH changes, with a 2-fold increase of ABA in the apoplast
in the dark. ABA can irreversibly conjugate to glucose forming ABA-glucoside.
This form is inactive and is restricted to the vacuole, and may be a sequestration
mechanism by which ABA is removed from the active pool. ABA has also been
shown to be a major factor in the control of stomatal opening through regulation of
guard cell turgor pressure (Zeevaart and Creelman, 1988). Guard cells under stress
appear to release ABA, initiating closure. Water-stressed roots can also produce
ABA that is translocated to the leaves and effects stomatal closure (Zeevaart and
Creelman, 1988).

14
Hartung (1983) reported the site of action of ABA in guard cells to be on the
outer surface of the plasmalemma. Raschke (1987) reported ABA prevented
stomatal opening by blocking H+ extrusion and K+ influx, and initiated rapid closure
by K+ release. Recent work suggested that ABA acts on a G-protein at the
membrane surface, activating phospholipase C, which catalyzes the hydrolysis of
phosphotidalinositol-pyrophosate to inositol-3-phosphate (IP3) and diglyceralhyde.
IP3 then causes intracellular Ca2+ release, blocking K+ channels and lowering
stomatal opening. There is also some evidence that internal ABA may have some
control over this process as well.
Abscisic acid is also thought to be involved in the process of turion and tuber
formation in hydrilla and other species. Tubers are among the most common form
of vegetative reproductive structures and are produced by several species including
potato (Solanum tuberosuml and jerusalem artichoke (Helianthus tuberosum!.
Tubers are swollen underground stems possessing multiple nodes and greatly reduced
leaf characteristics (Cutter, 1978). In potato, the morphology of tuber formation has
been well documented. Tubers are formed from stem tissue, generally at the ends
of basal stolons (Peterson, £t ah, 1985), and are initiated in the youngest elongating
intemode (Peterson, et ah, 1985). The pith cells in this area enlarge, followed by
rapid cell division of the perimedullary parenchyma and cortical cells. Cell division
occurs in early tuber development and final tuber size is achieved through cell
enlargement (Reeve et_ ah, 1973).

15
Turions are similar to tubers but are morphologically less complex, derived
from leaf tissue, with little differentiation of the stem tissue. Turion are often found
in freshwater aquatic species, such as green milfoil (Myriophyllum verticillatum L.)
(Weber and Nooden, 1976) and giant duckweed (Spirodella polvrhizal (Smart and
Trewavas, 1983). Unlike tubers, turions sprout from a single node at the tip of the
structure, although turions are comprised of several densely packed nodes (Yeo et
ah, 1984). The outer portion of the turion is made up of leaf tissue, which swells and
envelopes the terminal apex, giving the structure a scale-like appearance. These
leaves accumulate carbohydrates, with an abscission layer forming at the base of the
lowermost leaves. The turion detaches from the mother plant and remains dormant
until conditions favorable for growth arise.
Turion formation by dioecious hydrilla is promoted by short-days (Haller £t
ah, 1976). This response has been shown to be phytochrome mediated (Klaine and
Ward, 1984). Phytochrome has been implicated in several plant processes, including
flowering, seed germination and tuber formation (Quail, 1991). However, recent
work provided evidence that phytochrome may regulate more fundamental plant
processes (Lissemore et ah, 1987), (Smith and Whitelam, 1990), (Kendrick and
Nagatani, 1991), (Seeley et ah, 1992) and (Edgerton and Jones, 1992). Phytochrome
appears to be involved in the activation of K+ channels (Lew et ah, 1992) and has
been suggested to be a protein kinase (McMichael and Lagarias, 1990) (Doshi et ah,
1992). Recent research also indicated that at least two types of phytochrome [Type
I (Phytochrome A) or Type II (Phytochrome B] can be present within plant tissues

16
(Abe et ah, 1985) (Shimazaki and Pratt, 1985). Phytochrome A has been shown to
regulate stem elongation (Boylan and Quail, 1991), chloroplastic gene expression
(Sharrock et ah, 1988) and anthocyanin biosynthesis (Adamse et ah, 1988).
Phytochrome B has been linked to photoperiodic responses (Vince-Prue and
Takimoto, 1987; Takimoto and Saji, 1984) with respect to floral induction.
Changes in hormone levels occur soon after photoperiodic induction of tuber
formation. In potato, changes in ABA, gibberellin, and cytokinin levels have been
reported. Gibberellins are high Under long-day photoperiodic conditions and decline
under short days (Krauss, 1985), while the opposite effect is observed with abscisic
acid. Several researchers have reversed the effect of photoperiod with exogenous
applications of these hormones (i.e. preventing tuber formation under short-day
lengths with gibberellic acid while inducing tuberization with abscisic acid under long-
day lengths). However, a critical ratio or balance between these two hormones may
be needed for tuberization to occur (Menzel, 1980) (Vreugdenhill and Struik, 1989).
Cytokinins accumulate in response to tuberization but do not induce this process
(Krauss, 1985). In addition, increased nitrogen levels in the leaves have been shown
to delay or inhibit tuber formation in potato (Krauss, 1978), while increased
photosynthate concentration promotes this response (Wenzler et ah, 1989).
Exogenously applied abscisic acid also induced turion formation in hydrilla
(Van et ah, 1978), (Klaine and Ward, 1984). Klaine and Ward (1984) also reported
that turion formation in hydrilla was reduced by exogenous gibberellic acid, similar
to that observed for potato. However, cytokinin had no effect. Turion formation in

17
giant duckweed (Perry and Byrne, 1969) can also be induced by exogenous ABA
applications, suggesting that the role of abscisic acid in regulating vegetative
reproduction is similar for a variety of species.
The role of ABA and gibberellic acid in tuber formation is well documented
but specific changes related to initiation of this process is unclear. Hannapel et ah
(1985) showed exogenous gibberellin prevented the accumulation of patatin and two
other potato tuber proteins and correlated this with the inhibition of tuber formation.
Abscisic acid had an inhibitory effect on overall protein synthesis in Spirodela.
polvrhiza turion formation (Smart and Trewavas, 1984), and the authors suggested
that there were several novel proteins specific to induced tissue.
{
Quantification of abscisic acid has traditionally been accomplished through the
use of gas chromatography-mass spectrometry (Dumbroff et al.. 1983; Reymond £t
ah, 1987). However, the use of enzyme-linked immunoassays (ELISA) for ABA
analysis has increased over the years. The advantage of ELISA is that limited
sample purification and increased number of samples can be analyzed within a given
time frame.
: Several researchers have developed ABA antibodies (Weiler, 1980; Mertens
et ah. 1983; Ross et ah. 1987; Quarrie et ah. 1988). Most monoclonal antibody
immunoassays were able to use crude aqueous extracts without interference,
providing accurate quantification comparable to GC-MS analysis (Leroux et ah. 1985;
Quarrie et ah. 1988; Soejima et ah. 1990; Tahara et ah. 1991). Some
radioimmunoassays can detect free and conjugated ABA; this is dependant on Cj

18
or C4 ABA coupling to proteins for total or free ABA, respectively (Weiler, 1980).
However, ABA analysis with ELISA from plant tissues varies with plant
species and tissue type. Some extracts may require purification while other extracts
do not. Therefore, validation through internal standardization is recommended for
ABA analysis from a previously unvalidated tissue type.
Hydrilla is a perennial species, with regrowth from overwintering turions often
being the main source for re-establishment. Turions sprout in the spring, forming a
large, monotypic stand. The hydrilla then reproduces in the fall, replenishing the
turion supply within the hydrosoil. Therefore, turion depletion in the hydrosoil would
be the most effective means for long-term hydrilla control. However, herbicide
treatments during fall conditions (turion formation) would result in applications to
established hydrilla plants. However, fluridone has been shown to be very effective
for the control of hydrilla, and can reduce turion formation at concentrations greater
than 5 ppb. Fluridone will cause decreased ABA levels within plant tissues and this
may be the reason for the reduction in turion production.
The ability of hydrilla to reproduce by turions is the greatest restraint to the
control of this species. Therefore, a better understanding of the fundamental
processes involved in turion initiation and development would greatly assist in efforts
to effectively manage this species. To accomplish this goal the objectives of this
research were to 1) develop an ELISA based ABA analysis procedure for quantifying
ABA from hydrilla; 2) test the hypothesis that fluridone reduces turion formation
from a reduction in ABA content, not a lethal impact on plant growth; and 3)

19
compare the response (growth and turion development) of monoecious and dioecious
hydrilla biotypes cultured under controlled conditions to long and short-day
conditions, fluridone and exogenous plant growth regulators.

CHAPTER II
EVALUATION OF AN ENZYME LINKED IMMUNOASSAY PROCEDURE
FOR THE ANALYSIS OF ABSCISIC ACID IN HYDRILLA
Introduction
Abscisic acid (ABA) has been implicated in many plant developmental
processes, including control of gene expression, plasma membrane ion flux, and seed
dormancy (Neill et al. 1986; Barratt et al.. 1989). In addition, ABA plays a vital role
in plant water relations, through the regulation of stomata and growth under water
stressed conditions (Saab et al.. 1990; Saab et al.. 1992). ABA has also been
implicated in turion formation and heterophylly of aquatic macrophytes (Smart and
Trewavas, 1984; Kane and Albert, 1987).
Abscisic acid is a 15-carbon compound with a molecular weight of 264.3. It
exists in two forms: ’cis’ and ’trans’, which isomerize in light. Cis-ABA is the
biologically active form, therefore quantification of ABA should take this fact into
account. The acidic nature of abscisic acid (pKa of 4.8) has been utilized in analysis
protocols (Jones and Davies, 1991).
Abscisic acid is derived from the terpenoid biosynthetic pathway, but the exact
synthesis route has only recently been elucidated. Earlier work suggested ABA was
formed through two separate pathways: a direct and indirect pathway (Walton,
1980). The direct pathway was suspected to involve a C15 precursor derived from
farnesyl-pyrophosphate, which may itself be xanthoxin (Milborrow, 1983). Xanthoxin
20

21
was discovered in the late 1960’s (Taylor and Burden, 1973) as a precursor to ABA
and led to the indirect pathway hypothesis, as this compound is derived from the
breakdown of carotenoids. Therefore, ABA was also proposed to be derived from
carotenoid breakdown. However, elucidation of the exact biosynthetic pathway has
been extremely difficult due to the large number of potential ABA precursors versus
the minute amount of ABA (Cowan and Railton, 1986; Milborrow, 1983).
Subsequent research has provided the most definitive evidence that ABA is
produced via the indirect pathway of carotenoid breakdown. Biosynthetic inhibitors,
such as fluridone, have been shown to block carotenoid synthesis and also lower
ABA levels (Moore and Smith, 1984; Gamble and Mullet, 1986; Oishi and Bewley,
1990). In addition, carotenoid deficient mutants have lower levels of ABA (Wang
et al.. 1984; Neill et al„ 1986). Since these studies, most researchers have concluded
the synthesis of ABA involves epoxidation and isomerization of specific carotenoid
molecules, the formation of xanthoxin, and subsequent ABA formation (Zeevaart et
ah, 1989; Li and Walton, 1990; Parry and Horgan, 1991).
Abscisic acid can be extracted from plant tissue through a variety of methods.
Goliber and Feldman (1989) used 80% methanol for extraction from Hippuris
vulgaris while Gamble and Mullet (1986) found adequate extraction with 100%
acetone. Loveys and vanDijk (1988) found boiling water sufficient for extracting
ABA from grape (Vitis spp.l leaves and Tahara et al. (1991) found extraction with
tris-buffered saline to provide excellent extraction of ABA from wheat (Triticum
aestivum L. em. Thell.) leaves.

