Citation
Enzymatic and physiological studies of low temperature response in vegetative and somaclonal pangola

Material Information

Title:
Enzymatic and physiological studies of low temperature response in vegetative and somaclonal pangola
Creator:
Ackerman, Eugene Balliet, 1956-
Publication Date:
Language:
English
Physical Description:
x, 90 leaves : photos. ; 29 cm.

Subjects

Subjects / Keywords:
Callus ( jstor )
Cells ( jstor )
Cooling ( jstor )
Enzymes ( jstor )
Fluorescence ( jstor )
Freezing ( jstor )
Gels ( jstor )
Low temperature ( jstor )
Plants ( jstor )
Starches ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 75-88).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Eugene Balliet Ackerman.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
001575939 ( ALEPH )
AHJ9786 ( NOTIS )
22913685 ( OCLC )

Downloads

This item has the following downloads:


Full Text














ENZYMATIC AND PHYSIOLOGICAL STUDIES OF LOW TEMPERATURE
RESPONSE IN VEGETATIVE AND SOMACLONAL PANGOLA






By

EUGENE BALLET ACKERMAN


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







1990


ACKNOWLEDGEMENTS



I wish to convey my heartfelt appreciation to my major

professor, Dr. Sherlie H. West. His insight, guidance, and

assistance kindled a creative and exciting environment

throughout my graduate program. Combining his professional

stature with a very caring humanistic approach allows for a

meaningful multilevel relationship.

Sincere appreciation is expressed to the members of my

committee, Drs. Jerry M. Bennett, Robert H. Biggs, Dennis J.

Gray, and Rex L. Smith, for their technical guidance,

constructive criticism, and philosophical opinions.

I am also very grateful to the Institute of Food and

Agricultural Sciences, to the University of Florida, and to

the United States Department of Agriculture for support and

providing a vital professional community. I am indebted to

many of my fellows within this community. Among the scores

that have assisted me stands David Block. His detailed

support in electronics is genuinely appreciated.

My deepest gratitude is felt for the members of my

extended family. Their true commitment of heart and hand

enabled this period of study to become a reality. In the







light of such love, challenges are met and obstacles

overcome.

To my wife Amy, and our children, Jacob, Rachel, and

Taryn, I am truly grateful for their love which is tempered

with perception and patience.


iii














TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ...................... .............. ii

LIST OF TABLES. .................... ............ ...... vi

LIST OF FIGURES... ........ .......................... vii

ABSTRACT............................................. ix

CHAPTERS

I INTRODUCTION.... ............................ 1

II PRODUCTION OF PANGOLA SOMACLONES AND
SELECTION FOR INCREASED TOLERANCE TO FREEZING
AND CHILLING TEMPERATURE..................... 5

Materials and Methods...................... 17
Results and Discussion..................... 28
Conclusions................................ 41

III EVALUATION OF CHILLING TEMPERATURE AND
GIBBERELLIC ACID ON THE EXPRESSION OF
HYDROLYTIC ENZYMES............................ 42

Materials and Methods...................... 51
Results and Discussion..................... 57
Conclusions... .................... ........ 64

IV EVALUATION OF HYDROLYTIC ENZYMES IN FREEZING-
TEMPERATURE-TOLERANT SOMACLONES OF
PANGOLA ................................. ........ 63

Materials and Methods...................... 65
Results and Discussion..................... 66
Conclusions................................ 70

V SUMMARY AND CONCLUSIONS....................... 71







Page

REFERENCES..... ............ ........ ... .............. 75

BIOGRAPHICAL SKETCH.................................. 89













LIST OF TABLES


Table Page

1 Percentage of pangola callus surviving low
temperatures and supporting plant regeneration... 31

2 Percentage of regenerated pangola plants
with less electrolyte leakage than the
source plants .................................. 37

3 Freeze induced electrolyte leakage of selected
regenerated pangola plants and source
plants ........................................... 39

4 Percentage F, remaining in selected regenerated
pangolagrass plants and source plants after
exposure to chilling temperature................. 40

5 Reagents and gel preparation for native starch
PAGE slab gel electrophoresis..................... 53













LIST OF FIGURES


Figure Page

1 Decrease in the rate of fluorescent emission
(FR) from pangola chilled at 100C for
45 minutes. Peak fluorescence (P) is the
maximum fluorescent emission. ................... 18

2 Configuration of LaCroy transient recorder. ..... 26

3 Configuration of LaCroy transient recorder. ..... 27

4 Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 MM 2,4-D. X 7. ................................ 29

5 Pangola callus with embryogenic (e)
and nonembryogenic (n) callus. On MS media
containing 9 AM 2,4-D. X 58 ..................... 30

6 Longitudinal section of pangola callus
with nonembryogenic (n) callus, embryogenic (e)
callus, and young somatic embryo (em). On MS
media containing 2 MM 2,4-D. X 58. ............... 33

7 Longitudinal section of pangola callus
with embryogenic (e) callus supporting a
maturing somatic embryo; scutellum (sc),
coleoptile (co), and shoot apex (s). On MS
media without 2,4-D. X 75. ........................ 34

8 Regenerated pangola plant with plumule (p)
and young root (r). On MS media without 2,4-D.
X 7. ............................................. 35

9 Effect of temperature and GA3 on expression of
starch-degrading-hydrolytic enzymes in
pangola. White bands (bands 1-8) show
endoamylase activity, blue band (band 9)
indicates debranching enzyme activity. F refers
to the bromophenol blue front marker. Micrograms
protein refers to the total protein. ............. 59


vii









10 Effect of temperature and GA3 on production and
expression of starch-degrading-hydrolytic enzymes
in pangola. Zymograms (z) of hydrolytic
activity and coomassie protein stains (c) are
presented for each treatment. Micrograms protein
refers to the total protein. ..................... 61

11 Zymograms of starch-hydrolyzing enzymes isolated
from pangola plants that were exposed to
300C or 100C. White bands (bands 1-8) show
endoamylase activity, blue band (band 9)
indicates debranching enzyme activity. Lanes 1
and 3 represent the vegetatively propagated control
plants. Lanes 2 and 4 represent plants regenerated
from tissue culture. F refers to the bromophenol
blue front marker. ............................... 67

12 Zymograms of starch-hydrolyzing enzymes and total
protein profiles from pangola plants exposed
to 300C or 100C. Lanes 1 and 4 are zymograms. White
bands (bands 1-8) show endoamylase activity,
blue band (band 9) indicates debranching enzyme
activity. Coomassie blue stained proteins are
represented in lane 2 vegetativelyy propagated
control plants) and lanes 3 and 6 (plants
regenerated from tissue culture). ................ 69


viii


Figure


Page








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


ENZYMATIC AND PHYSIOLOGICAL STUDIES OF LOW TEMPERATURE
RESPONSE IN VEGETATIVE AND SOMACLONAL PANGOLA

by

Eugene Balliet Ackerman

May 1990

Chairman: Dr. S. H. West
Major Department: Agronomy

Conventional breeding programs are unable to generate

variation in 'pangola' digitgrass (Diqitaria decumbens

Stent.). Pangola is a highly sterile cultivar which resulted

from hybridization of closely related Diqitaria species.

Pangola is a triploid (2n = 27). A lack of variation causes

growth rates among individual plants to be uniformly reduced

by chilling and freezing temperatures. Shortages of forage

from pangola pastures occur throughout the winter months in

northern portion of central Florida.

The objectives of this research were to produce somatic

tissue cultures of pangola, regenerate plants, and select

variant plants exhibiting chilling and freezing-temperature

tolerance. Evaluation of freezing-temperature resistance was

based on in vitro survival and integrity of the plasmalemma

membrane. Evaluation of chilling-temperature resistance was

based on in vitro survival, integrity of the photosynthetic







electron transport chain, and the number of assimilatory

starch-hydrolyzing enzymes.

Somatic embryogenesis in pangola followed a pattern of

development similar to those described for other grasses.

Freezing or chilling temperature treatment did not

predispose plants that were regenerated from these calli to

be freeze tolerant. A range of freeze damage occurred

between regenerated plants with respect to plasma membrane

damage. A subset of plants selected for a decrease in

freeze-induced plasmalemma damage had no reduction in chill

damage to the photosynthetic electron transport chain.

Chill-induced alterations in assimilatory starch-

hydrolyzing enzymes resulted in the identification of two

chill-sensitive endoamylolytic enzymes. One debranching and

six endoamylolytic enzymes were unaffected by chilling

temperature. The number of hydrolytic enzymes was unaffected

by applying gibberellic acid (GA3) to the plant prior to

chilling temperature exposure.

All of the regenerated plants that were selected for an

increase in freezing tolerance were identical with respect

to the number of starch-hydrolyzing enzymes regardless of

temperature.

Somatic variation in freezing or chilling temperature

response of pangola was evident in vitro. Variant

regenerated plants that sustain less damage from freezing

temperature can be identified by a low-electrolyte leakage.













CHAPTER I
INTRODUCTION



Pastures that are established with 'pangola' digitgrass

(Diqitaria decumbens Stent.) produce an abundance of lush,

high-quality forage. Pangola can be used as a pasture, hay,

or silage crop (Hodges et al., 1975). During the early 1930s

pangola was introduced into the US Gulf Coast areas and

southern California (USDA, 1932). Pangola was registered by

the Crop Science Society of America (registration number 28)

in 1972 (Schank et al., 1972). The establishment of pangola

resulted in pastures that sustain abundant high-quality

yields in the hot summer months and drastically reduced

yields in the cooler months. Pangola is very freeze

susceptable as characterized by the death of grass crowns

(Hodges and Jones, 1958). Winterkilling results from a

combination of frost and freezing temperature.

The livestock industries in Florida, particularly the

beef industry, desire a pasture grass that provides a year-

round supply of abundant forage. Unfortunately, during the

cooler months, pangola production falls short of the

farmers' needs. Farmers are forced to purchase additional

feed stocks after their banked pangola pastures have been







2

depleted. Increasing the cost effectiveness of beef

production in Florida strongly depends on the year-round

production of abundant and inexpensive forage. Extending the

summertime growth characteristics of pangola into the cooler

months would substantially reduce the farm and ranch

investments in livestock feed.

Whole plant growth has been characterized by Burgess

(1985) as a natural process that is regulated by genetic,

biochemical, morphological, and physiological changes in

each cell. Multiplication and differentiation of plant cells

results from selective expression of parts of the genome.

Selective expression of the genome keeps the plant in

balance with its environment.

During the winter months, the most important

environmental factor restricting plant growth is chilling

(120C-00C) and freezing (0C and lower) temperatures

(Christiansen and St. John, 1984). Chilling and freezing

conditions are defined by these temperature ranges. A wide

range of tolerance to low-temperature stress often exists

within crop species due to the genetic diversity generated

by gene recombination during meiosis (Jones, 1985). Sterile

crops such as pangola are lacking in gene recombination and

therefore retain a high degree of genotypic and phenotypic

uniformity. Tissue culture methods have been used to

regenerate plants with increased genetic variation







3

(Scowcroft and Larkin, 1988) and increased tolerance to

unfavorable environments (Tal, 1983).

Many agronomically important plant species of tropical

origin are sensitive to chilling temperatures in the range

of 200C down to 0C. Below some critical temperature,

grasses are sharply restricted in growth (Gusta et al.,

1980). Several plant factors contribute to reduced growth

under chilling-temperature conditions. Membrane damage is a

universal manifestation of chill-temperature damage in

biological systems and is commonly regarded to be the

primary cause of injury (Lyons et al., 1979). Photosynthesis

is one of the first processes affected by chilling

temperatures (Oquist, 1987). Alterations in the light

reactions are evident at various stages in the

photosynthetic electron transport chain. Changes in the dark

reactions (i.e., carbohydrate anabolism) result from

alterations in the activity (Carter et al., 1972) and

synthesis (Jacobsen and Higgins, 1982) of amylolytic

enzymes. Despite having distinct genetical, biochemical, and

physiological factors, the action and interaction of all

these factors contribute to a common result: reduced growth.