22
Traditional methods of ABA quantification include high performance liquid
chromatography (HPLC) and/or gas-liquid chromatography (GC). These methods
provide excellent quantification but require a large amount of purification (Dumbroff
et al.. 1983; Leroux et al.. 1985; Soejima et al.. 1990). Recently, enzyme-linked
immunoassays (ELISA) have been developed for ABA detection, greatly reducing the
rigorous purification required prior to analysis (Raikhel et al.. 1987; Tahara et al..
1991). In addition, the number of samples that can be processed at a single time is
much greater with ELISA.
Hydrilla [Hydrilla verticillata (L.f.) Royle] is a submersed aquatic plant that has
become a weed problem throughout much of southeastern United States and
California (Langeland, 1990). Hydrilla is a highly aggressive species, forming large,
monotypic stands that interfere with flood control, navigation and many recreational
water activities including boating and fishing (Haller, 1976; Langeland, 1990).
Hydrilla persists within a given area primarily through the production of specialized
vegetative reproductive structures called turions. Turion formation in hydrilla and
several other aquatic species is thought to be directly affected by ABA but the exact
role of ABA is not known (Van et al.. 1978; Klaine and Ward, 1984). Therefore,
a better understanding of the affect of ABA on turion formation in hydrilla could
provide more effective management strategies for this noxious species.
Several researchers have found the enzyme-linked immunoassay (ELISA)
procedure specific enough to quantify ABA in crude extracts without purification
(Tahara et al, 1991). However, each individual plant species must be tested and

23
validated if crude extraction is to be used (Zeevaart and Creelman, 1988). In
addition, plant tissue types may also vary in the content of interfering compounds,
particularly between chlorophyllous and achlorophyllous tissue. Therefore, the major
impediment to the universal applicability of ELISA is the unpredictable presence of
compounds that cross-react with the ABA-specific antibody. A procedure known as
internal standardization must be conducted on tissue prior to analysis for ABA using
ELISA. This technique allows a determination of whether purification of the sample
is necessary before ELISA quantification.
Although ABA has been implemented in hydrilla turion development, ABA
quantification from hydrilla tissue by ELISA has not been widely used. Therefore,
studies were conducted to develop a purification procedure for detecting ABA from
hydrilla and to determine if such a procedure was warranted for quantification by
ELISA.
Materials and Methods
Elution profiles and column/ABA characterization. Reverse-phase cartridges,
particularly C18, have been widely used for abscisic acid purification (Hubick and
Reid, 1980). Therefore, initial studies were performed to characterize the
relationship between eluant volume, methanokwater ratio and pH on the retention
of ABA to this type of column. Columns1 were prepared prior to use by washing
1 PrepSep; Fisher Scientific, Pittsburgh, PA 15219.

24
once with methanol and twice with deionized water. All samples were passed
through the column under vacuum (90 kPa) and the resulting eluant was collected
in 20 ml glass vials. During elution these vials were placed within a vacuum
manifold .
In the first experiment, 100 ¡iL of 1 mM ABA was added to the top of the
column and eluted with varying concentrations of methanohwater (20:80, 50:50,
70:30, 80:20, 100:0) in 10 mL aliquots up to 50 mL. The elution pH was
approximately 7, with the majority of the ABA molecules in the protonated form.
The ABA content was then determined spectrophotometrically at 250 nm. Recovery
was expressed as a percent of ABA added.
i
A similar experiment was conducted with varying concentrations of
methanohwater (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 70:30, 80:20, and 100:0) in
10 ml aliquots up to 50 mL. In addition, the methanohwater elutions were acidified
with 1% formic acid to obtain a pH of less than 3.0. This resulted in most the ABA
in an unprotonated, neutral form. Recovery was expressed as a percent of ABA
added.
The preceding elution studies allowed the development of a purification and
concentrating procedure that was used in the following two experiments (see results
and discussion). An experiment was conducted to determine if the presence of
compound(s) in hydrilla extract would interfere with accurate quantification of ABA
2 PrepTorr; Fisher Scientific, Pittsburgh, PA 15219.

25
using UV spectroscopy. Tissue extract contained 20 mg-fresh weight hydrilla shoot
tips which were homogenized in 10 to 15 mL 100% methanol and the extract filtered
under vacuum. This extract contained three levels of plant tissue (0, 12.5, and 25 mg-
fresh weight/sample). Abscisic acid was added to the plant extracts at concentrations
of 0, 8, 13, or 18 nM prior to column extraction. ABA was detected
spectrophotometrically at 250 nm.
A study was also conducted to determine if components in hydrilla extract
would influence the recovery of ABA purified using column chromatography. This
study involved fortifying hydrilla extract with different concentrations of ABA before
or after purification. Tissue extract was prepared using the aforementioned methods
and three concentrations (150, 250, or 350 nM) of ABA were used for fortification.
Abscisic acid recovered was detected spectrophotometrically at 250 nm.
ELISA internal standardization for ABA in hydrilla. An internal
standardization experiment was conducted to determine if purification of hydrilla
tissue was necessary prior to ABA quantification with ELISA. Antibody and
antibody tracer specific for ABA were obtained directly from the manufacturer3.
Procedures for the preparation of ELISA plates, sample preparation and ABA
concentration determination are outlined in Appendices A-E.
Internal standardization was performed on crude extracts of hydrilla stem and
turion tissue utilizing ELISA. Due to the large range of values to be tested,
3 Phytodetekâ„¢, Idetek, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089.

26
standardization of each tissue type was performed in two experiments. In the first
experiment, 100 ¿iL of 0, 0.02, 0.04, 0.06, 0.08, 0.2, 0.4, or 0.6 pmol ABA was added
to dilutions of hydrilla stem tissue (1:2 and 1:4) and turion tissue (1:4 and 1:8).
Dilutions were based on 20 mg-dry weight/sample as a 1:1 dilution and ABA was
added prior to quantification by ELISA. Samples were extracted according to the
procedures outlined in the previous section. The methanolic extract was then dried
under a stream of nitrogen gas and reconstituted in tris-buffered saline prior to
quantification by ELISA.
In the second experiment, 100 /¿L of 0.0, 0.2, 0.5, 1.0, 2.0, or 5.0 pmol ABA
was added to dilutions of hydrilla stem tissue (1:1 and 1:3) and turion tissue (1:1 and
1:4). Dilutions were based on 12 or 8 mg-dry weight/sample as the 1:1 dilution) for
stem or turion tissue, respectively. ABA was added prior to quantification by ELISA.
Tissue extracts for both experiments were prepared in a manner similar to plant
material used in the column extraction procedure.
Results and Discussion
Abscisic acid under neutral pH conditions showed little affinity for the C18
column when the mobile phase contained greater than 30% methanol. The majority
of ABA was recovered with only 10 mL of 30% methanol (Table 2.1). Much higher
concentrations of methanol were necessary to elute ABA from the C18 column when
the mobile phase was acidic (Table 2.2). Under low pH conditions ABA retention
increased at similar methanol concentrations to the previous experiment. However,
complete retention was observed at concentrations below 20% methanol.

27
Table 2.1. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under neutral pH conditions.
Cumulative
Elution
Volume
0
10
20
Methanol Concentration (%)
30 40 50 70 80
100
mL
% ABA eluted
10
8+31
50±4
77+2
87±2
94 + 1
94±3
89±5
76±3
80+4
20
33+3
21 ±2
18±2
2+2
0
0
--
5+3
5+3
30
15+2
11±1
2±1
2+2
0
0
0
0
0
40
7±1
6±2
2±1
0
0
0
0
0
0
50
4 + 1
0
0
0
0
0
0
0
0
1 Means (four replications) followed by standard errors.

28
Table 2.2. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under low pH conditions.
Cumulative
Elution
Volume
0
10
20
Methanol Concentration (%)
30 40 50 70 80
100
mL
— % ABA eluted
10
0
0
0
2±ll
14+6 57±2
90±3
99±2
92±3
20
0
0
0
0
59±3 44+3
15 ±3
—
11 ±4
30
0
0
0
8±4
13+4 0
0
0
0
40
0
0
0
28 ±4
0 0
0
0
0
50
0
0
0
22 ±3
0 0
0
0
0
1 Means (four replications) followed by standard errors.

29
Concentrations greater than 50% removed ABA from the column after 20 mL of
elution volume. Extraction of ABA using Cig solid-phase columns is highly
dependent on pH. Low pH conditions allows greater retention of the ABA to the
column while neutral conditions do not. These results agree with the work of Parry
and Horgan, 1991 and other researchers.
The purpose of these studies was to develop a two-stage procedure for the
purification and concentration of ABA for analysis by ELISA. In the first
purification step, it was imperative that the appropriate solvent strength (%
methanol) be achieved to allow the retention on the column of as many extract
components as possible while allowing the elution of ABA. Because the volume of
solvent increased in this step, it was then necessary to concentrate the purified ABA
extract. This was accomplished by determining the appropriate solvent strength of
the mobile phase that would allow the retention of ABA by the column. This
technique is known as solid phase extraction and has many advantages over liquid
to liquid extraction, which results in large quantities of waste solvent. In addition,
solid phase extraction is cheap, rapid and allows for the simultaneous preparation of
numerous samples. The results provided information necessary to devise a clean-up
procedure that could be utilized for ABA analysis. This procedure provided over
92% recovery for all concentrations when ABA was extracted in the absence of plant
tissue (data not shown). Details of this procedure can be found in Appendix F.
When this procedure was used with the addition of plant tissue, it resulted in
greater than 89% recovery for all concentrations of ABA added (Table 2.3). Visual

30
Table 2.3. The effect of plant tissue and column extraction on the recovery of
abscisic acid. Abscisic acid was added before or after column purification.
ABA
Added
Plant + ABA
Before Column
Plant + ABA
After Column
Recovery
(nM)
0
0.276+0.401
- absorbance
0.276 ±0.40
150
0.580±0.20
0.632 ±0.34
92
200
0.703 ±0.09
0.752 ±0.45
93
250
0.786±0.18
0.880 ±0.42
89
1 Means (three replications) followed by standard errors.

31
observations indicated that the column purification procedure provided good
separation of chlorophyll and carotenoids. However, anthocyanins were extracted
with the ABA, as indicated by a pinkish color during the second column extraction.
Furthermore, the presence of interfering compounds in hydrilla tissue extract
was not detected using this procedure (Figure 2.1). An internal standardization
procedure allows for the detection of an interfering compound. If the addition of
ABA to various extract dilutions does not cause a change in the rate of absorbance
then interfering components are not present. The absence of such a compound
would be indicated by parallel lines as the rate at which ABA detected as a function
of ABA added was the same regardless of the extract dilution. Although different
amounts of ABA were detected, dilutions as a function of ABA added was the same.
The column clean-up procedure developed reported herein provided good
recovery of ABA and separation of possible interfering compounds. Although
additional purification could be necessary with chromatographic methods of
quantification, this procedure provided an excellent initial purification step.
Furthermore, this procedure provided adequate purification of hydrilla tissue for
quantification by ELISA, if purification was needed.
Internal standardization of ELISA allowed for the detection of an interfering
compound (s) by dilution of the interfering compound (s) with the addition of higher
levels of ABA. The absence of interference would be indicated by parallel lines of
the two levels of plant tissue and/or the lack of a significant interaction (P > 0.05).
The results from hydrilla stem tissue indicated the absence of interfering compounds

Figure 2.1. Internal abscisic acid standardization for column cleanup
procedure with hydrilla tissue.

33
(Figures 2.2 and 2.3). Good separation of the two dilutions was observed in the first
experiment at 0.6 and 0.8 pM ABA. However, the ability to detect differences in
dilution was limited to a range of 0.2 to 0.5 pM in the second experiment. Hydrilla
turion tissue also appeared to be free of interfering compounds (Figures 2.4 and 2.5).
Although the lines do not appear to be absolutely parallel, statistically there was no
interaction (P >0.05) between ABA added and ABA detected for the different
extract dilutions. This implies that there was a consistent change in the amount ABA
detected regardless of the amount of ABA added (i.e. the lines representing
independent and dependent variables for the different dilutions were parallel
although differing in magnitude).
Furthermore, the presence of interfering compounds would result in response
convergence at a higher added ABA, because the interfering compound (s) would
react with the antibody, over-estimating ABA present. However, these data do not
fit this scenario. In addition, preliminary experiments were conducted by adding
ABA prior to purification and the same relationship between ABA added and ABA
detected was observed (data not shown). Based on these results analysis of ABA can
be accurately determined from hydrilla tissue by analysis of crude extraction.
However, prudence should be exercised during analysis to ensure proper dilution of
the respective sample; only those readings within the linear range of the standard
curve are valid. The internal standardization reported herein provided an accurate
determination within the range of 0.02 to 5.0 pmol per sample, allowing a broad
range of sample ABA concentrations to exist. This also provides a large degree of

ABA ADDED
Figure 2.2 Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.02 to 0.8 pmol.

Figure 2.3. Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.2 to 5.0 pmol.

ELISA with hydrilla turion tissue. Range of values was 0.02 to 0.6 pmol.

o
LU
>
cc
LU
CD
Gü
O
<
GÜ
<
Q ■—i—i—i—i I i i i i l i i i i I i i i i l ■ ■ ■ ■
0 1 2 3 4 5
ABA ADDED
"igure 2.5. Internal standardization of abscisic acid quantification using
ELISA with hydrilla turion tissue. Range of values was 0.2 to 5.0 pmol.

38
flexibility when undertaking ABA analysis from hydrilla tissue. Using this
methodology, 8.0 pmol per sample for crown tissue (20 mg - dry weight) and 12 pmol
per sample for turion tissue (8 mg - dry weight) were detected from these hydrilla
tissue types. From these data, the appropriate amount of tissue needed for ABA
extraction would be 10 to 20 mg - dry weight.
However, this procedure should be used to correlate large differences in ABA
levels between tissue types, biotypes or seasonal hormone fluctuations. The minute
quantities of ABA found endogenously can be depleted by a multitude of factors
before actual analysis, therefore relative (large) differences only should be used when
determining the effect of ABA.

CHAPTER III
THE INTERACTIVE EFFECT OF PHOTOPERIOD AND FLURIDONE
ON THE GROWTH, REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
Introduction
Hydrilla is an exotic, submersed vascular plant that has become one of the
most troublesome aquatic weeds in the state of Florida. Hydrilla was first observed
in a Miami canal and in Crystal River around 1960 and is thought to have been
introduced from Asia by the aquarium plant industry (Blackburn et al. 1969; Haller,
1976). Since introduction, hydrilla has spread throughout Florida, the southeastern
United States, and California (Langeland, 1990). Infestations have also been
reported in areas of Maryland, Virginia and Washington D.C., but this is thought to
be a separate introduction involving a different hydrilla biotype (Stewart et al.. 1984).
Hydrilla is a major problem in many freshwater ecosystems, forming a dense,
entangled mat of shoots at the water surface (Haller and Sutton, 1975). This
extensive growth interferes with most recreational water activities, navigation, and
flood control. In addition, hydrilla effectively outcompetes most native vegetation,
changing the natural balance of the aquatic ecosystem. Changes in the native flora
and fauna occur that, ultimately impact the freshwater fishing industry of Florida.
Hydrilla regrows in the spring as the water temperature begins to rise (Haller,
1976). During the initial phases of growth, hydrilla shoots quickly reach the water
39

40
surface and begin to branch, forming a dense canopy (mat)(Van et al.. 1977). This
mat effectively absorbs nearly 95% of the available sunlight (PPFD), limiting light
penetration to understory vegetation and reducing competition. In addition, hydrilla
can adapt to extremely low light levels. Bowes et al. (1977) reported the light
compensation point of hydrilla to be 12 to 20 ^mol m^ sec'1. Further work by
Salvucci and Bowes (1983) showed that a change in photosynthetic metabolism, from
C3 to C4 - like photosynthesis occurred under certain conditions. This ability,
coupled with the low light compensation point, allows hydrilla to outcompete other
species and maintain the monotypic mat.
Hydrilla is difficult to control because spread can occur through a variety of
mechanisms including stem fragmentation and turion development. In hydrilla,
turions are produced at the tips of positively geotropic rhizomes, which extend from
the root crown into the hydrosoil (Sculthorpe, 1967). These structures, termed
subterranean turions or tubers, become detached from the mother plant and can
remain dormant for 1 to 5 years (Van and Stewart, 1990). Turions may also be
formed in leaf axils and are termed axillary turions (Yeo et al.. 1984). Axillary
turions are generally smaller than subterranean turions and are usually formed on
detached, floating plants (Haller, 1976; Miller et al.. 1993). Due to the smaller size,
axillary turions are thought to function in dispersal, whereas subterranean turions act
primarily in the persistence of hydrilla in a given area (Spencer et al.. 1987; Thullen,
1990).

41
Hydrilla forms turions during the late summer and early fall in North Florida
(Haller, 1976; Miller et al.. 1993). Van et al. (1978) reported that hydrilla responded
to photoperiod, with turions produced only under short-day conditions. Studies by
Klaine and Ward (1984) elucidated the role of phytochrome in controlling this
response. The level of ABA is thought to rise in response to short-day conditions,
preceding turion formation. ABA applied exogenously to hydrilla under long-day
conditions promotes turion production, but the differentiation between subterranean
and axillary turion formation has not been studied (Van et al.. 1978; Klaine and
Ward, 1984).
Management of hydrilla is difficult and results are variable (Schmidtz et al..
1987), with control efforts focusing primarily on herbicide use for logistical reasons.
Endothall, (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid), diquat, (6,7-
dihydrodipyridol[l,2-a:2’,r-c]pyrazinediium ion), and copper sulfate alone or in
combination (s) provide good initial control but hydrilla quickly regrows. A slow
acting herbicide, fluridone, (l-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(li/)-
pyridinone), was introduced for aquatic weed control during the 1980’s and has
shown great promise for the control of hydrilla (McCowan et al.. 1979). However,
proper exposure periods and concentrations are necessary for effective control
(Netherland et al.. 1993).
Fluridone inhibits carotenoid biosynthesis, causing photooxidation of
unprotected chlorophyll molecules (Bartels and Watson, 1978; Devlin et al.. 1978;
Maas and Dunlap, 1989). Fluridone has also been shown to inhibit the production

42
of ABA, which is thought to form from the carotenoid biosynthetic pathway (Li and
Walton, 1990; Parry and Horgan, 1991). Because of this, fluridone is used in
physiological experiments to study the effect of ABA on plant growth and
development (Hole et al.. 1989; Oishi and Bewley, 1990; Saab et al.. 1990).
Short-term control of hydrilla is often realized within a given area but long¬
term eradication is rarely achieved. Fluridone provides excellent control of hydrilla
and is generally applied in the spring as the hydrilla begins to regrow. Good initial
control is observed but regrowth from dormant turions occurs during the mid to late
summer months. This regrowth provides substantial biomass for reproduction in the
fall and turion levels in the hydrosoil are replenished. Thus the cycle of spring
treatment but regrowth and subsequent turion formation in the fall never results in
effective, long-term management.
One management strategy would be to treat hydrilla during the fall before or
during turion formation. However, hydrilla may not be sensitive to herbicides at this
time. Herbicides are most active on young, rapidly growing plants. Hydrilla growing
in the fall would be near maturity and should require a higher dose to achieve
control. Fluridone has been shown to reduce turion formation at concentrations _>
5 ppb (MacDonald et al.. 1993). However, the actual mechanism (i.e., reduction in
ABA levels and subsequent turion inhibition or turion inhibition as a result of plant
death), is not known. MacDonald et al. (1993) postulated that lower ABA levels
result from sublethal fluridone concentrations and indicated a potential growth
regulator phenomenon.

43
Clearly, the key in long-term hydrilla management is turion depletion in the
hydrosoil. Two strategies have been suggested concerning this issue; inhibition of
turion production or elimination of turion dormancy allowing precocious germination.
The objective of this study focused on the former strategy. Hydrilla was grown under
long (vegetative growth) and short-day (reproductive growth) conditions and
evaluated for changes in growth, turion formation and biochemical parameters in
response to varying rates of fluridone.
Materials and Methods
Hydrilla was initially planted on July 1, 1993 from apical stem segments and
grown under natural conditions in 900 L concrete vaults at the Center for Aquatic
Plants in Gainesville, FL. Stock hydrilla tissue was obtained from the Waccissa River
in north Florida; a pre-determined "fluridone-free" water system. Treatment plants
were grown in 10 x 10 cm plastic pots filled with organic potting soil4 and covered
with a 2 cm deep sand "cap" to prevent floating. Two 10 cm long apical hydrilla
sprigs were planted per pot and 6 pots were placed in 24 x 30 cm plastic dishpans.
On August 16, 1993 the plants were transferred to 540 L fiberglass vaults under short
(natural) or long (artificially extended) day greenhouse conditions. Environmental
conditions of the long-day greenhouse were 16 hr light/8 hr dark photoperiod, day
temperature of 30 ± 5 C, night temperature of 25 ± 5 C, with a mean quantum
4 Ace Hardware Corp., Oak Brook, IL 60521.

44
irradiance at noon of 1200 /xmoPm^sec-1 (PPFD). Photoperiod was extended for
4 hours after sunset with incandescent bulbs [100 /xmol'm^ sec'1 (PPFD)]. The
short-day greenhouse was maintained at similar conditions under natural (short-day)
photoperiodic conditions.
A second population of young hydrilla plants was established on August 18,
1993 in a manner similar to the older plants and placed under the same conditions.
The 8 week old population represented summer hydrilla growth while the younger
plants represented hydrilla regrowing from overwintering turions. Throughout the
remainder of this chapter, mature and young will be used to denote the 8 week old
and newly established hydrilla plants, respectively.
Each greenhouse (photoperiodic condition) contained 8 vaults, 4 comprising
mature hydrilla plants, 4 containing younger hydrilla. Each vault contained a total
of 10 dishpans. Fluridone concentrations of 0, 1, 5, and 10 ppb were established
within each population and photoperiodic regime beginning Sept. 2, 1993. Periodic
flushing and insecticide treatment were necessary to maintain optimum growth
conditions. After flushing fluridone was re-applied to each vault to achieve the
desired concentration.
Hydrilla (one dishpan) was harvested from each vault on Sept. 2, 1993 and
\
harvests continued on a weekly basis for 8 weeks. Each dishpan contained 6
i.
replications (1 replication = 1 pot). Parameters measured from both plant age
groups included fresh weight and subterranean turion number. Mature plants were
subsequently divided into apical stem, crown, and subterranean turion sections.