Several of the above factors were studied and reported

herein. The objectives of the study reported in Chapter II

were a) to generate somatic cell lines of pangola; b) to

select freezing and chilling resistant cell lines in vitro;

c) to regenerate whole plants from selected cultures; and d)







4

to test regenerated plants for increased tolerance to

freezing and chilling temperature at the organelle and

cellular level.

The studies reported in Chapter III were conducted with

the objectives a) to evaluate the effect of chilling

temperature on the expression of amylolytic enzymes in

pangola, b) to identify amylolytic proteins that may assist

in starch breakdown after exposure to chilling conditions,

and c) to determine the effect GA3 has on the synthesis of

these proteins.

The objectives of the study presented in Chapter IV

were a) to evaluate low-temperature-tolerant pangola

somaclones for variation in the expression of amylolytic

enzymes and b) to select plants that retain amylolytic

proteins which may assist in starch breakdown after exposure

to chilling conditions.












CHAPTER II
PRODUCTION OF PANGOLA SOMACLONES AND SELECTION
FOR INCREASED TOLERANCE TO FREEZING AND CHILLING TEMPERATURE



This study tested the tentative assumption that

somaclonal variation may be obtained in pangola plants that

are regenerated from tissue culture. Hypothetically, somatic

variants may be selected for increased low-temperature

tolerance.

To test this hypothesis, the objectives of this study

were a) to generate somatic cell lines of pangola, b) to

select freezing and chilling resistant cell lines in

culture, c) to regenerate plants from the selected callus,

and d) to test for increased tolerance to freezing and

chilling temperature at the whole plant level.

Pangola establishes excellent summertime pastures but

is an unproductive fall and winter forage crop in Florida.

Chilling temperatures (00C-120C) encountered by pangola

during the cooler seasons reduce growth rates and eventually

stop productive growth (West et al., 1968).

The genetic diversity that permits a fertile plant

species to adapt to temperature extremes is not present in

pangola. Pangola is highly sterile due to irregularities in

both microsporogenesis and macrosporogenesis (Sheth, 1955).






6

Sheth et al. (1956) and Schank et al., (1972) reported that

pangola produced very few viable seed. This genetic crossing

barrier eliminates any genetic variation that is acquired

from independent gene segregation and gene recombination.

An increase in genetic variability, from sterile

plants, may be obtained by subjecting the tissue to somatic

cell culture (Reisch, 1983). The concept of somaclonal

variation as a new source of genetic variability was

presented by Larkin and Scowcroft (1981) and has been

reviewed a number of times (Scowcroft, 1985; Ahloowalia,

1986; Larkin, 1987, Ryan et al., 1987). Variation arising as

a consequence of tissue culture has been reported in the

following forage grasses: ryegrass (Lolium multiflorum L.)

(Ahloowalia, 1983; Torello and Symington, 1984; Skene and

Barlass, 1983), tall fescue (Festuca arundinacea L.)

(Kasperbauer and Eizenga, 1985), guineagrass (Panicum

maximum L.) (Bajaj et al., 1981), and crabgrass (Digitaria

sanquinalis L.) (Elmore et al., 1988). This suggests that

somatic variation in pangola could be used to generate

variation.

A fundamental requirement for obtaining somatic

variation is the ability to support in vitro callus growth

and plant regeneration. Urata and Long (1968) were first to

report the regeneration of grass plants from tissue culture.

Pangolagrass was one of the several tropical grasses that

they regenerated through organogenesis. Recently, using






7

pangolagrass, Marousky and West (1988) reported shoot

development from ovarian explant tissue and somatic embryo

formation from callus.

Until recently, it was thought that the only way to

obtain useful nonchimeral mutants was by regeneration of

somatic embryos (Gamborg et al., 1970). Both organogenic

shoots (Horsch et al., 1985) and somatic embryos (Williams

and Maheswaran, 1986) can initiate from single cells. This

indicates that both modes of regeneration reported in

pangola may give rise to the more useful nonchimeral

regenerated plants.

Regenerated plants expressing altered phenotypes are

not necessarily expressing altered genes but may be

expressing epigenetic changes. Epigenetic changes arising

due to stress placed on the cultured cell may result in

alterations of either transcription and/or translation from

an unaltered genome.

Epigenetic variation may be useful in an asexually

propagated crop such as pangola, provided that the

characteristic remains stable. Meins (1974) stated that this

type of variation is usually, but not necessarily

reversible. Secor and Shepard (1981) reported that potato

(Solanum tuberosum L.) somaclones, which had gone through a

number of vegetative generations in the field, still

retained selected characteristics.







8

Mitra and Steward (1961) were among the first to report

the existence of true genetic change in somatic cell

culture. They observed chromosomal abnormalities, polyploidy

and aneuploidy in carrot (Daucus carola L.) cell cultures.

These genetic changes were gross chromosomal mutations and

are not stably inherited or useful. According to Orton

(1984), stably inherited or Mendelian variation accounts for

chromosomal changes that are heritable and segregate in

crosses with individuals exhibiting a distinct phenotype.

Using this criteria, any heritable phenotypic alteration

caused by a base change, deletion, or rearrangement in the

DNA is included. The DNA may be located in the nucleus,

mitochondria, or chloroplast. The site of mutation may be at

loci that results in either major qualitative effects or

subtle quantitative effects.

Larkin et al. (1985) cited reports of 14 different

species where chromosomal modifications had been induced and

then inherited in a Mendelian manner. The DNA alterations

included point mutations, deletions, interchanges,

inversions, and amplifications. In two of the species, corn

(Zea mavs L.) and alfalfa (Medicago sativa L.), increased

transposable element activity during tissue culture has been

reported by Benzion et al. (1986) and Gross and Bingham

(1986), respectively.

The reason why tissue culture results in an increased

frequency of chromosomal aberration is yet to be explained.







9
Benzoin et al. (1986) hypothesized that the late replicating

heterochromatin may occasionally replicate so late that

bridge formation and subsequent chromosome breakage occurs

at anaphase. The resulting breakage-fusion-bridge cycle

would lead to exchanges, interchanges, or translocations.

Larkin et al. (1984) studied heritable somaclonal

variation in wheat (Triticum aestivum L.) and concluded that

two mechanisms, one operating to create a mutant gene and a

second to make the mutant homozygous, accounted for the

somaclonal variation. Their conclusions were based on the

observations that variation was evident in morphological and

biochemical traits, traits under simple genetic control

(grain color), quantitatively inherited characteristics

(heading date), and a number of traits within a single

somaclone having subsequent independent assortment.

Homozygous and heterozygous mutants were present at

different loci within a single somaclone. Chromosomal loss

or addition was not attributed to have been the primary

cause of variation.

Phenotypic expression may be altered by a variety of

structural changes in chromosomes. An understanding of how

to relate specific cultural conditions to favor one class of

mutation is beginning to emerge. The following is a partial

review of reports on the use of culture variables to affect

phenotypic changes: explant source (Demarly, 1986), media

constituents (Van Harten et al., 1981; Hughes, 1983), cell







10

cycle duration (Muller and Grafe, 1978; Bayliss, 1976), and

regeneration (Hughes, 1983; Mahfouz et al., 1983).

The length of time in culture influences the cultures

ability to regenerate. Matthews and Vasil (1975) reported

that increasing the length of time in culture favors variant

cells and results in more precocious regeneration. This

decline has been attributed, at least partly, to genetic

mutations (Smith and Street, 1974). In pangola, Marousky and

West (1988) reported that after 5 weeks in culture

embryogenesis occurred at a 90-100% frequency. Genetic

mutations in in vitro pangola cells may be favored by

extending the length of time in culture beyond 5 weeks while

not totally compromising the regenerative capacity.

Isolation of variant cells can be achieved by positive

selection, i.e., the application of a stress, followed by

the harvesting of surviving cells. The stress may be exerted

either stepwise or gradually. Larkin et al. (1985) reported

that a stepwise application of pressure is more apt to favor

novel genotypes which arise from either gene amplification

and/or gene mutation. Variant phenotypes produced by this

procedure have resulted in regenerated plants that are

resistant to disease (Foroughi-Wehr et al., 1986; Larkin and

Scowcroft, 1983), mineral toxicity (Ahloowalia, 1982; Oono,

1981), mineral deficiency (Qureshi et al., 1981), and

herbicide damage (Chaleff and Bascomb, 1987).







11

The few reported attempts to select chilling or

freezing-temperature-resistant cell lines that maintain

resistance in culture have had mixed results. The earliest

attempts to select chilling or freezing-temperature-

resistant callus was reported by Steponkus (1972). Steponkus

(1972) obtained cell lines of ivy (Hedera helix L.) which

survived the selection procedure of freezing, but none of

the lines maintained a stable enhanced resistance to the

freezing temperature (Dix, 1980). Based on respiration rates

in mitochondria, Dix and Street (1976) obtained some cell

lines of tobacco (Nicotiana sylvestris L.) and pepper

(Capsicum annum L.) that maintained chill resistance and

other lines that lost chill resistance. Templeton-Somers et

al. (1981) selected freezing-temperature resistant carrot

callus. Resubmission of the resistant callus to freezing

conditions resulted in considerable variation in the ability

of the cell lines to tolerate the selection conditions. Chen

et al. (1982) selected chilling-tolerant sugarcane

(Saccharum spp.) cell lines, most of which retained chill

tolerance in culture.

It has not been determined if the chilling or freezing-

temperature resistance of the selected callus will be

expressed in regenerated plants. Cell lines, selected for

chilling or freezing-temperature tolerance, often lose their

ability to differentiate and regenerate plants (Dix and

Street, 1976; Chen et al., 1982). Dix (1977) sexually







12

crossed plants that were regenerated from chill-sensitive

callus. Callus, obtained from progeny plants, had lost their

chill sensitivity. He concluded that the resistance was not

heritable. Malmberg (1979) showed that culturing leaves from

a tobacco plant, that was regenerated from a chilling-

temperature-sensitive callus, resulted in callus that

retained chill sensitivity. Templeton-Somers et al. (1981)

reported that chilling-temperature resistance in selected

callus is not always expressed during embryo development.

They stated that although there is no concrete evidence for

increased chill resistance in the somatic embryos, further

work is necessary to determine the expression in more mature

plants.

A visual symptom of freezing-temperature injury in

mature plant tissue is the infiltration of tissue

intercellular space with water. Chen et at. (1976) used this

soaked appearance, along with loss of turgor, as a criterion

for evaluating freezing injury. Another common result of

freezing injury is the leakage of ions from cells. Until

recently, reports such as Levitt (1972) and Sukurman and

Weiser (1972) attributed the efflux of ions from frozen and

thawed tissue to the breakdown of the semipermeable property

of the cell membrane. Palta et al. (1977a) has shown that in

onion (Allium cepa L.) bulb cells the semipermeable

properties remain intact during the process of freezing,

whereas the active transport properties of the cell






13
membranes are damaged. They reported that inactivation of

the active transport system results in a large passive

efflux of ions and sugars. During thawing, as ice melts in

the extracellular space, ions and sugars move down

concentration gradients vacuolee to extracellular solution),

as the active transport system is unable to pump them back

into the vacuole. As a result of osmotic equilibrium between

the outside solution and the cell sap, the cell is unable to

absorb water. The tissues become flaccid due to the

infiltration of the tissue with extracellular solution and

lose of turgor.