45
Subsamples were a composite from all replications. Immediately following
measurements and/or subsampling, young plants (entire plant) and mature plant
subsamples were frozen in liquid nitrogen and stored at -20 C. Samples were
lyophilized at -SO'C and dry weights recorded. The tissue ground was then to 0.5 mm
fineness and stored at - 20 C prior to biochemical analyses.
Chlorophyll/carotenoid analyses. Approximately 10 mg dry apical stem tissue
was placed in a 20 ml glass scintillation vial and filled with 10 ml of
chloroforrmmethanol (2:1 v/v). Vials were placed on a rotary shaker (150 rpm) for
12 hours at 4 C. The homogenate was filtered and the crude extract dried under a
stream of nitrogen gas at room temperature under dim light (< 5 /onolm'^sec'1).
The residue was resuspended in 80% acetone and absorbances at 470, 646, and 663
nm were measured spectrophotometrically. Total chlorophyll concentration was
calculated using the formula: 17.32 x - 7.18 x A663. Total carotenoid content
was determined using the formula: 1000 x A470 - 3.27 x Chla - 104 x Chlb)/229
(Lichtenthaler and Wellburn, 1983). Chlorophyll a (Chla) and chlorophyll b (Chlb)
concentrations (fig/mL) were calculated from the following formulas: 12.21 x A^ -
2.81 x A^4b and 20.13 x A^ - 5.03 x A^, for Chi a and b, respectively. Three
replications of the composite apical stem section subsample from mature plants only
(described above) were analyzed for chlorophyll and carotenoid content.
Anthocvanin analysis. Approximately 10 mg dry apical stem tissue was placed
into a 20 ml scintillation vial filled with 5 ml of 1% acidic methanol (v/v). Tissue
was extracted for 12 hours on a rotary shaker (150 rpm) at 4 C. The resulting

46
mixture was allowed to settle and absorbance of the supernate was measured
spectrophotometrically at 530 and 657 nm. Chlorophyll has some absorbance at 530
nm in acidic methanol, therefore corrections were made using the formula: A530 -
0.25 x Ag57, which assumes an extinction coefficient of 34,000 M^cm'1 (Jonsson et
al, 1984; Mancinelli, 1990). Anthocyanin content was determined from apical stem
section subsamples from mature plants only (described above) with 3 replications per
subsample.
Abscisic acid analysis. Tissue (10 to 20 mg dry weight) was extracted with
100% methanol for 12 hours on a rotary shaker (150 rpm) at 4 C. Methanol was
evaporated at -50 C and the residue resuspended in 0.5 to 1.0 ml of tris-buffered
saline and re-extracted as described previously. Following extraction, the
homogenate was centrifuged at 9000 rpm for 4 minutes. Analysis for ABA was
performed on the resulting supernate using an enzyme-linked immunoassay specific
for ABA. Details of the immunoassay procedure are presented in Appendices A-E.
A single sample from composite apical stem segments and crown subsamples of
mature were analyzed for ABA content. A single sample from 3 replicates of
younger plants 0, 2,4, 6, and 7 weeks after treatment (WAT) were analyzed for ABA
content.
Statistical analysis. Data were initially analyzed by analysis of variance to test
for treatment effects (WAT, photoperiod, and fluridone concentration) and
interactions. The effect of photoperiod was separated with Fisher’s protected LSD
test at the 0.05 level of significance. The effect of fluridone concentration was

47
compared to the untreated control using Dunnett’s "T" test at the 0.05 level of
significance. Means are presented with standard errors.
Results and Discussion
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod and fluridone concentration on the dry weight of mature and
young hydrilla plants. The dry weight of untreated plants increased over time in both
photoperiodic regimes for both mature and young hydrilla populations (Tables 3.1
and 3.2). Young plants showed a greater increase in biomass than the mature
hydrilla over the duration of the study. Seven WAT, the biomass of young plants
increased by 83 and 90% under long and short-day conditions, respectively.
However, mature plants only showed an increase of 39 and 25% for long and short
day photoperiods, respectively. The dry weight of mature plants was reduced by
fluridone concentrations of 5 and 10 ppb by more than 63%, regardless of
photoperiod at 7 WAT. Young hydrilla plant biomass was reduced by fluridone
concentrations of 5 and 10 ppb greater than 95% under short-day conditions and
greater than 68% under long days. Fluridone at 1 ppb under long-day conditions
caused 50 and 42% reduction in dry weight of mature plants compared to the
untreated control after 6 and 7 weeks of treatment, respectively. Short-day grown
mature hydrilla, however, did not show significant biomass reduction at 1 ppb
fluridone, while younger plants were significantly reduced at 5 and 7 WAT under
short day conditions.

48
Table 3.1. The effect of photoperiod and fluridone on the dry weight of mature
hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
4.6
3.9
11.9
g/plant
9.6
3.4
7.4
7.5
1
4.4
4.6
8.0
5.3
3.6
3.7*3
3.2*
5
4.3
4.3
4.0*
3.5*
1.7
2.2’
1.1*
10
4.2
3.9
5.6*
bo
*
4.1
2.8*
2.8*
Short Day
0
5.3
4.7
5.5
4.8
4.6
6.9
7.1
1
4.3
4.1
5.1
3.5
4.1
6.0
4.3
5
3.9
3.5
3.8
2.3*
1.7*
1.9*
1.4*
10
4.9
3.3
4.5
1.6*
1.9*
2.3*
1.7*
1 Average hydrilla plant dry weight at the initiation of the study was 4.25 g.
LSD0 os = 2.3 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s Y test 0 os)-

49
Table 3.2. The effect of photoperiod and fluridone on the dry weight of young
hydrilla plants. '
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4 5 6 7
Long Day
0
0.17
0.17
— g/plant
0.40 0.36 0.58 0.75 0.66
1
0.20
0.26
0.37
0.44 0.57 0.51*30.71
5
0.20
0.25
0.21*
0.19* 0.10* 0.07* 0.03*
10
0.30*
0.44*
0.20*
0.17* 0.23* 0.12* 0.21*
Short Day
0
0.28
0.43
0.55
0.60 0.90 1.38 2.97
1
0.25
0.34
0.41
0.57 0.49* 0.86 0.81*
5
0.33
0.43
0.42
0.42* 0.29* 0.27* 0.31*
10
0.28
0.23*
0.18*
0.14* 0.07* 0.07* 0.05*
1 Average hydrilla plant dry weight at the initiation of the study was 0.18 g.
1 LSD0 os = 0 .17 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s Y test q.os)-

50
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod, and fluridone concentration on the subterranean turion
production of mature and younger hydrilla plants. Turions were not produced during
the first 4 weeks of treatment for either mature or young plants (data not shown).
Mature hydrilla grown under short-day conditions produced a greater number of
turions at 0 and 1 ppb fluridone 5 WAT or longer than under long-day conditions
(Table 3.3). Subterranean turion production was reduced by more than 80% in
mature hydrilla by fluridone at 5 and 10 ppb at 4 to 7 WAT under both
photoperiods. Fluridone at 1 ppb also reduced turion production of mature plants
at 4 to 7 WAT under long and short day conditions, with the exception of 5 and 7
WAT under short days.
Subterranean turion production by younger hydrilla plants was reduced under
long day conditions and 5 and 10 ppb fluridone by nearly 100% (Table 3.4).
Fluridone at 1 ppb reduced turion production in the younger plant 6 and 7 WAT
under short and long photoperiods, respectively.
Young and mature hydrilla plants responded similarly to photoperiod and
fluridone in terms of growth and reproduction. Fluridone is very effective for
hydrilla control and rates of 5 and 10 ppb reduced dry weight and nearly eliminated
turion production when hydrilla was exposed for greater than 5 weeks, similar to
previous research (MacDonald et al„ 1993). At 1 ppb fluridone, hydrilla growth was
arrested in the mature plant population, while young plants at this treatment
continued to accumulate biomass. Both mature and young plants gained biomass

51
Table 3.3. The effect of photoperiod and fluridone on the subterranean turion
production of mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment
period1 (ppb)
4
5
6
7
Long Day
0
3.5
turions/plant
2.2 2.0
4.0
1
0.3*2
0.3*
0*
0*
5
0.2*
0*
0*
0.2*
10
0.2*
0*
0.2*
*
©
Short Day
0
4.3
6.3
8.0
11.7
1
3.0
5.8
3.0*
9.0
5
0.2*
0.2*
0.3*
2.3*
10
0.2*
0*
0*
0.8*
1 LSD0 os = 1.7 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s T test q.os)-

52
Table 3.4. The effect of photoperiod and fluridone on the subterranean turion
production of young hydrilla.
Photo- Fluridone
Weeks After Treatment
period1 (ppb)
4
5
6
7
Long Day
0
0
turions/plant
0 1.7
0.7
1
0
0.7
0*
0.7
5
0
0
0*
0
10
0
0
0*
0
Short Day
0
0.8
4.0
6.2
12.0
1
3.3
4.2
5.0
4.0*
5
0.2
0*
0*
0*
10
0*
0*
0*
0*
1 LSD0 os = 1.0 to separate the effect of photoperiod within week and fluridone
concentration.
9 • •
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s ’t’ test q.qs)-

53
in untreated conditions but the younger, more rapidly growing plants should have
been more susceptible than mature plants to fluridone treatment.
A possible explanation for this apparent contradiction may be related to light
intensity. Mature hydrilla plants have the majority of photosynthetic tissue near the
water surface, exposed to a greater amount of light and potential photooxidative
stress versus younger plant tissue emerging through the water column. In addition,
the removal of apical dominance would cause branching at the water surface for the
mature plant (Van et al.. 1977). The new tissue produced by the mature hydrilla
plants would then be more susceptible to the affect of fluridone due to the higher
light intensity at the surface. This would result in a reduction of growth. However,
the loss of apical dominance in young hydrilla resulting from fluridone treatment
would cause branching near the hydrosoil, where light intensity and potential
photooxidative stress is much lower. Hydrilla can survive under very low light
intensities (Bowes et al.. 1977) and this could explain the ability of the younger plants
to increase in dry matter over the study period. Even though carotenoid levels would
be lower as a result of fluridone treatment, so would potential photooxidative stress.
Another explanation could be the rapid growth of the younger plants as compared
to the mature hydrilla plants. The rapid growth could overwhelm the lethal affects
of fluridone, with continued growth before fluridone began to take affect.
Subterranean turion production appeared to be directly related to growth in
terms of dry matter for both young and mature plants, where reduction in growth
also resulted in a reduction of turion number. There was turion formation under

54
long-day conditions by the mature hydrilla plants. However, the number of turions
produced did not increase over time as in the short-day grown plants, which indicated
a breach in long-day extension, or pre-induction before transfer to greenhouse
conditions.
There was a significant (P < 0.05) three-way interaction between weeks after
treatment, photoperiod and fluridone concentration for total chlorophyll, carotenoid
and anthocyanin content. Chlorophyll and carotenoid content of the mature hydrilla
plants was decreased at 5 and 10 ppb fluridone after 2 weeks of treatment in both
long and short-day photoperiodic conditions (Tables 3.5 and 3.6). Chlorophyll and
carotenoid content under long-day conditions was also decreased by 1 ppb fluridone,
but the effect under short days was variable. There was also a general decline in
these pigment levels as a function of weeks after treatment, with greater decreases
observed at higher levels of fluridone. Conversely, anthocyanin content increased
over time and short-day conditions (Table 3.7). The effect of fluridone on
anthocyanin content was highly variable and anthocyanin content appeared to
decrease anthocyanin content at higher fluridone rates.
Fluridone decreased carotenoid levels at concentrations of 5 and 10 ppb after
1 week of exposure as expected, as the mechanism of action of this compound has
been demonstrated to be an inhibition of carotenoid biosynthesis (Bartels and
Watson, 1978). Chlorophyll content also decreased in a similar fashion. Earlier
work by Doong et al.. 1993 demonstrated a ’lag’ in the chlorophyll content of hydrilla
at 5 ppb fluridone following carotenoid decrease but this was not evident from the

55
Table 3.5. The effect of photoperiod and fluridone on the chlorophyll content of
mature hydrilla plants.
Weeks After Treatment
l
Photo- Fluridone
pb)
1
2
3
4
5
6
7
0
673
847
679
695
543
450
489
1
981 *3
529*
322*
398*
170*
364*
—
5
498*
310*
91*
94*
75*
198*
219
10
299*
167*
134*
25*
—
62*
207
0
616
613
527
465
438
269
486
1
552
603
320*
489
278*
254
292
5
534*
357*
207*
225*
46*
52*
128
10
200*
85*
63*
278*
32*
29*
28*
Long Day
Short Day
1 Average hydrilla chlorophyll content at the initiation of the study was 904 ^g/g
fresh wt.
9
LSDo.05 = 58 to separate the effect of photoperiod within week and fluridone
concentration.
a , ,
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s Y test Q 05).