Using onion bulb cells Hansteen-Cramer (1922) first

reported that Ca is needed for the stability of natural

membranes. Palta et al. (1977b) reported a small but

increasing efflux of Ca from onion bulb cells as a result of

freezing injury. They concluded that such removal of Ca from

the membranes causes instability of the membrane structure

and finally results in breakdown of the membrane system.

Based on the leakage of ions, Dexter et al. (1932)

reported that measurement of electrical conductivity of the

ions can provide a quantitative evaluation of freezing

injury. Electrolyte leakage has been used by Patterson et

al. (1976) to measure low-temperature sensitivity in species

of passionflower (Passiflora) having a range of climactic

requirements. Wiltbank and Oswalt (1984) reported the use of







14

electrolyte leakage to determine changes in the killing-

point temperature of several citrus cultivars.

Use of electrolyte leakage to measure variation in low-

temperature stress should be applicable in assessing

variations in low-temperature sensitivities of regenerated

pangolagrass plants.

Damage to the photosystem apparatus occurs at

temperatures above those which cause breakdown of cellular

integrity (Berry and Bjorkman, 1980). This damage can be

used as an indication of the amount of low-temperature

stress in the plant. Drake and Salisbury (1972) reported

that growth inhibition in C4 species is proportional to the

severity of chilling and to the intensity of light received

during chilling. Chatterton et al. (1972) attributed reduced

growth in pangola, during chilling, to damage in the

photosynthetic apparatus.

Early indicators of reduced photosynthetic activity in

chill-sensitive plants were believed to be thylakoid

membrane phase transitions (Murata and Fork, 1975; Shneyour

et al., 1973) and increases in stomatal resistance to CO2

flux (Crookston et al., 1974; Drake and Salisbury, 1972).

However, work by Low et. al. (1984) has indicated that both

chilling-sensitive and chilling-tolerant plants exhibit

equivalent thylakoid phase transitions. Low et al. (1984)

and Martin (1986) have reported that there is insufficient

thylakoid membrane damage to account for the impairment of







15

photosynthesis. Further, Hallgren et al. (1982) reported

that stomatal closure does not necessarily inhibit CO2

uptake because intercellular CO2 pressure can remain

constant despite a decreased stomatal conductance.

Chilling corn in light decreased quantum yield of CO2

assimilation, rate of 02 evolution, and variable fluorescent

emission of photosystem II (Long, 1983). Hillard and West

(1970) reported that pangola exhibited decreased rates of

CO2 assimilation after chilling in the dark. Chill-stressed

pangola plants also had a reduction in the Hill reaction

(West, 1970). The extent of low-temperature alterations in

variable fluorescent emission of photosystem II in pangola

is unknown.

Steinback and Mall (1986) reported that the loss of

photosynthetic competence in low temperature is due to

photoinhibition of photosystem II activity. Chill-induced

damage to photosystem II has been associated with altered

energy flows in the electron transport chain (Percival et

al., 1987; Heinrich and Lasch, 1987). The active center of

photosystem II is not immediately affected by chilling

(Smillie, personal communication). Chilling-temperature

inhibition of the water-splitting side of photosystem II was

reported by Margulies (1972) and 6quist (1983), as assayed

by electron transfer and fluorescent excitation of

chlorophyll a. No chilling temperature effects on

photosystem I were observed by Oquist (1983). Hetherington







16

et al. (1983) and Yung-ling et al. (1987) reported that the

damaged water-splitting enzyme systems produce thermal

rather than chemical energy. Chilled leaves having an

inhibited water-splitting protein complex sustained a lower

yield of fluorescent emission from chlorophyll a

(Papageorgiou, 1975).

The lower yield of chlorophyll fluorescent emission due

to chilling temperature is evident in FR measurements. A FR

measurement is defined as the linear increase of chlorophyll

a fluorescent emission per unit surface area as a function

of time between I and P, where the fluorescence at time I is

the initial fluorescent emission and the fluorescence at

time P is the maximum fluorescent emission (Figure 1).

Lawlor (1987) stated that high FR values are indicative of

an intact electron transport chain. The quantity of

fluorescent emission at time I is regulated by the redox

state of the quinone protein (Q), the primary electron

acceptor of photosystem II. The more that Q and

plastoquinone (PQ) are in the reduced states the higher will

be the initial fluorescence. Munday and Govindjee (1969)

reported that the amount of variable fluorescence,

fluorescent emission from I to P, is limited by the

photoreduction of Q via the photosystem II reaction linked

to the water-splitting enzyme system.

Smillie and Hetherington (1983) used FR as a chilling

stress measurement. Hetherington et al. (1983) found that







17

chill stress reduced the FR value in corn. Their work with

corn led to the identification of chill-tolerant corn

populations based on FR values.

Machado et al. (1984) rated increased atrazine

resistance using somatic variants regenerated from tissue

culture, by testing differences in the emission of

chlorophyll fluorescence. To date, no reports on somatic

variation in chilling or freezing-temperature tolerance have

been presented using fluorescent emission measurements.



Materials and Methods

Plant Material and Growing Conditions

A 'pangola' digitgrass (Digitaria decumbens Stent.)

plant was obtained from the University of Florida's Agronomy

Seed Laboratory in September of 1986. Eleven vegetatively

propagated source plants were grown in a greenhouse with

natural lighting. The average daily temperature range was

300C to 350C. The source plants were grown in 15-cm pots

containing Metro-mix 350 growing medium. Rapid growth was

maintained by supplying six grams of Osmocote (W. R. Grace,

Cambridge, MA) a slow-releasing fertilizer (14-14-14, N-

P205-<20) to each pot. The plants received adequate water on

a daily basis and were clipped to 15 cm above the soil

surface every two weeks. Care was taken to prevent root

binding by vertically halving the entire plant and repotting

when needed.





















RELATIVE

FLUORESCENCE


FR,
!


FR
/


27C


TIME


Fig. 1. Decrease in the rate of fluorescent emission
(F,) from pangolagrass chilled at 100C for 45
minutes. Peak fluorescence (P) is the maximum
fluorescent emission.










Cell Culture Initiation

Source plants were no longer clipped after the first

of May, 1987, in order to promote floral development. In

early June of 1987 immature inflorescence 10 to 15 cm in

length were excised from culms at approximately 8:00 AM.

Entire inflorescence was surface sterilized in 0.8% (v/v)

sodium hypochlorite (15% Clorox in water). A drop of Tween-

20 (Fisher Scientific Company, Fair Lawn, NJ) was added per

100 mL of solution. Flower heads were totally immersed in

the sterilizing solution for 45 min while being shaken at 35

rpm on a New Brunswick Scientific G10 gyrotory shaker

(Edison, NJ). The remaining decontamination procedures and

transferring to culture media was done under an Edgeguard

laminar flow hood (The Baker Co., Sanford, ME). The flower

heads were rinsed four times with sterile deionized water

(dHO2) and then cut longitudinally by making an incision

with a scalpel through the leaf sheaths. The immature

florets were exposed by spreading the tissue apart on either

side of the incision with a pair of curved tip microforceps.

The spikes were excised with smooth tip microforceps and cut

into 5-mm segments. Ten to 15 segments, or explants, were

cultured in each of 100 X 15 mm petri dishes containing a

solid MS media (Murashige and Skoog, 1962). The MS media

contained premixed MS salts (Sigma Chemical Co., St. Louis,

MO), 8% T C agar (Carolina Biological Supply Co.,







20

Burlington, NC), 4% sucrose, and 9 jIM 2,4-

dichlorophenoxyacetic acid (2,4-D). The petri dishes were

wrapped in parafilm (American Can Co., Greenwich, CT) and

placed in a Percival growth chamber (Boone, IA) at 270C with

a 12 h light/dark cycle. Three weeks later, the explant

material was discarded after being separated from callus

tissue. Calli were transferred to fresh media of the same

composition at three-week intervals, for three cycles.



Temperature Treatments

After the third transfer cycle, calli were subjected to

different temperature treatments. Compact white embryogenic

callus was sectioned into 1 cm2 pieces and four of these

calli were placed in each petri dish. The petri dishes

contained the same MS media composition as used for callus

initiation. A minimum of 30 plates (containing a total of

120 calli) were placed in each temperature treatment. The

treatments were 270C, 20C for 12 d, 20C for 24 d, -120C for 4

h, -12C for 8 h, -120C for 23 h. All cultures were

maintained in the dark. The 20C treatments were placed in a

Percival growth chamber whereas the -120C treatments were

kept in a Whirlpool freezer (Benton Harbor, MI).

Callus viability was estimated by the ability to regrow

on fresh MS media containing 2 pM 2,4-D at 270C in 12 h

light/dark for 42 d.









Plant Regeneration

After exposure to temperature treatments the callus was

transferred to a MS media containing 2 jM 2,4-D and placed

at 270C with a 12 h light/dark period. Chilling or freezing

temperature damage and subsequent developmental morphology

of the callus were observed by using an Olympus SZ-Tr stero

microscope (Tokyo, Japan). Viable embryogenic calli and

developing embroids were separated from necrotic calli and

transferred to fresh MS media of the same composition every

other week, for three cycles. The number of surviving calli

were recorded.

Subsequent embryo maturation and plantlet regeneration

was advanced by transferring cultures to MS media devoid of

2,4-D. Cultures were transferred to fresh media of the same

composition every other week, for three cycles. After the

third transfer, individual regenerated plants that had a

well-defined root system consisting of several adventitious

roots and leaf lengths of 2 to 4 cm were separated and

counted. The regenerated plantlets were transferred to septic

6-cm pots. The pots contained Metro mix 350 growing media

which was kept constantly moist. The plants were withheld

from direct sunlight for seven days, after which time the

plants were exposed to full sun and fertilized with Peters

fertilizer (20-20-20, N-P205-K20) (W. R. Grace and Co.,

Foglesville, PA). The plants were watered as needed for the







22

next three weeks. The plants were then transferred to 15-cm

pots and maintained as previously described.



Scanning Electron Microscope

Micrographs of embryo development were obtained with a

Hitachi S-450 scanning electron microscope (Tokyo, Japan).

Selected tissue was fixed in FAA and dried through an

alcohol dehydration series. Some of the tissue was sectioned

with a single-edge razor blade. The tissue was dried in a

Balzer CPD 030 critical point dryer (Furstentum,

Liechtenstein) and coated with gold in a Samsputter 2a

automatic coating apparatus (Tousimis Research Corp.,

Rockville, MD).



Plant Selection: Electrolyte Leakage

A total of 263 regenerated plants and three

vegetatively propagated source plants were tested for

freezing- temperature damage using modifications in the

electrolyte leakage procedure presented by Patterson et al.

(1976) and Wiltbank and Oswalt (1984).

Tissue was obtained from plants maintained in the

greenhouse. Six young, fully expanded leaves from each plant

were cut from the plant and washed three times in deionized

water. Leaf tips and 6 cm of leaf tissue were immersed in a

55 mL test tube containing 20 mL of deionized water. Care

was taken to avoid submerging cut tissue. The temperature of






23

the leaf tissue and water was decreased by approximately 2C

per min to a final temperature of -5C for 30 min in a Lauda

refrigerated circulator (Brinkmann Instruments Co.,

Westburry, NY) containing ethylene glycol. After 30 min at -

50C the supercooled water was crystallized by seeding with a

small deionized ice crystal. Leaf tissue remained in the ice

for an additional 30 min. The test tubes were then

readjusted to 250C for 90 min. Leaf material was removed

from the water and electrical conductivity of the water was

measured with an ASA 610 electrical conductivity bridge

(Agro Sciences Inc., Ann Arbor, MI).