56
Table 3.6. The effect of photoperiod and fluridone on the carotenoid content of
mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
68
74
- /xg/g-fresh weight -
86 92 87
65
92
1
91*3
56*
44*
54*
24*
72
70
5
49*
36*
15*
15*
12*
61
39*
10
26*
21*
23*
3*
—
11*
42*
Short Day
0
67
67
74
84
77
48
106
1
52*
63
44*
76
55*
46
47*
5
54*
50*
25*
44*
9*
12*
26*
10
19*
10*
10*
—
7*
9*
6*
1 Average hydrilla carotenoid content at the initiation of the study was 89 fig/g fresh
wt.
LSD0 05 = 10 to separate the effect of photoperiod within week and fluridone
concentration.
a
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s Y test o.os)-

57
Table 3.7. The effect of photoperiod and fluridone on the anthocyanin content of
mature hydrilla plants.
Photo- Fluridone
Weeks After Treatment1
period2 (ppb)
1
2
3
4
5
6
7
Long Day
0
58
76
- ¿ig/g-fresh weight -
69 76 123
76
113
1
70*3
69
75
128*
68
66
156*
5
52
41*
62
101*
94*
80
—
10
54
53*
86*
141*
—
30*
—
Short Day 0
63
75
73
143
117
*
329
419
1
56
55*
85
131
206*
346
300
5
70
61*
143*
110*
95
81*
216
10
51
*
o
U1
83
109*
94
99*
—
1 Average hydrilla anthocyanin content at the initiation of the study was 63 fxg/g
fresh wt.
9
LSDo.05 = 39 to separate the effect of photoperiod within week and fluridone
concentration.
Values within a week and photoperiod followed by * are significantly different from
the control (Dunnett’s Y test 0 05).

58
present study. A possible reason for this effect could be that high light intensity
caused a greater degree of photooxidative stress and thus, more rapid chlorophyll
breakdown. Chlorophyll content of untreated plants declined throughout the
duration of the study in both photoperiodic regimes. Berg (1977) and Kar and
Choudhuri (1987) reported a loss of chlorophyll content in hydrilla during late
summer and hypothesized that a senescence had occurred. However, a decline in
chlorophyll was also observed under long-day conditions, when senescence should not
have been a factor.
The effect of fluridone on anthocyanin content was highly variable and did not
cause an increase in the level of this pigment, as demonstrated by earlier studies
(Doong et al.. 1993). However, the level at which Doong et al.. reported elevated
anthocyanin content was 50 ppb fluridone, far above the levels tested in this study.
Interestingly, anthocyanin levels increased dramatically under short-day conditions,
with higher fluridone concentrations suppressing the level of this pigment. Berg
(1977) reported an increase in anthocyanin content as hydrilla began to decline
during late summer and this was probably the case for the short-day grown hydrilla
plants. The decline in anthocyanin content from fluridone treatment probably
resulted from tissue deterioration.
There was no significant differences in the ABA content of crown tissue from
mature plants (P > 0.05 - see Appendix G). The average ABA content of these
structures was 25 and 204 pmol/g - fresh weight for crown and turion tissue types,
respectively. There was a significant interaction between weeks after treatment and

59
fluridone concentration for apical stem tissue but not a significant (P >0.05) effect
of photoperiod (Appendix H). Abscisic acid content increased in control and 1 ppb
fluridone treated plants 1 and 2 weeks after study initiation, respectively, while
fluridone at 5 and 10 ppb decreased ABA content (Table 3.8).
ABA content in younger plant tissue was only affected by photoperiod (P <
0.05), not by fluridone concentration and there was not an interaction (Appendix I).
Abscisic acid content in young plant tissue was highest under short-day conditions
when averaged over time and fluridone: 0.53 + 0.07 and 0.71 ±0.05 pmol/g-fresh
weight, long and short days, respectively.
The results of the ABA analyses performed in this study are highly variable
but follow similar findings of other researchers (Van et al.. 1978; Klaine and Ward,
1984; Anderson et al.. 1990 [unpublished]). ABA content was shown to rise soon
after treatment, but the increase was transient. In addition, fluridone reduced ABA
content and turion production in mature plants at 5 and 10 ppb, but this could also
be the indirect consequence of plant death. ABA was higher under short-day length
conditions, which favor turion formation. However, direct evidence linking
turionformation and increased ABA content was not established. Although ABA is
probably involved in some aspect of turion formation and/or development, the exact
mechanism is still unknown.
The results from this study provided further evidence that hydrilla can be
effectively managed by late summer to early fall applications of fluridone, which
prevent turion formation. Mature hydrilla tissue was shown to be as susceptible to

60
Table 3.8. The effect of fluridone concentration on the abscisic acid content of
mature hydrilla apical stem segments, averaged across photoperiodic regime.
Weeks After
Treatment1
0
Fluridone (ppb)
1 5
10
pmol/g-fresh
weight —
1
18
36
13
13
2
39
25
13
15
3
—
18
11
5
4
19
13
11
11
5
—
11
7
—
6
26
19
12
3
7
15
16
10
15
1 Average abscisic acid content at the initiation of the study was 15 pmol/g fresh wt.
9
LSDg.05 = 13 to separate the effect of fluridone concentration within week.

61
fluridone as younger tissue and fluridone concentrations of 5 ppb or higher will
dramatically reduce subterranean turion production. This contradicts the earlier
hypothesis of MacDonald et al. (1993) who proposed that sublethal concentrations
of fluridone could block turion formation. However, the present studies support the
argument that lowering ABA concentration sufficiently to block reproduction would
require a lethal reduction in carotenoids. Therefore, the reduction in turion
formation is probably a coincidental result of plant death. Prudence should be
exercised when applying these results to real-world situations, as factors such as
exposure time and critical concentrations have yet to be evaluated.

CHAPTER IV
THE INFLUENCE OF FLURIDONE, PHOTOPERIOD AND
PLANT GROWTH REGULATORS ON THE GROWTH AND
REPRODUCTION OF MONOECIOUS AND DIOECIOUS
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
Introduction
Hydrilla is a submersed aquatic vascular plant that has become a major
aquatic weed problem in the southeastern U.S. (Haller, 1976; Langeland, 1990). This
species is particularly important in Florida, where it was introduced in the late 1950’s
by the aquarium plant industry (Blackburn et al.. 1969). Since introduction hydrilla
has spread throughout the southeastern United States and California.
There are both monoecious and dioecious biotypes of hydrilla which noth can
be found in the United States. Until recently, each biotype was believed to grow in
distinct regions, but monoecious and dioecious have been reported in a common lake
in North Carolina (Coley et al.. 1993). Dioecious hydrilla exists primarily in those
areas mentioned above, and consists of only the female plant. Monoecious hydrilla
was introduced during the late 1970’s and can now be found in areas of Virginia,
Maryland, Washington D.C., and North Carolina (Stewart et al.. 1984; Coley et al..
1993).
Hydrilla causes problems in many freshwater ecosystems, displacing most
native vegetation. This highly aggressive species exists in large, monotypic stands,
which form a dense mat at the water surface (Haller and Sutton, 1975; Bowes et al..
62

63
1977). Hydrilla’s extensive growth interferes with navigation and flood control, and
severely limits most water recreation activities. Furthermore, hydrilla growth will
alter the flora and fauna of a given area, impacting freshwater fish populations.
Hydrilla possesses several physiological and morphological adaptations
including a low light compensation point, C4-like photosynthetic metabolism, and
specialized vegetative reproduction - [turions] (Bowes et al.. 1977; Van et al.. 1976;
Salvucci and Bowes, 1983). Turions are specialized vegetative structures that
function in persistence and dispersal of many aquatic macrophytes (Sculthorpe, 1967).
In hydrilla, turions may be formed at the tips of rhizomes that penetrate into the
hydrosoil (subterranean turions), or in leaf axils (axillary turions)[Yeo et al.. 1984],
Subterranean turions are slightly larger and function to insure persistence of hydrilla
within a given area, while the smaller axillary turions are thought to function in
dispersal (Haller, 1976; Spencer et al.. 1987; Thullen, 1990).
Hydrilla turion production varies between biotypes, from both a physiological
and morphological standpoint. Dioecious hydrilla forms turions only under short-day
(< 12 hours light) conditions, while monoecious hydrilla appears to be day-neutral
(Klaine and Ward, 1984; Van et ah. 1978; Anderson and Gee, 1986). Monoecious
hydrilla will produce a greater number of smaller turions than dioecious hydrilla
(Spencer and Anderson, 1987; Van, 1989). This size characteristic is true for both
axillary and subterranean turions. Turion formation in the dioecious biotype is
believed to be regulated by phytochrome (Klaine and Ward, 1984; Van et al.. 1978).
This response has been associated with increased levels of abscisic acid (ABA) and

64
exogenous applications of this hormone have resulted in turion formation under non-
photoinductive conditions (Klaine and Ward, 1984; Van et al.. 1978). However,
delineation between axillary and subterranean turion formation was not addressed.
Furthermore, the effect of ABA on monoecious hydrilla was not studied.
ABA has been associated with a number of plant processes including water
relations, growth and development, and seed dormancy (Neill et al.. 1986; Zeevaart,
J.A.D. et al.. 1988; Saab et al.. 1990). ABA has also been shown to cause
heterophylly in some aquatic plants (Kane and Albert, 1987; Goliber and Feldman,
1989).
Turion formation is similar to tuber formation in terrestrial species, such as
potato (Solanum tuberosum! and Jerusalem artichoke (Helianthus tuberosum!.
Tuber formation in potato involves changes in abscisic and gibberellic acid content,
with an increase in ABA during tuberization and a subsequent decline in gibberellic
acid levels (Krauss, 1985). Similar to turion formation in hydrilla, ABA will promote
tuber formation under non-photoinductive conditions while gibberellic acid will
inhibit tuberization under inductive conditions. However, most researchers feel there
exists a critical ratio between these two hormones, not simply an ’on-off
phenomenon (Menzel, 1980; Vreugdenhill and Struik, 1989). This interactive effect
has not been evaluated on either biotype of hydrilla.
Plant growth regulator experiments are usually performed under laboratory
conditions to limit variability. Axenic culture is sometimes employed to study various
plant processes under highly controlled conditions. Axenic culture has proven to be

65
a valuable technique for studying the physiology of aquatic plants (Kane and Albert,
1987). In addition, axenic culture permits plant growth under heterotrophic
conditions. This allows for the potential use of a compound that influences
photosynthesis and other autotrophic processes. Fluridone is often used to regulate
ABA biosynthesis, but is lethal under autotrophic conditions.
Monoecious and dioecious hydrilla biotypes differ in the process of turion
formation but little is known regarding the processes involved. Therefore, the
objectives of these studies were to evaluate the growth and reproductive development
of monoecious and dioecious hydrilla in axenic culture to 1) long and short-day
photoperiod, 2) fluridone under long-day conditions, and 3) exogenous abscisic acid
and gibberellic acid applications.
Materials and Methods
Axenic plant stock material was obtained from Dr. Michael E. Kane,
Environmental Horticulture Dept., University of Florida. From this material, stock
cultures of both biotypes were established from 3 to 5 cm long apical stem segments.
Stock cultures were maintained in aluminum foil capped 0.94 L glass vessels filled
with 150 cm quartz silica sand (as a rooting medium) + 500 ml of liquid growth
medium. Basal medium consisted of half-strength Murashige and Skoog mineral
salts1 and 0.75% (w/v) sucrose, adjusted to pH 6.5 with 1.0 N KOH prior to
1 Murashige and Skoog Basal Salt Mixture (1962); Sigma Chemical Co.,
St. Louis, MO 63178.