Mean electrolyte conductivity of the solute leakage

from individual plants, 263 regenerated plants and three

vegetatively propagated source plants, was calculated based

on two replications. The proportion of regenerated plants

with electrolyte conductivity less than the mean electrolyte

conductivity the standard deviation (S.D.) of the four

source plants are reported for each in vitro temperature

treatment. Ten regenerated plants were selected,

irrespective of in vitro temperature treatment, based on

their low quantity of electrolyte leakage relative to the

electrolyte leakage of the source plants.

Six replications of the 10 selected regenerated plants

and 4 vegetatively propagated plants were tested in a

completely randomized design for analysis of mean

electrolyte leakages. MSTAT, a PC statistical program, was







24

used for data analysis. Means were calculated and data were

analyzed by Duncan's New Multiple-range Test. The same

plants were used for chlorophyll fluorescence

determinations.



Plant Selection: Chlorophyll Fluorescence

Experiments were conducted during March, 1988. The 10

previously selected plants were removed from the greenhouse

at 8:00 AM and dark adjusted for 60 min at 270C before

obtaining initial fluorescent emission measurements. The top

third of the first fully extended leaf was excised from the

plant and placed in a 270C Hansatech LD2 leaf chamber

(King's Lynn, Norfolk, UK). The leaf was positioned above a

pad wetted with a 1 M sodium bicarbonate solution. The

sodium bicarbonate maintained an adequate supply of CO2 for

photosynthesis (Walker, 1987). A template was constructed

which limited the light exposure to 50 mm2 of leaf tissue.

The template did not interfere with gas flow within the

chamber. Excitation energy was applied two minutes after the

leaf tissue was placed in the leaf chamber. A quantum flux

density of 60 W m-2 (660 nm) was supplied by a Hansatech LS1

light source (King's Lynn, Norfolk, UK) for photosynthesis.

The LD2 unit was attached to a Lauda RM6 refrigerated

circulator (Brinkmann, Westbury, NY) and maintained at 27C.

Chlorophyll fluorescent emission was detected by a Hansatech

fluorescence detector that was fitted with a 740 nm







25
interference filter. Data points were obtained every 6.9 ms

with a LeCroy transient recorder (Figs. 2 and 3) (Spring

Valley, NY). The signals were relayed to an IBM PC for

operating the Waveform-catalyst software program version 2

(LeCroy Research Systems Corp., Spring Valley, NY), storage

of data, and retrieval of data.

Fluorescent emission from chill-stressed plants was

obtained after measuring the initial fluorescent emission.

Chilling treatment was imposed by placing the plants in a

darkened 100C Percival growth chamber for 45 min. The top

third of the first mature leaf was excised from the plant

and placed in the 270C leaf chamber for two minutes before

excitation energy was applied. Measurements were obtained as

previously described.

Measurements were expressed as FR as described by

Hetherington et al. (1983). The effect of chilling on

chlorophyll fluorescence was gauged from the chilling

induced decrease in FR (Smillie and Hetherington, 1983). FR

was measured before chilling (Reading 1) and again 45 min

after beginning chilling (Reading 2). Percentage FR

remaining after chilling was calculated as (Reading

2/Reading 1) X 100.

Data means + S.D. of individual plants (10 regenerated

plants and four source plants) were calculated on six

replicates.





















*~;'~~


b .


Fig. 2. Configuration of LeCroy transient recorder.
a 8013A crate.
b 8100 gain differential amplifier, in crate
slot #2.
c 8800A memory, in crate slot #5.
d 8210 waveform analyzer, in crate slot #6.
e 8901A GPIB interface, in crate slot #12.














































Fig. 3. Configuration of LaCroy transient recorder.
a 8100 gain differential amplifier.
control lock.
filter in.
gain 1.
multiply 1.
input to fluorescent detector.
output to 8210 waveform analyzer.
b 8210 waveform analyzer.
post trigger samples 7.
sample interval external clock.
input channels 4.
display channel 4.
input to 8100.










Results and Discussion

Somatic embryogenesis in pangola followed a pattern of

development similar to that reported by Marousky and West

(1988). Within 10 d of initiation on MS media containing 9

AM 2,4-D, soft friable callus formed from the pedicils and

ovarian tissue. Prolific growth was observed. After two

weeks of culture, two types of callus were produced (Figures

4, 5). Embryogenic callus was pale yellow to white in color,

nodular and compact in appearance, and was composed of

small, densely cytoplasmic cells. Alternatively, non-

embryogenic callus was friable and contained loose cells

which were often elongated and highly vacuolated.

Isolation of cell lines with an increased tolerance to

freezing or chilling temperature was achieved by a stepwise

exertion of low temperature and positive selection. Larkin

et al. (1985) reported that a stepwise exertion of selective

pressure tends to select the novel genotypes that arise from

either gene amplification and/or gene mutation. Freezing and

chilling-temperature stress adversely affected the viability

of embryogenic callus (Table 1). Callus viability decreased

when the temperature severity or duration of exposure

increased. An LD 50 was approximated when chilling (20C)

temperature was imposed for 24 d or when freezing (-120C)

temperature was imposed for 23 h. Damaged and necrotic

callus became apparent several days after the temperature
















































Fig. 4. Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 gM 2,4-D. X 7.





















































Fig. 5. Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 AM 2,4-D. X 58.









Table 1. Percentage of pangola callus surviving low
temperatures and supporting plant regeneration.



Treatment Pangola callus


Tempera-
ture Duration+ Total Viable Supporting regeneration

C d or h % %

27 constant 125 74 14
2 12d 101 73 12
24d 437 45 9
-12 4h 234 63 7
8h 284 65 2
23h 198 48 0


+Period of exposure to temperature treatment.

Percentage of viable callus from which plants regenerated.







32

treatments ended. The affected callus appeared dark brown in

color, softer in consistency, and discolored the surrounding

media. The surviving embryogenic calli retained their

characteristic appearance. Theoretically the surviving cells

had a higher degree of chill tolerance than the chill-

susceptible cells. Selection procedures reported by Dix and

Street (1976), Breidenbach and Waring (1977), Dix (1980),

Templeton-Somers et al. (1981), and Chen et al. (1982) have

successfully isolated cell lines that tolerated chilling or

freezing temperatures.

Somatic embryos were obtained from the surviving calli

by reducing the level of 2,4-D in the medium was reduced to

2 MM (Figure 6). Maturing embryos contained the

characteristic organs of a grass embryo: scutellum,

coleoptile, and shoot apex (Figure 7). Gross abnormalities

in embryo development were absent. Variation in the degree

of typical embryo development was not assessed. Vasil (1983)

reported embryo development ranging from atypical to typical

in somatic embryos of different grasses.

Eleven weeks after calli were initiated plant

regeneration from somatic embryos was advanced on MS media

without 2,4-D (Figure 8). Matthewes and Vasil (1975)

reported that increasing the length of time in culture

favors variant cells and results in more precocious

regeneration. Table 1 shows that after 11 weeks in culture,

















































Fig. 6. Longitudinal section of pangola callus with
nonembryogenic (n) callus, embryogenic (e) callus,
and young somatic embryo (em). On MS media
containing 2 jM 2,4-D. X 58.












































Fig. 7. Longitudinal section of pangola callus with
embryogenic (e) callus supporting a maturing somatic
embryo; scutellum (sc), coleoptile (co), and shoot
apex (s). On MS media without 2,4-D. X 75.

















































Fig. 8. Regenerated pangola plant with plumule (p) and
young root (r). On MS media without 2,4-D. X 7.







36

at 270C, 14% of the viable embryogenic calli supported plant

regeneration. Marousky and West (1988) observed

embryogenesis in pangolagrass callus at a 90-100 percent

frequency after five weeks of culture. The percentage of

viable embryogenic callus capable of supporting plant

regeneration decreased when the temperature severity or

duration of exposure increased. Callus surviving -120C for

32 h was unable to regenerate plants. Dix and Street (1976)

and Chen et al. (1982) found that tobacco cell lines which

were selected for low-temperature resistance were unable to

support plant regeneration.

An observed increase in the concentration of Ca in the

efflusate of freeze-injured leaf tissue (data not shown)

agreed with Palta et al. (1977a) and implied a loss of

plasmalemma integrity. The percentage of regenerated plants

exhibiting electrolyte conductivity values less than the

vegetatively propagated source plants is given in Table 2.

The percentage of regenerated plants ranged from a high of

approximately 50 % (270C, 20C for 12 d) to a low of 12%

(-120C for 8 h). Increasing the severity of the in vitro

temperature treatment on calli resulted in fewer regenerated

plants with less electrolyte leakage (Table 2). Steponkus

(1972) reported a total loss of chilling-temperature

tolerance in cells during tissue culturing. Other

researchers reported a partial loss of chilling or freezing-

temperature tolerance in cells during culturing (Dix and











Table 2. Percentage of regenerated pangola plants with
less electrolyte leakage than the source plants.



Callus treatment Regenerated plants


Temperature Duration+ Total Electrolyte conductivity
less than source plant

C d or h %

27 constant 89 47
2 12d 16 50
24d 65 14
-12 4h 51 29
8h 42 12


+Length of time for callus exposure to temperature
treatment.

Percentage of regenerated plants with lower electrolyte
conductivity than the mean electrolyte conductivity of three
source plants + S.D.







38

Street, 1976; Templeton-Somers et al., 1981; Chen et al.,

1982). Templeton-Somers et al. (1981) reported that

chilling-temperature resistance expressed in carrot culture

cells are not necessarily expressed in the somatic embryo

stage of development. These observations are supported by

the present study where freeze-tolerant cell lines supported

the regeneration of freeze-susceptible plants.

Electrolyte conductivity of the 10 selected freeze-

tolerant-regenerated plants was less than that for the

source plants (Table 3). Six of the regenerated plants had

significantly reduced electrolyte leakage when compared to

the vegetatively propagated source plants.

Data presented in Table 3 supported the hypothesis that

somatic variation in pangolagrass was obtainable and that

variant regenerated plants were able to have been selected

for an increase in freeze tolerance.

The percent FR remaining in regenerated plants and

source plants after chilling-temperature exposure is

presented in Table 4. No variation in the percent FR

remaining after chilling was detected between regenerated

plants or between regenerated plants and source plants.

These results do not support the hypothesis that somatic

variants may be selected for chilling tolerance using

chlorophyll fluorescence. Hetherington et al. (1983)

reported that chill stress reduced the FR value in corn, a











Table 3. Freeze-induced electrolyte
regenerated pangola plants


leakage of selected
and source plants.


Plant No. Mean conductivity

gamps

Source plant
1 78.2 a
2 65.0 ab
3 63.0 abc
4 61.2 a b c
Regenerated plant
59 48.4 a b c d
91 40.4 b c d e
40 40.0 b c d e
8 32.6 cde
103 29.6 d e
84 29.0 de
76 22.8 de
24 15.2 e
4 14.8 e
65 8.8 e



Means not followed by same letter are significantly
different at P < 0.05 (DMRT).









Table 4. Percent F, remaining in selected regenerated
pangola plants and source plants after
exposure to chilling temperature.


Regenerated plants Source plants


Plant No. F, remaining* Plant No. FR remaining*

% %

59 39 +15 2 57 + 9
91 40 + 8 3 56 + 9
40 58 + 9 4 51 +10
8 51 +11 1 51 +11
103 48 + 7
84 56 + 7
76 70 + 9
24 70 +10
4 48 + 8
65 35 +12


+Percent F, remaining = (Fg 230C / FR after 45 min at 100C)
100. Mean of six replications + S.D.






41

C4 grass. Their work with corn led to the identification of

chill-tolerant corn populations based on FR values.



Conclusions

Callus growth and subsequent somatic embryogenesis was

obtained in pangolagrass. Increasing the in vitro chilling

or freezing-temperature treatments reduced callus

survivability and competency for supporting embryogenesis.

Regenerated pangolagrass plants sustained a range of

freeze-induced damage between individuals as evidenced by

plasmalemma integrities. The ability of in vitro cells to

survive freezing or chilling temperatures did not predispose

plants, regenerated from those cells, to be freeze tolerant.

Apparently, increasing the chilling or freezing temperature

treatments in culture reduced the proportion of regenerated

plants that were freezing-temperature tolerant.

Somaclones with increased freeze tolerance relative to

vegetative control plants were obtained. Within this group

of plants, no variation in the extent of chill damage to the

photosynthetic electron transport chain within the thylakoid

membrane was observed.













CHAPTER III
EVALUATION OF CHILLING TEMPERATURE AND GIBBERELLIC ACID
ON THE EXPRESSION OF HYDROLYTIC ENZYMES



The hypothesis tested in this section was that chilling

temperatures reduce the number of debranching enzymes,

endoamylases, and exoamylases that break down starch.

Hypothetically, gibberellic acid (GA3) may nullify the

influence of low temperature on the reduction in the number

of starch-degrading enzymes.

The objectives of this study were a) to evaluate the

influence of low temperature on the expression of starch-

hydrolyzing enzymes; b) to identify hydrolytic enzymes which

may assist in starch breakdown under low- temperature

conditions; c) to evaluate the effect of GA3 on the

expression of starch-hydrolyzing enzymes during low-

temperature conditions; and d) to identify GA3 induced

isozymic forms of hydrolytic enzymes which may assist in

starch breakdown under low-temperature conditions.

The severe growth depression of pangola that results

from low nighttime temperatures has been associated with the

accumulation of starch granules in mesophyll cells and

bundle sheath cells (Hillard and West, 1970; Chatterton et

al., 1972). Hillard (1975) reported that cultivars of







43
Digitaria whose growth was least affected by chilling

temperature were those that retained the least amount of

starch in their chloroplasts after chilling nights.

The accumulation of large assimilated starch grains in

the chloroplast has been shown to reduce growth rates and

net photosynthesis via physical damage of the chloroplast

membrane (West, 1970), and disruption of photophos-

phorylation and electron transport (Pearson and Derrick,

1977).

Garrard and West (1972) determined that low leaf

temperature drastically reduces starch anabolism and

translocation, even when nearby sinks are maintained at high

temperatures. Jones (1985) concurred that translocation of

photosynthate to the growing region and utilization of

photosynthate by this region was limited by chilling

temperature. Kleinedorst and Brouwer (1972) reported that

restricting chilling to the leaves had little effect on

meristematic cell division. They observed a reduction in

cell elongation and postulated that this was due to reduced

carbohydrate availability for cell wall formation in the

region of cell elongation.

The biosynthesis of chloroplastic starch is well

characterized and has been reviewed by Preiss (1982), Preiss

and Levi (1979), and Preiss et al. (1987).

Most starches are a composite of two different types

of polysaccharides (Meyer and Gibbons, 1951). One is







44

unbranched and composed of long chains of a-1,4 linked

glucose units; the other is branched and consists of shorter

chains of a-1,4 linked glucose residues which are joined

through the a-1,6 positions to form a large molecule. Meyer

called the linear component amylose and the branched one

amylopectin. The number of glucose units in various

amylopectins ranges from 2,000 to 200,000 whereas amylose

may contain a few thousand glucose units (Salisburry and

Ross, 1985b).

Assimilatory starch granules in photosynthetic tissue

are located in the chloroplast. Viewed under a scanning

electron microscope, starch granules in pea (Pisum sativum

L.) are consistently surrounded by thylakoid membranes

(Beck, 1985). Beck (1985) reported the appearance of the

granules to be dramatically different between illuminated

and darkened pea leaves. The starch granules from an

illuminated leaf, unlike a darkened leaf, appeared pasty and

had a lack of sharp contours. The pasty starch mantle

surrounded

a crystalline starch core (Beck, 1985; Steup et al., 1983).

Beck (1985) investigated the water content of the starch

granule and reported that the mantle had a higher water

content than the core. Accompanying the higher water

content, the mantle also contained more a-1,6 branching than

the core.







45

Confusion in the literature concerning the mode of

starch degradation reflects the fact that the predominant

pathway of starch degradation within the chloroplast is

unresolved. Until recently, many researchers believed that

starch phosphorolysis by phosphorylase (Stitt and Heldt,

1981; Levi and Preiss, 1978; Stitt and Rees, 1980) was the

predominant pathway of starch degradation. Other researchers

believed that starch hydrolysis by endoamylase, exoamylase,

R-enzyme (debranching activity), and D-enzyme

(transglycosylase) (Herold et al., 1981; Peavy et al., 1977;

Chang, 1982) was the predominant pathway of starch

degradation. Assimilated starch is now believed to be

degraded by the combined action of hydrolysis and

phosphorolysis (Lin et al., 1988; Kakefuda et al., 1986;

Robinson and Preiss, 1987; Echeverria and Boyer, 1986).

Starch phosphorylase catalyses the phosphorolysis of

a-1,4 glucosyl chains yielding glucose-l-phosphate. a-

amylase attacks a-1,4 linkages of amylose or amylopectin

yielding glucose and small amounts of maltose (Salisburry

and Ross, 1985a). P-amylase attacks only the nonreducing

ends of chains, splitting off pairs of glucosyl units as

maltose (Salisburry and Ross, 1985a). R-enzyme attacks the

1,6 branching points of amylopectin making this component of

starch susceptible to further attack by hydrolysis or

phosphorolysis (Salisburry and Ross, 1985a). D-enzyme can

transfer groups of glucosyl units between short chain







46

dextrins producing a mixture of longer and shorter chain

dextrins and glucose (Salisburry and Ross, 1985a).

Dextrins were described by Meyer and Gibbons (1951) as

starch breakdown products that were composed of mixtures of

chain fragments of low but varying molecular weights.

Dextrins are routinely analyzed using a potassium-iodide-

iodine reaction (Jensen, 1962). Kakefuda and Duke (1984)

reported that electrophoretic gels containing amylopectin

stain dark violet with iodine and reveal areas of enzyme

activity as unstained or differentially stained regions

according to the type of starch-degrading enzyme present.

Unstained areas indicate complete amylopectin breakdown due

to the action of endoamylases, and pink regions reveal the

degradation of amylopectin to P-limit dextrin by

exoamylases. Debranching enzyme debranches amylopectin to

form amylose, which stains blue with iodine (Ziegler and

Beck, 1986).

Beck (1985) noted that during the nocturnal breakdown

of the starch granule, the mantle was degraded more rapidly

than the core. Endogenous hydrolytic enzymes accounted for

the degradation of the pasty mantle. Beck et al. (1981) and

Steup and Schachtele (1981) reported that assimilatory

starch is a very poor substrate for the chloroplastic

phosphorylase in spinach (Spinacia oleracea). They found

that amylolysis of assimilatory starch resulted in

degradation products that were acted on by phosphorylase






47
three to four times more rapidly than starting materials.

These results demonstrate the cooperativity and not a

competition between the amylolytic system and phosphorylase

in assimilatory starch breakdown.

Beck (1985) stated that, although amylases cooperate

with phosphorylases in the degradation of assimilatory

starch, the overall cellular location and physiological role

of these enzymes in leaves is far from clear. For example,

spinach leaf chloroplasts contain endoamylase, debranching

enzyme, phosphorylase, and D-enzyme (Okita and Preiss, 1980;

Preiss et al., 1980). In contrast, pea leaf chloroplasts

contain phosphorylase, R-enzyme, D-enzyme (Kakefuda et al.,

1986), and a small amount of P-amylase (Levi and Preiss,

1978). The a-amylase is either absent or in very low

activity in the chloroplast (Jacobsen and Beach et al.,

1985; Kakefuda et al., 1986). Beers and Duke (1988) reported

that the location of extrachloroplastic a-amylase is

primarily apoplastic.

Although amylase and phosphorylase activities

constitute the major degradative activity in the

chloroplast, only endoamylase has been demonstrated to

attack assimilated starch granules (Steup et al., 1983). The

extrachloroplastic location of a-amylase presents a logistic

problem. No extrachloroplastic homoglucans which could act

as substrates for such amylases are known and no







48

explanations have been presented to account for these

enzymes outside the chloroplast.

In corn, Echeverria and Boyer (1986) reported starch

accumulation in the chloroplasts of bundle sheath cells.

Starch was absent from the chloroplasts of mesophyll cells.

Analysis of starch-degradative enzymes showed that 30% of

the degradative enzymes were located in mesophyll cells,

where no starch accumulated. The remaining 70% of starch-

degradative enzymes were located in the bundle sheath cells.

In bundle sheath cells, 60% of the activity of these enzymes

were chloroplastic and 40% were located in the cytosol.

Starch phosphorylase was limited to bundle sheath cells with

only 70% of phosphorylase activity being chloroplastic.

In pangola, low temperature causes an excessive

accumulation of assimilated starch and a decrease in the

activity of starch-degrading enzymes (Carter et al., 1974).

Hillard (1975) reported that cultivars of weeping lovegrass

(Eragrostis curuvla L.) and digitgrass whose growth was

least affected by low temperature retained the least starch

in their chloroplasts after chilling nights and had the

highest amyolytic enzyme activities at chilling

temperatures. Karbassi et al. (1972) reported a reduction in

starch degrading activity when pangolagrass plants were

pretreated at chilling night temperatures or when the enzyme

assay was conducted under chilling conditions.







49

Carter et al. (1972) reported the localization of two

bands of amyolytic enzymes by polyacrylamide gel

electrophoresis using amylose containing gels. The relative

activity of both bands were decreased when exposed to 100C

incubation temperatures with the band nearest the origin

decreasing the least under low temperature. Carter et al.

(1973) modified the electrophoretic procedure to separate

the amyolytic enzymes into seven bands. The bands separated

into a fast migrating pair (bands 1 and 2) and a slow

migrating group (bands 3 to 7). Chilling temperature

decreased the activity of band 2 the most, while band 7 was

decreased the least. No change in the number of bands was

detected under the different temperature treatments.

Production of a-amylase in the barley (Hordeum vulgare

L.) aleurone layers has been shown to respond to GA3

(Jacobsen and Higgins, 1982). Addition of the hormone to the

de-embryonated seed or isolated aleurone layers results in a

large increase in a-amylase activity (Atzorn and Weiler,

1983) and debranching enzyme activity (Bewley and Black,

1985). GA3 acts at the transcriptional level increasing a-

amylase mRNAs(Bewley and Black, 1985). That increase in a-

amylase activity is the result of production of multiple

isozyme forms (Callis and Ho, 1983). Nolan and Ho (1988) and

Nolan et al. (1987) reported that GA3 differentially

controls the expression of two a-amylase genes or a group of







50

genes giving rise to two groups of a-amylase isozymes with

different properties.