66
autoclaving. Growth medium was autoclaved at 22 psi and 250 F for 20 minutes.
Deionized water was added to the vessels containing sand prior to autoclaving to
prevent breakage. After autoclaving, medium was added to the vessels and plant
material transferred. All transfers were performed under sterile conditions. Stock
cultures were maintained on a monthly basis and sterility was checked by indexing,
according to the procedures of Kane (unpublished) and Knauss (1976). Stock
cultures were maintained in an environmentally controlled growth chamber set at the
following conditions: 11 + 1 hr light/6 + 6 hr dark photoperiod (lhr light
interruption at the middle of the dark cycle to maintain long-day conditions); 150
9 1
/xmol'm sec (PPFD); 30 ± 5 C. The light source consisted of fluorescent and
i
incandescent bulbs.
Experimental plant material was grown in 38 x 300 mm glass tubes2 filled with
50 cm3 quartz-silica sand and 175 ml growth medium. Plant material was obtained
from stock plants. Establishment of hydrilla was accomplished by transferring a 3 to
5 cm long (0.005 g dry wt) apical stem section into each tube and forcing the basal
end into the sand 1 to 2 cm deep to enhance rooting. Each tube was considered a
replication. Experimental cultures were maintained in an environmentally controlled
growth chamber set at the following conditions: 9 + 1 h light/7 + 7 h dark
photoperiod (1 h light interruption at the middle of the dark cycle to maintain long-
day conditions); 600 ¿imol m'^sec'1 (PPFD); 30 ± 5 C mean light temperature and
2 Bélico Glass, Inc., Vineland, N.J. 08360.

67
25 ± 5 C mean dark temperature. Short-day grown plants were cultured in a
chamber maintained at: 10 h light/14 h dark photoperiod -- 200 /zmol m'^sec'1
(PPFD) and 25 ± 5 C mean light temperature and 20 + 5 C mean dark
temperature. Lighting in both chambers consisted of a mixture of incandescent and
fluorescent bulbs.
Photoperiodic response. Monoecious and dioecious hydrilla biotypes were
grown under long or short-day conditions for 8 weeks with a minimum of 6
replications. The experiment was repeated once. After 8 weeks, the plants were
harvested and subterranean and axillary turion number was determined.
Subterranean turion development was determined by the formation a rhizome
penetrating the sand substrate. Axillary turion formation was distinguished by a
swelling of shoot tissue, compact leaf development and the lack of prominent
internodes. The plants were then dried at 60 C for 48 h and dry weights recorded.
Effect of fluridone. A preliminary experiment was conducted to determine
if hydrilla could be grown heterotrophically using axenic techniques with
supplemental sucrose. Monoecious and dioecious hydrilla biotypes were grown under
long-day conditions for 12 weeks. The experiment was repeated once with 4
replications. Fluridone was prepared as a concentrated stock in water from technical
grade material3 and filter-sterilized (0.22 ¿un)4 prior to addition to the tubes.
Fluridone was added at concentrations of 0, 0.5, 1.0, 5.0, or 10 ppb at the time of
3 DowElanco, Inc., Indianapolis, IN 46268.
4 MSI, Fisher Scientific, Pittsburgh, PA. 15219.

68
establishment. After 12 weeks, the plants were harvested and the number of axillary
turions produced was determined. The plants were then dried at 60 C for 48 h and
dry weights recorded.
Effect of exogenous abscisic acid and gibberellic acid. Monoecious and
dioecious hydrilla biotypes were grown under long-day conditions for a total of 8
weeks. Biotypes were run as separate experiments and each experiment conducted
twice with a minimum of three replications. After 4 weeks of growth the growth
medium was exchanged and hormone treatments were applied. Abscisic acid (99%
mixed isomers)5 and gibberellic acid (90% GA3)5 were prepared as concentrated
stocks in water and filtered-sterilized prior to treatment. Small amounts of ethanol
was used to facilitate dilution and the appropriate ethanol concentration was added
to the control plants. Abscisic acid was applied at 0, 0.1, 1.0, or 10 ¿íM and
gibberellic acid at 0, 5, 50, or 500 ¿iM to establish a matrix of 16 total treatments.
Four weeks after treatment, the plants were harvested and the number of axillary
turions, subterranean turions, and flowers produced were counted. Axillary turions
were subsampled and the plants dried at 60 C for 48 h and dry weights recorded. No
attempt was made to distinguish between male and female flowers of the monoecious
biotype.
Statistical analysis. Data were initially analyzed by analysis of variance to test
for treatment effects (biotype, photoperiod, fluridone concentration, ABA or GA
content) and interactions. Treatment means were separated with Fisher’s
5 Sigma Chemical Co., St. Louis, MO 63178.

69
protected LSD test or Dunnett’s ’T test at the 0.05 level of significance. Means are
presented with standard errors.
Results and Discussion
Although plant material was obtained from axenic stocks, a certain degree of
contamination, bacteria and/or fungal, occurred in each of the experiments. Because
of the relatively short duration of the experiments, this contamination did not cause
plant death. There was no observed trend for any treatment to be more
contaminated than the others. Therefore, it is not the author’s intent to claim axenic
culture was used or maintained, and conclusions drawn from these studies are not
intended to reflect that premise.
There was a significant (P < 0.05) interaction between biotype and
photoperiod for subterranean and axillary turion production and dry weight. Both
biotypes produced greater biomass under long-day conditions (Table 4.1). Dioecious
hydrilla grown under short days, produced less than 70% of the biomass produced
by long-day grown plants. There was a 20% difference for monoecious hydrilla.
Axillary turion production was not observed for dioecious hydrilla, while monoecious
hydrilla produced these structures under both photoperiods (Table 4.2). Dioecious
hydrilla produced subterranean turions only under short-day conditions, while the
monoecious hydrilla produced subterranean turions under both photoperiods (Table
4.3).
In this study, photoperiod had a tremendous effect on plant growth and
subterranean turion formation for dioecious hydrilla. Dioecious hydrilla has been

70
Table 4.1. The. effect of photoperiod on the growth of 8 week old dioecious and
monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
— g dry/plant
Long-Day
0.92 ± 0.091
1.21 ±0.05
Short-Day
0.28 ±0.04
0.95 ±0.05
1 Means followed by standard errors.

71
Table 4.2. The effect of photoperiod on the axillary turion production of 8 week old
dioecious and monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
turions/plant -
Long-Day
0
0.88+0.401
Short-Day
0
2.71 ±1.41
1 Means followed by standard errors.

72
Table 4.3. The effect of photoperiod on the subterranean turion production of 8
week old dioecious and monoecious hydrilla.
Photoperiod
Dioecious
Monoecious
turions/plant -
Long-Day
0
0.75 ± 0.491
Short-Day
1.29 ±0.42
1.00 ±0.53
1 Means followed by standard errors.

73
shown to undergo ’annual decline’ (Berg, 1977), which is characterized by a loss of
biomass prior to the onset of winter. In these experiments short days may have
caused this phenomenon in dioecious hydrilla, thus limiting biomass production.
Several species of plants cease vegetative growth during the onset of reproduction
(Gardener et al„ 1985). However, this does not seem to be the case with
monoecious hydrilla. Another possible explanation could be the lower light intensity
in the short-day chamber. Monoecious hydrilla also displayed significantly less
biomass in this environment, but to a much lesser extent than dioecious hydrilla.
Dioecious hydrilla has been reported to be a short-day plant in terms of turion
formation, with a critical photoperiod of 11 to 12 hours (Van et al„ 1978; Miller et
ah, 1993). This characteristic response was also observed in these studies. However,
axillary turion development was not observed under either day length. Haller (1976)
and Miller et al. (1993) reported axillary turion formation occurred on floating
hydrilla plants. Plants used in these experiments were intentionally rooted which
possibly precluded axillary turion formation. Monoecious hydrilla produced axillary
and subterranean turions under both photoperiods. This contrasts with earlier
research, which showed no turion formation under 14 to 16 hour daylengths or photo¬
interruption of long nights (Anderson and Spencer, 1986; Spencer and Anderson,
1987).
Monoecious and dioecious hydrilla biotypes were adversely affected by
increasing concentrations of fluridone (Table 4.4). Ten ppb fluridone caused 91 and
82 % reduction in shoot biomass of dioecious and monoecious hydrilla, respectively.

Table 4.4. The effect of fluridone on the growth of dioecious and monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.
Fluridone
Dioecious
Monoecious
(PPb)
g dry/plant
0
0.56 + 0.181
0.63 ±0.26
0.5
0.45±0.16
0.71 ±0.08
1.0
1.03 ±0.18
0.60±0.08
5.0
0.26±0.10
0.32±0.10
10
0.05±0.01*2
0.11±0.04*
LSDq.05
0.383
1 Means followed by standard errors.
Values within a given biotype followed by * are significantly different from the
control (Dunnett’s Y test0 0s)-
a
To separate the effect of photoperiod within a given level of fluridone.

75
One ppb fluridone caused a 46 % increase in dioecious shoot biomass. Fluridone
decreased turion production at higher concentrations (5 and 10 ppb), while
stimulating turion production at 0.5 ppb in monoecious hydrilla (Table 4.5). This
observation is similar to results reported from earlier field experiments with
dioecious hydrilla (MacDonald et ah. 1993). This indicates that these biotypes
behave similarly to fluridone in response to turion formation.
Fluridone is a very effective herbicide, inhibiting carotenoid biosynthesis and
causing subsequent photooxidation of unprotected chlorophyll molecules (Bartels and
Watson, 1978; Devlin et al.. 1978). In this study, plant tissue became whitish in color,
indicating pigment loss. Monoecious hydrilla appeared to ’recover’ as indicated by
green coloration, but still showed a decrease in biomass over the course of the study.
The lethal effect of fluridone has been demonstrated to be an inhibition of
photosynthesis and plant death due to starvation. However, plants grown with
sucrose amendments should have been able to survive fluridone treatment. If
hydrilla could have been grown heterotrophically then fluridone could have been
used to regulate ABA formation without the lethal effects from reduced
photosynthesis. This could have allowed a determination of whether fluridone acts
as a growth regulator at low concentrations or a herbicide at higher concentrations.
Hydrilla could not be grown heterotrophically and therefore separation of the growth
regulator and herbicidal effects of fluridone could not be ascertained.
In this experiment, fluridone decreased the growth of both monoecious and
dioecious hydrilla biotypes, with monoecious being affected to a lesser degree than

76
Table 4.5. The effect of fluridone on the axillary turion production of monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.
Fluridone
Turions/Plant
(PPb)
0
2.010.71
0.5
5.0+1.I*2
1.0
2.711.7
5.0
0.310.3
10
0*
1 Means followed by standard errors.
2 Values followed by * are significantly different from the control (Dunnett’s ’t’
testaos).