Atkin et al. (1973) reported that chilling-temperature

exposure of corn plants reduces the amount of gibberellin in

the xylem exudate. They suggested that slower growth rates

during chilling-temperature exposure may be related to a

decrease in the gibberellin concentration in the leaf

tissue. Foliar application of GA3 to 'Tifdwarf,' a

bermudagrass (Cynodon dactylon L.) that is damaged by

chilling temperature, stimulates growth during chilling

temperatures (Dudeck and Peacock, 1985). GA3 has been shown

to improve growth rates of tropical forage grass when

applied 10 hours prior to chilling (Karbassi et al., 1971;

Whitney et al., 1973).

Foliar application of GA3 on pangola increases the

amylolytic activity in the plants at either ambient or

chilling temperatures (Carter et al., 1973; Karbassi et al.,

1972). Karnok and Beard (1986) showed that GA3 application

on chill-stressed bermudagrass reduced the number of starch

granules in the bundle sheath chloroplast.

Carter et al. (1973) reported that the application of

GA3 increased the relative activity of the seven amylolytic

enzymes that were separated by gel electrophoresis. The

activity of band 2 was increased the most while.the activity

of band 7 was least influenced. Of the 3-7 group, the enzyme

of band 3 was more responsive and those of bands 5 and 7







51

were the less enhanced by GA3. GA3 application did not cause

the production of additional amylolytic isozymes. This

response is unlike that in the aleurone layers of barley

seed and may be unique to chloroplasts.



Materials and Methods

Plant Material and Treatment Application

Experiments were conducted during the months of July

and August, 1989, at which time the photoperiod decreased

from 14 h to 13 h and the quantum flux density averaged

1,500 to 1,700 Meinsteins m-2 s1 (400-700 nm) at midday.

Average daily temperature in the greenhouse was maintained

between a range of 300C to 350C. Vegetatively propagated

source plants were grown in the greenhouse as described in

chapter II. Gibberellic acid was applied to a vegetatively

propagated source plant following the procedures outlined by

Karbassi et al. (1971) with the following modifications. A

10 uM GA3 solution, adjusted to pH 6.8 with KOH, was

sprayed to the drip point on all vegetative parts of the

plant 10 h before the 100C temperature treatment was

imposed. Temperature treatments were initiated at the end of

the photoperiod. One vegetatively propagated source plant

was placed in a darkened 300C Percival growth chamber. A

second source plant and the GA3 treated source plant were

placed in an identical growth chamber at 100C. Plants

remained in the growth chambers for 12 h until sampling.







52

Treatment application and subsequent analysis of starch-

hydrolyzing enzymes were replicated 18 times using different

plants.



Whole Leaf Extract Preparation

One gram fresh weight of the first fully extended leaf

was obtained at the end of the temperature controled 12 h

dark period. The tissue was washed with deionized water and

cut into 0.5 to 1.0 mm segments with a straight edge razor

blade. Segments were ground for 3 min in a chilled mortar

containing 3 mL of ice cold extraction buffer (Table 5). 3-

mercaptoethanol and polyvinylpolypyrolidone were added to

the extraction buffer just prior to grinding in a porcelain

mortar, Coors 522, size No. 6 having an unglazed grinding

surface. Ground glass was added to facilitate a fine grind.

The slurry was maintained at 20C during centrifugation at

20,000 g for 20 min. The supernatant was maintained at 20C

and used as samples for electrophoresis.

Protein controls were prepared for electrophoresis by

diluting a-amylase from bacillus (Sigma Chemical Company,

St. Louis, MO), E-amylase from sweet potato (Sigma Chemical

Company, St. Louis, MO), and pullanase a debranching enzyme

from Enterobacter aerogenes (Sigma Chemical Company, St.

Louis, MO) with extraction buffer.











Table 5. Reagents and gel preparation for native starch PAGE
slab gel electrophoresis.


Extraction buffer
0.5 M Tris, pH 6.8
Glycerol
B-Mercaptoethanol
polyvinylpolypyrolidone


Electrode buffer
Trizma base
(Tris[hydroxymethyl]aminomethane)
Glycine
pH 8.3

Separating gel 7.5% gel, 0.375 M Tris
dH20
Soluble starch
1.5 M Tris, pH 8.8
Acrylamide/bis (30%T, 2.67%C)
Ammonium persulfate (10%)
TEMED
N,N,N',N'-Tetramethylethylenediamine

Stacking gel 4.0% gel, 0.125 M Tris
dHO2
0.5 M Tris, pH 6.8
Acrylamide/bis (30%T, 2.67%C)
Ammonium persulfate (10%)
TEMED

Marker buffer
0.5 M Tris, pH 6.8
Glycerol
B-Mercaptoethanol
Bromophenol blue


0.5
11.0
0.1
20.0


M
%
%
mg/mg fw
plant


24.8 mM

191.8 mM



24.8 mL
100.0 mg
12.5 mL
12.5 mL
250.0 pL
50.0 gL



6.1 mL
2.5 mL
1.3 mL
50.0 gL
10.0 AL


0.1 M
11.0 %
0.1 %
0.002 %


The pH of preparations were adjusted with HC1


at 12'C.








Slab Electrophoresis

Reservoir, stacking, and separating gel buffers for SDS

PAGE gels as described by Laemmli (1970) were modified for

native PAGE gel preparations (Table 5). Native proteins are

separated based on their charge, molecular weight, and

configuration. The separating gel was amended by

substituting the water phase with a soluble starch solution.

A SE600 vertical slab gel electrophoresis unit (Hoeffer

Scientific Instruments, San Francisco, CA) was operated with

a discontinuous buffer system using two 160 mm long, 180 mm

wide, and 1 mm thick gels.

The 7.5% acrylamide separating gel (Table 5) was

prepared by boiling 100 mg of soluble starch in 24.8 mL of

dHO2 for two min and allowed to cool. Then 12.5 mL of 1.5 M

TRIS (pH 8.8) and 12.5 mL of acrylamide/bis (30%T, 2.67%C)

were added. The solution was degassed for 10 min before

adding 250 jL of ammonium persulfate (10%) and 25 pL of

TEMED. Twenty two and a half milliliters of the acrylamide

solution were immediately transferred to between the glass

sandwich with a pipette and overlayed with a thin layer of

butanol. After 15 min a sharp gel/butanol interphase was

visible. The butanol was poured out of the glass sandwich

and the remaining butanol was rinsed with dH2O before drying

the glass.

The stacking gel solution (Table 5) was prepared by

adding 6.1 mL dHO2 and 2.5 mL of 0.5 M TRIS (pH 6.8) to 1.3






55

mL of acrylamide/bis. The solution was degassed for 10 min

before adding 50 lL of ammonium persulfate (10%) and 10 pL

TEMED. The stacking gel solution was then pipetted into the

remaining space, between the glass sandwich, until 2 mm from

the top of the glass. The comb was inserted into the

stacking gel solution and removed after polymerization.

Wells, formed by the comb, were each loaded with 90 pL

of an appropriately dilute enzyme preparation. Forty

microliters, 60 ML, and 70 AL of enzyme preparation were

adjusted to 90 iL with the marker buffer (Table 5) before

loading into separate wells. After loading, the

electrophoresis was conducted at 35 volts for 2.5 hours at

120C. Each sample was replicated twice on each gel so that

after electrophoresis half the gel could be used for protein

staining and half for enzyme activity staining.



Protein and Enzyme Activity Staining

The quantity of protein loaded into each well for

electrophoresis was determined spectrophotometrically using

Bio-Rad's protein assay (Bio-Rad, Richmond, CA). A protein

standard curve was constructed ranging from 20-140 Mg

protein using bovine serum albumin (United States

Biochemical Corporation, Cleveland, OH). Absorbance at 595

nm was measured with a Hewlett Packard 8451A diode array

spectrophotometer (United States). Twenty microliters, 40

ML, and 60 AL of enzyme preparation were adjusted to 100 ML







56

with extraction buffer and subjected to the standard assay

procedure. The quantity of protein in the extracts was

determined by comparison to the protein standard

curve.

Gels were stained for protein using the method of

Schuler and Zielinski (1989). The gel was placed in 300 mL

of a fixative containing 40% methanol, 10% glacial acetic

acid, and 0.1% coomassie brilliant blue for 30 min and

gently shaken. The gel was then destined in 300 mL of 40%

methanol and 10% glacial acetic acid. The destaining

solution was changed twice over a three hour period. After

clearing the background stain, the gels were photographed as

quickly as possible and dried on a slab gel dryer (Hoeffer

Scientific Instruments SE 1160).

Staining of the gel for starch-degrading enzymes

followed the procedure outlined by Vallejos (1983). The gel

was incubated in a solution consisting of 50 mM sodium

acetate and 1 M calcium chloride adjusted to a pH of 5.6

with HC1 at 350C for 60 min. After incubation, the gel was

rinsed thoroughly with dHO2 and stained in 10 mM iodine and

14 mM potassium iodide. When the zymogram developed a dark

purple background it was rinsed in dHO2 and photographed

quickly before being dried on a slab gel dryer (Hoeffer

Scientific Instruments SE 1160).







57

Results and Discussion

Native starch PAGE gels stained differentially

according to the type of hydrolytic enzyme (data not shown).

a-Amylase activity resulted in a clear band, P-amylase

activity resulted in a red band, and debranching activity

produced a blue band. These results support previous

reports. Ziegler and Beck (1986) and Kakefuda and Duke

(1984) reported the identification of the type of hydrolytic

enzyme activity based on the color of bands after iodine

staining of starch impregnated native gels.

Following electrophoresis, incubation, and staining of

gels containing pangola leaf extracts distinct banding

colors and banding patterns were evident. Interpretation of

the type of enzyme activities were based on the band color

as related to the known a-amylase, P-amylase, and pullanase

samples and Ziegler and Beck (1986) and Kakefuda and Duke

(1984) reports. For reference purposes, zymograms will be

identified by plant treatment with regards to night

temperature and GA3 application as follows: 300C(control),

100C, 100C+GA.

Red bands resulting from exoamylolytic enzyme activity

were not detected regardless of temperature or GA3 treatment

(Figure 9). Enoamylolytic enzyme activity was evident in the

300C treatment by the appearance of eight clear bands. The

bands separated into a fast migrating group (bands 1-5), a

slower migrating pair (bands 6 and 7), and a single slowly







58

migrating band (band 8) (Figure 9). Carter et el. (1972)

previously found one fast migrating band and a slower

migrating pair of bands in pangola. By altering the

electrophoretic conditions, they further separated

endoamylolytic enzymes into a fast migrating pair of bands

and a slower migrating group of five bands (Carter et al.,

1973). The results from this study differ from the

previously cited literature in that an additional

endoamylolytic enzyme was resolved and bands on the gels did

not form similar migratory patterns.

Treatment at 100C reduced the number of visable

endoamylolytic bands. Bands 6 and 8 were not visable when

compared to the 300C treatment (Figure 9). Based on

densitometer tracings, Carter et. al. (1972) and Carter et

al. (1973) reported a reduction in the enzyme activity

within the bands and not a reduction in the number of bands.

Approximately doubling the total protein load in the 10C

treatment relative to the 300C treatment, 83 pg to 42 Ig

respectively, did not result in the appearance of band 6 or

8. This indicates the presence of two chill-sensitive-

endoamylolytic isozymes.

Data presented in Figure 9 supported the hypothesis

that chilling temperature reduced the number of starch-

degrading enzymes; specifically endoamylolytic isozymes. The

remaining endoamylolytic isozymes may not be able to

satisfactorily hydrolyze starch, particularly if the









Treatment


30C


10C


10"C+GA


5-
4-
3-
2-
1-


Protein


9g
42 83 55 73 48

Fig. 9. Effect of temperature and GA3 on expression of
starch-degrading-hydrolytic enzymes in pangola.
Areas of endoamylase activity show up as white
(bands 1-8) that of debranching enzyme activity blue
(band 9) against the dark background of the
amylose-amylopectin containing gel. F refers to
the bromophenol blue front marker. Micrograms
protein refers to the total protein.







60

remaining endoamylases have a lower activity as reported by

Carter et al. (1972) and Carter et al. (1973). The

consequence of is a starch buildup.

Coomassie stain for total protein did not reveal a

specific protein band at endoamylase enzyme position 6 or 8

respective of protein load or treatment (Figure 10). The

mechanism of sensitivity remains unclear, i.e.

continued protein synthesis with reduced enzyme activity or

termination of protein synthesis.

The number of hydrolytic enzyme bands in the 100C

treatment zymogram were not altered as a result of treatment

with GA3 (Figure 9). Approximately doubling the total

protein load in the 100C+GA treatment relative to the 10C

treatment did not increase the number of detectable bands

(Figure 9). Carter et al. (1973) and Karabassi et al. (1971)

reported that relative activities of amylolytic enzymes and

not the number of isozymes were increased by applying GA3.

In both studies 10 AM GA3 was applied to pangolagrass 10

hours prior to low-temperature treatment.

The data presented in Figure 9 did not support the

hypothesis that GA3 nullifies the effect of low temperature

on the reduction in number of starch-degrading enzymes.

A slow moving blue band (band 9), indicative of

debranching enzyme activity, was evident in the 30C

treatment. Kakefuda et al. (1986) identified a debranching








61


Treatment


10'C


7 C C


Protein


42 42 82


55 55 83
55 55 83


48 48 73


Fig. 10. Effect of temperature and GA3 on production and
expression of starch-degrading-hydrolytic enzymes
in pangola. Zymograms (z) of hydrolytic
activity and coomassie protein stains (c) are
presented for each treatment. Micrograms protein
refers to the total protein.


30"C


7. .


1 -C+GA







62

enzyme in crude pea leaf extract using electrophoresis as

described by Kakefuda and Duke (1984). The slow moving blue

band remained unchanged regardless of temperature or GA3

treatment (Figure 9).



Conclusions

Current studies indicate that anabolism of assimilated

starch in chloroplasts is accomplished by the cooperation of

hydrolytic and phosphorolytic enzymes. The cellular location

and physiological role of these enzymes remain unclear and

need to be identified for the type of plant in question.

Eight endoamylolytic enzymes and one debranching enzyme

have been identified in pangola leaf tissue by native starch

gel electrophoresis. Two temperature-sensitive-

endoamylolytic enzymes were identified. The mechanism of

sensitivity remains unclear i.e., continued protein

synthesis with reduced enzyme activity or termination of

protein synthesis.

The number of starch-degrading-hydrolytic enzymes was

unaffected by applying GA3 to the plant prior to low-

temperature exposure.












CHAPTER IV
EVALUATION OF HYDROLYTIC ENZYMES IN FREEZING-TEMPERATURE-
TOLERANT SOMACLONES OF PANGOLA



This study tested the tentative assumption that chill-

tolerant-endoamylolytic isozymes may be present in low-

temperature-tolerant somaclones of pangolagrass.

The objectives of this study were a) to evaluate low-

temperature-tolerant-pangola somaclones for variation in the

expression of hydrolytic enzymes and b) to select plants

that retain hydrolytic enzyme activity which may assist in

starch breakdown after exposure to low temperatures.

The rapidity of screening and simple inheritance make

isozymes a powerful tool in the analysis of variation.

Variation in isozyme patterns of starch-hydrolyzing enzymes

has been studied extensively during the germination process

in seeds (Bewley and Black, 1985; Jacobsen and Beach, 1985;

Atzorn and Weiler, 1983; Nolan and Ho, 1988).

Starch-hydrolyzing isozyme patterns have been used to

show induced variation within the same seed type. Henke

(1981) used a half-seed a-amylase assay capable of screening

thousands of half-seeds in a short time. After screening

100,000 mutagenized barley seeds for the ability to express

a-amylase in the absence of GA3 or in the presence of GA3







64

plus an inhibitor, he found 1% of the seeds to be variant.

Embryos of the variant half-seeds were grown to maturity and

selfed. The resulting seed provided evidence for inheritance

of a mutant trait. Ho et al.(1980) used a comparable

approach and obtained similar results.

Few studies used starch-hydrolyzing enzymes for the

identification of somatic variants. A somaclonal variant

which effects the isozyme pattern of one of the seed protein

complexes of wheat, namely a-amylase, was reported by Larkin

et al. (1984). Seeds from 68 somaclones were assayed to

identify eight somatic variants. Of the variant plants, four

were GA3-insensitive, one was GA3-supersensitive, and four

were ABA-insensitive. The characteristics were heritable

through two seed generations.

Ryan and Scowcroft (1987) obtained somatic variation

within P-amylase isozymes in wheat seeds. One variant seed

phenotype was observed from among 149 regenerated plants.

This variant was characterized by five new isozyme bands, as

well as an increased intensity of two previously existing

bands. The variant segregated in a mendelian manner, without

recombination between bands, and mitosis and meiosis were

cytogenetically normal in both the homozygous variant and in

the F1 backcross. This new variant has not been previously

observed in the 10 known p-amylase phenotypic groups and

gives the appearance of being a new gene.







65

To date no reports have been made of variation in the

starch-hydrolyzing isozyme banding patterns in

photosynthetic tissue of somatically regenerated plants.

Exposing photosynthetic tissue of regenerated plants to low-

temperature treatments may be useful in identifying variant

plants which express an increased number of hydrolytic

enzymes under low-temperature conditions.



Materials and Methods

Plant Material and Treatment Application

Sixteen vegetatively propagated plants were produced

from each of the 10 previously selected low-temperature

tolerant regenerated plants. Vegetatively propagated

regenerated plants and vegetatively propagated source plants

were maintained in the greenhouse as described in chapter

II. Experiments were conducted during the month of August,

1989. Temperature treatments were imposed at the end of the

photoperiod. One vegetatively propagated regenerate plant,

from each of the 10 selected regenerated plants, and one

vegetatively propagated source plant was placed in a

darkened 300C Percival growth chamber. An identical

combination of plants was placed in a darkened 10C Percival

growth chamber. Plants remained in the growth chambers for

12 hours until sampling.







66

The application of temperature treatment and subsequent

analysis of starch-hydrolyzing enzymes were replicated eight

times.



Whole Leaf Extract Preparation

Protein extracts were prepared for native starch PAGE

slab gel electrophoresis as described in Chapter III.



Slab Electrophoresis

Electrophoretic separation of the protein extracts was

preformed as presented in Chapter III.



Protein and Enzyme Activity Staining

The quantity of total protein, coomassie blue staining

procedure for the total protein, and I2-KI stain for starch-

degrading enzyme activity was followed as described in

Chapter III.



Results and Discussion

Zymograms of the 10 regenerated plants from Chapter II

contained the characteristic hydrolytic banding pattern

found in the vegetatively propagated control plants. Figure

11 shows the comparison of a typical zymogram from the

regenerated plants to that of a vegetatively propagated

plant. No variation in the isozyme banding pattern was







67

Treatment


30C


10"C


1 2 3 4


7-

6-



5-
4-
3-
1--













F-


Fig. 11. Zymograms of starch-hydrolyzing enzymes isolated
from pangola plants that were exposed to 300C
or 10 C. White bands (bands 1-8) show endoamylase
activity, blue band (band 9) indicates debranching
enzyme activity. Lanes 1 and 3 represent the
vegetatively propagated control plants. Lanes 2 and
4 represent plants regenerated from tissue culture.
F refers to the bromophenol blue front marker.







68

observed between the regenerated plants or between

regenerated plants and vegetatively propagated control

plants respective of temperature treatment.

Nine hydrolytic bands were evident in zymograms from

the 300C treatment (Figure 11). Exoamylolytic activity was

not detected. Endoamylolytic activity resulted in eight

clear bands which separated into a fast migrating group

(bands 1-5), a slower migrating pair (band 6 and 7), and a

single slowly migrating band (band 8). Debranching enzyme

activity resulted in a single extremely slow migrating blue

band.

The 100C treatment resulted in a banding pattern

identical to the 300C treatment except for the elimination

of bands six and eight (Figure 11). Data do not support the

hypothesis that somatic variation in low-temperature

tolerant somaclones can be used to obtain chill-tolerant

endoamylolytic isozymes. Amplifying the number of test

plants would increase the probability of detecting a desired

variation.

Coomassie staining of the protein did not show specific

banding for the endoamylolytic protein at band number six or

eight for regenerated plants or vegetatively propagated

control plants in either temperature treatment (Figure 12).

Evidently coomassie stain was not sensitive enough to detect

the quantity of protein at band six or eight position.

Silver stain which detects 10-50 ng of protein and is







69



Treatment

300C 100C

1 2 3 4 5 6

9-

8-



7- .
6-



5-
4-
3-
2-
1-












F-

Fig. 12. Zymograms of starch-hydrolyzing enzymes and total
protein profiles from pangola plants exposed
to 300C or 100C. Lanes 1 and 4 are zymograms. White
bands (bands 1-8) show endoamylase activity, blue
band (band 9) indicates debranching enzyme
activity. Coomassie blue stained proteins are
presented in lane 2 vegetativelyy propagated
control plants) and lanes 3 and 6 (plants
regenerated from tissue culture).







70

approximately 100 times more sensitive than coomassie stain

may detect protein at band six or eight position.



Conclusions

All of the regenerated plants that were selected in

chapter II for freezing-temperature tolerance were similar

in that they showed no somaclonal variation in starch-

hydrolyzing enzymes or total protein banding patterns. The

characteristic banding patterns of hydrolytic enzymes in the

30C treatment (eight endoamylolytic and one debranching

enzyme) and the 100C treatment (six endoamylolytic and one

debranching enzyme) were consistent between regenerated

plants and between regenerated plants and vegetatively

propagated control plants. The regenerated plants and the

vegetatively propagated control plant had two similar

temperature-sensitive-endoamylolytic enzymes.












CHAPTER V
SUMMARY AND CONCLUSIONS



A large number of factors contribute to the reduction

of growth of pangola under freezing and chilling-temperature

conditions. Several of the major factors are loss of

plasmalemma integrity, reduction in photosynthetic electron

transport, and decreased activity of assimilated starch-

hydrolyzing enzymes. Irrespective of being distinct factors,

the action and interaction of all these factors contribute

to a common result: reduced growth resulting from freezing

or chilling-temperature exposure.

Several experiments were conducted to induce phenotypic

variation and select variant plants for a reduction in low-

temperature damage. The studies reported in Chapter II were

conducted with the objectives: a) to generate callus lines

of pangola; b) to select freezing or chilling-temperature

resistant cell lines in vitro; c) to regenerate whole plants

from selected cultures; and d) to test regenerated plants

for increased tolerance to freezing and chilling temperature

at the organelle and cellular level. Somatic embryogenesis

in pangola followed a pattern of development similar to

those described for other grasses. Increasing the in vitro







stress reduced callus survivability and competency for

supporting embryogenesis. Regenerated pangola plants

sustained a range of freeze-induced damage to their

plasmalemma membranes. Selection of in vitro cells that

survived freezing or chilling temperature did not result in

plants, regenerated from these cells, that were freezing-

temperature tolerant. Apparently, increasing the freezing or

chilling-temperature selection pressure in culture reduced

the proportion of regenerated plants that were freezing-

temperature tolerant. Somaclones with increased freeze

tolerance relative to vegetative control plants were

obtained. Within this group of plants, no variation in the

extent of chill damage to the photosynthetic electron

transport chain within the thylakoid membrane was observed.