77
dioecious hydrilla. Although the goal of fluridone treatment in this experiment was
to lower ABA levels thus precluding turion formation, it is possible that lowered
ABA may be causing the reduction in growth. Studies with ABA-deficient mutants
have demonstrated lower growth rates, epinasty and wiltiness associated with these
types of mutations (Koornneef et al„ 1982).
Fluridone at 5 and 10 ppb caused a deep purple coloration in dioecious
hydrilla. Fluridone at high rates has been shown to increase the anthocyanin content
of hydrilla but only in mature tissue (Doong et al.. 1993). This phenomenon was
thought to be in response to oxidative stress due to the loss of carotenoids.
Anthocyanin production is also associated with high levels of carbohydrate (Creasy,
1968), present in mature tissue, and which was also present in this study.
There was not a significant (P > 0.05) effect of experiment for exogenous
applications of abscisic and gibberellic acid, therefore experiments for each biotype
were pooled. In dioecious hydrilla, exogenous applications of ABA alone induced
axillary turion formation at all concentrations (Table 4.6). The greatest number of
turions were observed following treatments of 0.1 and 1.0 /¿M ABA. The effect from
ABA was reversed by applications of GA, but not completely. ABA also stimulated
axillary turion production in monoecious hydrilla, but only at concentrations of 1.0
and 10 ¿xM (Table 4.7). Similarly, GA at 50 and 500 ¿¿M completely reversed this
effect at 1.0 /xM ABA.
Total subterranean turion weight per plant reflected the same trend observed
from turion number per plant (Tables 4.8 and 4.9). The size of dioecious turions
were always larger than monoecious hydrilla regardless of treatment (0.005 and 0.002

78
Table 4,6. Axillary turion production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (¿xM)1
0
Gibberellic Acid (/xM)1
5 50
500
-- turions/plant
0
0
0 0
0
0.1
15.6±5.12
6.43±2.8 5.0+2.6
0
1.0
27.0+6.2
0.9±0.9 17.7±9.4
4.6±3.2
10
11.4 ±4.0
7.6±4.3 5.4+2.6
4.4±1.3
1 LSD0 os = 10.6 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.

Table 4,7. Axillary turion production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (¿1M)1
0
Gibberellic Acid (^M)1
5 50
500
- turions/plant
0
0
0
0
0
0.1
0
0
0
0
1.0
42.6±14.72
9.3 ±6.1
0
0
10
52.3 ±17.1
35.6 + 10.6
22.4 + 10.2
4.1 ±3.1
1 LSD0 05 = 4.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.

80
Table 4.8. Axillary turion weight per plant by 8 week old, long day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic Gibberellic Acid (¿iM)1
Acid (mM)1 0 5 50 500
mg dry/plant
0
0
0
0
0
0.1
74±312
33 ±15
20±12
0
1.0
128 ±45
3±3
65 ±40
5±3
10
52 ±22
52±3
31 ± 15
15 ±8
1 LSD0 os = 0.06 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.

81
Table 4.9. Axillary turion weight per plant by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic
Acid (¿xM)1
0
0.1
1.0
10
Gibberellic Acid (/xM)1
5 50 500
g dry/plant
0
0
0
0 0 0
0.231+0.0762 0.020+0.013 0
0
0
0.146+0.052 0.057+0.025 0.042+0.03 0.006 + 0.005
1 LSD0 05 - 0 .07 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.

82
g'1 dry wt turion for dioecious and monoecious hydrilla, respectively). The
percentage of total plant weight represented by turions also reflected the same
response as described for turion number (Tables 4.10 and 4.11). ' nterestingly,
monoecious hydrilla appears to partition a greater percentage of biomass for
reproduction than dioecious hydrilla, producing a greater number of smaller turions.
The effect of ABA and GA on the shoot dry weight of both hydrilla biotypes
was different than the effects on turion formation (Tables 4.12 and 4.13). Both ABA
and gibberellic acid inhibited shoot growth of dioecious hydrilla with GA being the
more inhibitory compound. At the highest concentration of both compounds, ABA
ameliorated the activity of GA. Monoecious hydrilla was inhibited equally by ABA
and GA in terms of shoot growth. However, these two compounds appeared to act
additively to inhibit shoot growth. Although a reduction in shoot growth was
observed on a dry weight basis, the plants treated with GA elongated to the top of
the tube. It is probable that the reduction in dry weight was caused by decreased
leaf production by the plants.
Dioecious hydrilla did not produce subterranean turions or flowers (data not
shown). Monoecious hydrilla produced an average of 5 subterranean turions per
tube only in the absence of hormone applications (untreated control). In addition,
monoecious hydrilla produced an average of 44 flowers per tube the addition of
gibberellic acid at 50 ¿*M. Floral production has been reported to be influenced by
nitrogen and phosphorus (Pietrse et al.. 1984). Flowering has been associated with
short day conditions in dioecious hydrilla, suggesting turion formation and flower

83
Table 4.10. Percent weight of axillary turions produced by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic
Acid (¿íM)1 0
0
0
0.1
12.0±6.22
1.0
22.5 ±8.1
10
0.65 ±0.10
Gibberellic Acid (/xM)1
5 50 500
% of total dry weight
0 0 0
0.8 ±0.09 0.72 ±0.08 0
0.41 ±0.07 0.58±0.14 0.42±0.12
0.62 ±0.13 0.44 ±0.10 0.50+0.12
1 LSD0 os - 8 .95 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.

84
Table 4.11. Percent weight of axillary turions produced by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.
Abscisic Gibberellic Acid (/¿MJ1
Acid (/iM)1 0 5 50 500
% of total dry weight
0
0
0
0
0
0.1
0
0
0
0
1.0
31.2+9.32
2.5 ±1.7
0
0
10
29.0±9.6
13.3+3.9
8.7±4.8
1.2+0.8
1 LSDq q5 = 9.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.

Table 4.12. Shoot biomass production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic
Acid (/xM)1
0
Gibberellic Acid (¿xM)1
5 50
500
g dry/plant
0
0.80+0.042
0.73 ±0.15
0.66±0.10
0.35 ±0.14
0.1
0.58±0.11
0.80+0.09
0.72 ±0.08
0.50 ±0.04
1.0
0.69+0.13
0.41 ±0.07
0.58±0.14
0.42 ±0.12
10
0.65±0.10
0.62±0.13
0.44 ±0.11
0.50 ±0.12
1 LSD0 05 - 0 .08 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
2 Means followed by standard errors.

86
Table 4.13. Shoot biomass production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.
Abscisic Gibberellic Acid (¿1M)1
Acid (/iM)1 0 5 50 500
g dry/plant
0
0.68±0.092
0.53 ±0.12
0.72 ±0.07
0.49 ±0.07
0.1
0.42 ±0.03
0.58 ±0.07
0.54±0.10
0.34 ±0.06
1.0
0.58 ±0.11
0.47±0.12
0.42 ±0.11
0.31 ±0.05
10
0.46 ±0.05
0.37 ±0.05
0.33 ±0.06
0.32 ±0.06
1 LSD0 os = 0.23 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.
Means followed by standard errors.

87
production are linked to a common physiological process. However, floral
stimulation by exogenous gibberellic acid, implied a different, separate mechanism
was involved in this process.
Several researchers have demonstrated the effect of exogenous ABA
applications on turion formation in dioecious hydrilla but little work has been done
with the monoecious biotype (Klaine and Ward, 1984; Van et al„ 1978). Dioecious
hydrilla appeared to be more sensitive to ABA applications than monoecious hydrilla,
as evidenced by turion formation at 0.1 /¿M and reduced sensitivity to GA
applications. ABA has been shown to induce tuber formation in potato and this
effect can be reversed by application of GA (Vreugdenhill and Struik, 1989). The
increased sensitivity of the dioecious biotype to exogenously applied ABA could be
related to photoperiodic responses, whereby applications of ABA could induce a
cascade of reactions that lead to turion formation. However, this should have
resulted in subterranean turion formation, not in the production of axillary turions.
Interestingly, monoecious hydrilla produced subterranean turions without
treatment, similar to those plants in the photoperiod experiment. If ABA was
directly responsible for the formation of these structures, then exogenous applications
should have induced this process. This suggested an indirect role of ABA in turion
development, with the formation of the rhizome being the initial step and not related
to changes in ABA. ABA could be produced after rhizome differentiation to
maintain dormancy and induce swelling of the rhizome tip. In a normally developing
hydrilla plant, the amount of ABA produced during subterranean turion development

88
is sufficiently high that ABA could begin to accumulate in other tissues. These
tissues could be an active meristem, such as the tips of stems or buds in the leaf axils.
As ABA begins to accumulate, the tissue begins importing carbohydrate, forming a
turion. ABA has been shown to regulate assimilate flow in plants (Porter, 1981) and
could be acting to insure proper carbohydrate levels of the turion.
However, there appears to be critical ratio between ABA and GA, as
evidenced by turion size. Turions produced under any influence of GA, were
extremely small and probably would not have been able to regenerate a new plant.
Therefore, ABA must accumulate in a limited number of sites, ensuring proper
development of the propagule. This could be regulated by GA. Several researchers
feel there exists a critical ratio of ABA to GA at all times within the plant, similar
to the roles of auxin and ethylene (Menzel, 1980; Vreugdenhill and Struik, 1989).
Therefore, the effect of exogenous ABA is probably not direct, but rather alters the
ratio of ABA:GA. The relatively lower GA level induces the desired phenomenon
(personal communication, Dr. Anwar Khan, Cornell University).
Collectively, these studies provided evidence that hydrilla cultured under
controlled conditions responds similarly to hydrilla grown under field conditions. The
exogenous applications of hormones indicated that ABA alone does not control
hydrilla turion formation but rather a critical ABA:GA balance may be the key to
the regulation of this phenological process. These hormones also appear to regulate
floral production in monoecious hydrilla, and subterranean turion formation.
However, the role of ABA and GA in these processes warrants further research.

CHAPTER V
SUMMARY AND CONCLUSIONS
These studies provided knowledge about the growth and turion formation
process in hydrilla which is a submersed aquatic plant found throughout Florida,
where it causes severe problems in infested areas. Insight into the turion formation
process of this species could provide a basis for improving management programs.
The abscisic acid content of hydrilla tissue was determined by enzyme-linked
immunoassay. Accurate quantification could be obtained from plant dilutions of 10
to 20 mg dry weight hydrilla tissue containing 0.02 to 5.0 pmol of abscisic acid.
Although an effective solid-phase purification and concentration procedure was
developed, no purification of the hydrilla extracts was necessary prior to
quantification of abscisic acid with ELISA.
Short days promoted subterranean turion formation but this effect was
reduced by long days and 5 and 10 ppb fluridone. Fluridone caused a significant
reduction in chlorophyll and carotenoid levels but the effect on anthocyanin content
was variable. Short days caused elevated anthocyanins and this effect was diminished
by fluridone. Fluridone reduced the abscisic acid content of mature plant apical
stems and abscisic acid content was greater under short days in younger plants.
These studies provided further evidence that fluridone can be used in a fall herbicide
treatment to reduce turion production but, the ability of fluridone to inhibit turion
89

90
production appears to be a coincidental result of plant death.
The growth of monoecious and dioecious hydrilla was reduced under short-day
conditions. Monoecious hydrilla formed turions under long and short day conditions,
while dioecious hydrilla formed turions only under short-day conditions. Fluridone
reduced the growth of both biotypes and reduced turion formation in monoecious
hydrilla. Exogenously applied abscisic acid at 0.1 /¿M or higher induced turion
formation of dioecious hydrilla under long day conditions, but this could be partially
reversed by exogenously applied gibberellic acid. Abscisic acid also induced turion
formation in monoecious hydrilla, but concentrations >.1.0 ¿iM were necessary to
achieve results similar to those for dioecious hydrilla. This study indicated abscisic
acid alone does not control hydrilla turion formation, but rather a balance between
abscisic acid and gibberellic acid is the key to regulation of this phenological process.

APPENDIX A
ELISA PLATE COATING PROCEDURE
1. Dissolve 10 mg of rabbit anti-mouse IgG antibodyla in 40.5 mis of
sodium bicarbonateA buffer and wait 5 minutes. Dispense 200 ¿iL into
each well of two - 96 well microtiter plates2*.
2. Cover with plastic wrap or aluminum foil and incubate at 4 C for 24
hours.
3. After 24 hours, decant the solution in each well and wash each well
with 200 /¿L of wash solution6. Repeat this step for a total of 3
washes, decanting each wash at a time. After the last wash dry by
inverting on an absorbent paper toweling or large Kimwipes .
4. Dissolve 2 mg of monoclonal ABA antibody3 into 40.5 mis of sodium
bicarbonate bufferA and wait 3-5 minutes. Dispense 200 /¿L in each
well of the above plates. Incubate for 36 to 48 hours at 4 C.
5. After incubation, decant the solution and repeat the wash and drying
procedure in step 3.
6. Dissolve 400 mg of rabbit serum albuminlb in 41 mis of TBS bufferc
and add 200 ¡xL to each well. Cover (keep dark) and incubate for 1
hour at room temperature.
7. Decant the unbound albumin and wash the plate again, repeating the
wash procedure in step 3. Dry the plate and it is ready for use.
NOTE: The plate must be used within 30 minutes of this step.
1 Sigma Chemical, St. Louis, MO 63178.
a Catalog # M-9637 b Catalog # A-0639
2 Fisher Scientific, Pittsburgh, PA 15219.
a Catalog # 08-757-23 b Catalog # 06-666-IB
3 Phytodetekâ„¢, Idetek, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089. Catalog
No. 8015.
91

APPENDIX B
ELISA SAMPLE QUANTIFICATION
1. Mix tracer solution by reconstituting 4 vials of tracer4, each with 1 ml
of D.I. water. Mix gently and wait 5 minutes. Transfer the contents
of the vials to 17 mis of tracer bufferE and mix. Be sure to rinse the
tracer vials with the buffer solution to remove all tracer.
2. Add 100 nL of sample or standard to the wells on the plate.
3. Next add 100 ¿iL of tracer solution to each well and mix gently by
tapping the sides of the plate. Cover and incubate for 3 hours at 4 C.
4. Prior to the end of the incubation period (5 - 10 minutes), dissolve 8
pNPP5 tablets in 41 mis of substrate bufferD (1 tablet/5 mis buffer).
5. After incubation, decant the sample or standard and repeat the wash
procedure in step 3. Dry the plate and add 200 /iL of substrate buffer
solution to each well. Incubate the plate for 45 to 60 minutes at 37 C.
6. After 45 minutes, check the absorbance of the zero ABA standard6 at
405 nm. The absorbance for the zero (TBS alone) should be just over
1.0 (actually from 1.0 to 1.2). If the absorbance is lower, allow the
plate to incubate for a while longer.
7. After the above criteria is met, add 50 yiL of 1 N KOH to each well to
stop the reaction. DO NOT DECANT THIS SOLUTION. Wait 5
minutes, measure and record the absorbances.
Phytodetek, Idetek, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089 Catalog
No. 8019.
Sigma Chemical, St. Louis, MO 63178. Catalog # 104-105.
Hyperion Micro-Reader, Model 4025. Hyperion, Miami, FL. 33186.
92

APPENDIX C
ELISA STOCK SOLUTIONS AND STANDARDS
1. Dissolve 0.0264 g of abscisic acid into 100 ml of HPLC grade methanol
to make a 1 mM ABA solution.
2. Add 100 /xL of ImM ABA to 100 ml methanol to make a 1 ¿iM ABA
solution.
3. Add 100 nL of 1 ¿tM ABA to 100 ml methanol to make a 1 nM ABA
solution.
4. Quantification is based on pmol per 0.1 ml, therefore, a 1 nM ABA
solution contains 100 pM of ABA per 0.1 ml. Base further dilutions
on this premise and make several standards (0.02, 0.05, 0.1, 0.2, 0.5,
1.0, 2.0, 5.0 and 10 pM/0.1 ml). Save all dilutions.
5. Add 1 ml of standard prepared in methanol (step 4) into a scintillation
vial and evaporate the solvent. Reconstitute with 1 ml of TBS buffer
and vortex for 30 seconds - making the standard for the plate.
NOTE: Make all dilutions using volumetric flasks, bring all solutions up to
volume.
93

APPENDIX D
ELISA BUFFERS AND SOLUTIONS
NOTE: Use only distilled, deionized water when making solutions.
A. Sodium bicarbonate (50 mM), pH 9.6.
B. Wash Buffer, pH 7.0
- 0.85% NaCl
- 0.05% TWEEN 20
- 0.1% Sodium Azide
C. Tris-Buffered-Saline (TBS buffer)
- 100 mM Tris-HCl stock (pH 8.5)
- 100 mM NaCl
- 1 mM MgCl2
- 0.1% Sodium Azide
Adjust to pH 7.5.
D. Substrate Buffer (pH 9.6)
- 9.6% diethanolamine (98%)
- 0.5 mM MgCl2
E. Tracer Buffer - TBS buffer (step C) + 0.1 % gelatin.
94

APPENDIX E
ELISA STANDARD CURVE AND SAMPLE ABA DETERMINATION
1. Take the average of the zero ABA (Bo term) and the 100 pM (NSB)
replications. The Bo should range from 1.0 to 1.3 while the NSB will
be from 0.07 to 0.115 in absorbance values.
2. Use the following formula: ((standard - NSB) / (Bo - NSB)) * 100, to
determine % binding for the standards.
3. Calculate the logit of % binding term using the following formula: LN
(%binding/(100 - %binding)).
4. Using the logit term generated above as the independent variable and
the natural log of the ABA standard (concentration added) as the
dependent variable, generate a regression equation for the standards.
5. Substitute the logit term for x and solve, generating the natural log of
the predicted ABA concentration. Take the anti-nlog of the above
value (e*) to produce the predicted concentration.
7. To obtain sample ABA content, substitute the sample absorbance for
the standard absorbance in step 2. Perform step 3 and skip to step 5,
entering the logit value into the standard curve regression equation
and take the anti-nlog to obtain sample ABA content.
8. The above value generated is the amount of ABA per the 100 /xL,
therefore divide by 0.1 and multiply by the dilution factor used to
obtain sample ABA content.
95

APPENDIX F
ABSCISIC ACID COLUMN EXTRACTION PROCEDURE
1. extract sample with 10 mis of 100% methanol in glass homogenizer,
filter under suction.
2. dilute filtered extract to 20% MeOH with acidic water (1% formic
acid).
3. pass through Cj8 column, ABA and polar compounds will be retained
on the column, water soluble compounds will elute off, discard eluant.
4. elute column with 15 mis of 40% MeOH (unacidified), retain eluant;
ABA will elute off but most polar compounds will remain on the
column.
5. dilute the above eluant to 10-20% MeOH with acidic water (50-60 mis)
and pass through another C18 column. ABA will again be retained on
the column.
6. elute with 10 mis of 100% MeOH or 100% acetone, collect eluant for
quantification.
7. if large quantities of samples are to be extracted, the second column
used in steps 5 & 6 can also be used as the initial column in step 3.
96

APPENDIX G
THE EFFECT OF PHOTOPERIOD AND FLURIDONE ON THE ABSCISIC
ACID CONTENT OF MATURE HYDRILLA ROOT CROWNS.
Weeks After Treatment
Photo- Fluridone
period (ppb)
1
2
3
4
5
6
7
- pmol g'
1 fresh weight
Long Day
0
16
95
13
29
33
20
19
1
15
29
21
17
16
25
19
5
24
16
23
—
18
30
10
10
17
28
29
17
19
22
14
Short Day
0
24
27
13
30
44
23
16
1
123
46
42
21
22
25
—
5
36
14
20
24
25
19
11
10
2
42
27
10
23
12
9
97

APPENDIX H
THE EFFECT OF PHOTOPERIOD AND FLURIDONE ON THE ABSCISIC
ACID CONTENT OF MATURE HYDRILLA APICAL STEM SEGMENTS.
Photo- Fluridone
Weeks After Treatment
period (ppb)
1
2
3
4
5
6
7
Long Day
0
11
47
- pmol g'
11
1 fresh
12
weight
1
14
10
1
42
28
13
8
8
16
12
5
14
11
9
8
7
—
12
10
14
19
1
11
—
3
—
Short Day
0
25
31
4
26
5
38
20
1
30
23
22
18
14
21
21
5
12
15
12
15
—
12
10
10
11
11
9
—
—
4
15
98

APPENDIX I
THE EFFECT OF PHOTOPERIOD AND FLURIDONE ON THE ABSCISIC
ACID CONTENT OF YOUNG HYDRILLA.
Photo- Fluridone
period (ppb)
Weeks After Treatment
2
4
6
7
Long Day
0
0.30
0.62
—
0.76
1
0.16
0.32
0.96
0.58
5
0.87
0.27
—
—
10
0.80
0.44
0.82
0.78
Short Day
0
1.15
0.52
1.70
0.62
1
0.77
0.26
1.28
0.29
5
0.67
0.41
0.65
1.14
10
1.02
0.41
—
—
99

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BIOGRAPHICAL SKETCH
Gregory E. MacDonald was born on October 14, 1963, in Geneva, New York.
During his youth he spent many hours working his relatives’ farms, milking cows and
repairing farm machinery. He graduated from Geneva High School in 1981 and
received an Associate of Applied Science in agricultural engineering from Alfred
State University. In May of 1986, he received a Bachelor of Science from Cornell
University in vegetable crop production.
In April of 1987, he enrolled at the University of Florida and was awarded a
Master of Science in agronomy in 1991. He continued his career at the University
of Florida in 1991, to pursue a Doctor of Philosophy under the supervision of Dr.
Donn Shilling. He is active in the Southern Weed Science Society, Weed Science
Society of America, Florida Weed Science Society, and Aquatic Plant Management
Society.
On October 30, 1993, he married Miss Michelina Carter and the couple plan
to move to Tifton, Georgia, upon completion of his degree. His hobbies include
bowling, camping, fishing, and antique agricultural machinery restoration.
113

I certify that I have read this study and that in my opinion it conforms to
acceptable standard of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degreexif-Beetox^pf Philosophy,
T)onn G. Shilling, Chair
Associate Professor of
Agronomy
I certify that I have read this study and that in my opinion it conforms to
acceptable standard of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
William T. Haller
Professor of Agronomy
I certify that I have read this study and that in my opinion it conforms to
acceptable standard of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy
Geptge Boíles
rofessor of Botany
I certify that I have read this study and that in my opinion it conforms to
acceptable standard of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
dL £. <7^^.
íchaél E. Kane
Associate Professor of
Horticultural Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standard of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Thomas A. Bewicl
Associate Professor of
Horticultural Science

This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfilment of
the requirements for the degree of Doctor of Philosophy.
August 1994
Dean, College of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 5605