The objectives reported in Chapter III were a) to

evaluate the influence of chilling temperature on the

expression of starch-hydrolyzing enzymes; b) to identify

hydrolytic enzymes which may assist in starch breakdown

after chilling-temperature exposure; and c) to evaluate the

effect of GA3 on the expression of starch-hydrolyzing

enzymes during chilling-temperature conditions.

Current studies indicate that anabolism of assimilated

starch in chloroplasts is accomplished by the cooperation of

hydrolytic and phosphorolytic enzymes. The cellular location

and physiological role of these enzymes remain unclear and

need to be identified for the type of plant in question.







73

Eight endoamylolytic and one debranching enzyme have been

identified in pangola leaf tissue by native starch gel

electrophoresis. Two temperature-sensitive-endoamylolytic

enzymes were identified. The mechanism of sensitivity

remains unclear, i.e., continued protein synthesis with

reduced enzyme activity or termination of protein synthesis.

The number of starch-degrading-hydrolytic enzymes was

unaffected by applying GA3 to the plant prior to chilling-

temperature exposure.

Chapter IV reports on research conducted with the

objectives: a) to evaluate the chill-temperature-tolerant

pangola somaclones for variation in the expression of

hydrolytic enzymes and b) to select plants that retain

hydrolytic enzyme activity which may assist in starch

breakdown after exposure to chilling temperature. All of the

regenerated plants that were selected for Freezing-

temperature tolerance in Chapter II showed no somaclonal

variation with respect to the number of starch-hydrolyzing

enzymes or total protein banding patterns. The

characteristic banding patterns of hydrolytic enzymes in the

300 treatment (eight endoamylolytic enzymes and one

debranching enzyme) and the 100C treatment (six

endoamylolytic enzymes and one debranching enzyme) were

consistent between regenerated plants and between

regenerated plants and vegetatively propagated control

plants. The regenerated plants and the vegetatively







74

propagated control plant had two temperature-sensitive-

endoamylolytic enzymes.

Tissue cultured pangola circumvents sterility barriers

which prevent plant improvement via conventional breeding

programs. Somatic variants of pangola can be regenerated

from tissue culture and selected for increased tolerance to

freezing-temperature conditions. The selection factor for

freeze tolerance is plasmalemma integrity.

Direction for future research should be threefold.

First, field evaluations of the selected freeze-temperature

tolerant plants should be continued. Forage characteristics

such as plant spread, leaf area, yield, flowering date, rust

resistance, crude protein, organic matter, and percent

organic matter that is digested by rumen microorganisms are

being studied. Second, densitometer tracings of gels should

be obtained for analyzing relative activities of the

identified amylolytic enzymes. Temperature and GA3

treatments should be imposed as presented in these studies.

Third, a larger number of regenerated plants (larger than

263 regenerates) should be produced and screened for

freezing and chilling-temperature tolerance. This may lead

to the identification of individuals with increased

tolerance in all three selection factors.












REFERENCES


Ahloowalia, B. S. 1982. Plant regeneration from callus
culture in wheat. Crop Sci. 22: 405-410.

Ahloowalia, B. S. 1983. Spectrum of variation in somaclones
of triploid ryegrass. Crop Sci. 23: 1141-1147.

Ahloowalia, B. S. 1986. Limitation to the use of somaclonal
variation in crop improvement. p. 14-27. In J. Semal
(ed.) Somaclonal variations and crop improvement.
Maritnus Nijhoff Publishers, Boston.

Atkin, R. K., G. E. Barton, and D. K. Robinson. 1973. Effect
of root-growing temperature on growth substances in
xylem exudate of Zea mavs. J. Environ. Bot. 24:475-487.

Atzorn, R., and E. W. Weiler. 1983. The role of endogenous
gibberellins in the formation of a-amylase by aleurone
layers of germinating barley caryopses. Planta 159:
289-299.

Bajaj, Y. P. S., B. S. Sidhu, and V. K. Dubey. 1981.
Regeneration of genetically diverse plants from tissue
cultures of forage grass Panicum sp. Euphytica 30: 135-
140.

Bayliss, M. W. 1976. Variation of cell cycle duration within
suspension cultures of Daucus carota and its
consequence for the induction of ploidy changes with
colchicine. Protoplasma 88: 279-285.

Beck, E. 1985. The degradation of transitory starch granules
in chloroplasts. p. 27-44. In R. L. Heath, and J.
Preiss (eds.) Regulation of carbon partitioning in
photosynthetic tissue. American Soc. Plant
Physiologists. Rockville, MD.

Beck, E., P. Pongratz, and I. Reuter. 1981. The amylolytic
system of isolated chloroplasts and its role in the
breakdown of assimilatory starch. p. 529-538. In G.
Akoyunoglou (ed.) Photosynthesis. Proc. 5th Int. Cong.
Photosynth., Balaban Int. Science Service,
Philadelphia.









Beers E. P., and S. H. Duke. 1988. Location of a-amylase in
the apoplast of pea (Pisum sativum) stems. Plant
Physiol. 87: 799-802.

Benzion, G., R. L. Phillips, and H. W. Rines. 1986. Case
histories of genetic variability in vitro: oats and
maize. p. 73-89. In I.K. Vasil (ed.) Cell culture and
somatic cell genetics of plants. Academic Press, New
York.

Berry, J., and 0. Bjorkman. 1980. Photosynthetic response
and adaptation to temperature in higher plants. Annu.
Rev. Plant Physiol. 31: 491-543.

Bewley, J. D., and M. Black. 1985. Control of the
mobilization of stored reserves. p. 305-325. In J. D.
Bewley and M. Black (eds.) Seed physiology of
development and germination. Plenum Press, New York.

Breidenbach R. W., and A. J. Waring. 1977. Response to
chilling of tomato seedlings and cell in suspension
cultures. Plant Physiol. 60: 190-192.

Burgess, J. 1985. The regulation of development. p. 207-233.
In J. Burgess (ed.) Plant cell development. Cambridge
University Press, Cambridge, UK.

Callis, J., and T.-H. D. Ho. 1983. Multiple molecular forms
of the gibberellin induced a-amylase from the aleurone
layer of barley seeds. Arch. Biochem. Biophys. 224:
224-234.

Carter, J. L., L. A. Garrard, and S. H. West. 1972. Starch
degrading enzymes of temperate and tropical species.
Phytochemistry 11: 2423-2428.

Carter, J. L., L. A. Garrard, and S. H. West. 1973. Effect
of gibberellic acid on starch degrading enzymes in
leaves of Digitaria decumbens. Phytochemistry 12: 251-
254.

Carter, J. L., L. A. Garrard, and S. H. West. 1974.
Amylolytic activity of orchardgrass and starch and
sucrose contents of orchardgrass vs pangola digitgrass
leaf blades as influenced by night temperature and
gibberellic acid. Crop Sci. 14: 384-387.

Chaleff, R. S., and N. F. Bascomb. 1987. Genetic and
biochemical evidence for multiple forms of
acetolacetate synthase in Nicotiana tabacum. Mol. Gen.
Genet. 210: 33-39.









Chang, C. W. 1982. Enzymatic degradation of starch in cotton
leaves. Phytochemistry 21: 1263-1269.

Chatterton, N. J., C. E. Carlson, W. E. Hungerford, and D.
R. Lee. 1972. Effect of tillering and cool nights on
photosynthesis and chloroplast starch in pangola. Crop
Sci. 12: 206-208.

Chen, P. M., M. J. Burke, and P. H. Li. 1976. The frost
hardiness of several solanum species in relation to the
freezing of water, melting point depression, and tissue
water content. Bot. Gaz. 137: 313-317.

Chen, W.-H., W. Cockburn, and H. E. Street. 1982. Cell
plating and selection of cold-tolerant cell lines in
sugarcane. p. 485-486. In A. Fujiwara (ed.) Plant
tissue culture 1982.Proc. 5th International Congress of
Plant Tissue and Cell Culture, Lake Yamanaka, Japan.
July 11-16. 1982. Abe Photo Printing Co., Tokyo,
Japan.

Christiansen, M. N., and J. B. St. John. 1984. Chemical
modification of plant response to temperature extremes.
p. 235-244. In R. L. Ory and F. R. Rittig (eds.)
Bioregulators chemistry and use. 186h Meeting of
American Chemical Society, Washington, DC. 28 Aug.-2
Sept. American Chemical Society, USA.

Crookston, R. L., J. O. O'Toole, J. L. Ozbun, and D. H.
Wallace. 1974. Photosynthetic depression in beans after
exposure to cold for one night. Crop Sci. 14: 457-464.

Demarly, Y. 1986. Experimental and theoretical approach of
in vitro variations. p. 84-99. In Somaclonal variation
and crop improvement. Martinus Nijhoff, Boston.

Dexter, S. T. 1932. Studies of the hardiness of plants: a
modification of the newton pressure methods for small
samples. Plant Physiol. 7: 721-726.

Dix, P. J. 1977. Chilling resistance is not transmitted
sexually in plants regenerated from Nicotinia
svlvestris cell lines. Z. Pflanzenphysiol. 84: 223-226.

Dix, P. J. 1980. Environmental stress resistance. selection
in plant cell cultures, p. 183-186. In F. Sala, B.
Parisi, R. Cella, 0. Ciferr (eds.) Plant cell cultures
results and perspectives. Elsevier/North-Holland
Biomedical Press, New York.









Dix, P. J., and H. E. Street. 1976. Selection of plant cell
lines with enhanced chilling resistance. Ann. Bot. 40:
903-910.

Drake, B. G., and F. B. Salisbury. 1972. Aftereffects of low
and high temperature pretreatment on leaf resistance in
Xanthium. Plant Physiol. 50: 572-575.

Dudeck, A. E., and C. H. Peacock. 1985. 'Tifdwarf'
bermudagrass growth response to carboxin and GA3 during
suboptimum temperatures. HortScience 20: 936-938.

Echeverria, E., and C. D. Boyer. 1986. Localization of
starch biosynthetic and degradative enzymes in maize
leaves. Amer. J. Bot. 73: 167-171.

Elmore, J. A., C. I. Franklin, and R. A. Gonzales. 1988.
Economically important somaclonal variants of a forage
grass (Diqitaria sungunalis L.). In vitro cellular and
developmental biology. 24: 37A.

Foroughi-Wehr, B., W. Friedt, R. Schuchmann, F. Kohler, and
G. Wenzel. 1986. In vitro selection for resistance. p.
35-44. In J. Semal (ed.) Somaclonal variations and crop
improvement. Martinus Nijhoff Publishers, Boston.

Gamborg, O. L., F. Constable, and R. A. Miller. 1970.
Embryogenesis and plant production of albino plants
from cell cultures of Bromus inermis. Planta 95: 353-
358.

Garrard, L. A. and S. H. West. 1972. Suboptimal temperature
and assimilate accumulation in leaves of 'Pangola"
digitgrass (Digitaria decumbens Stent.). Crop Sci. 12:
621-623.

Gross, R. W., and E. T. Bingham. 1986. An unstable
anthocyanin mutant recovered from tissue culture of
alfalfa (Medicago sativa). Plant Cell Rep. 5: 104-107.

Gusta, L. V., J. D. Butler, C. Rajashekar, and M. J. Burke.
1980. Freezing resistance of perennial turfgrass.
HortSci. 15: 494-496.

Hallgren, J. E., E. Sunbom, and M. Strand. 1982.
Photosynthetic responses to low temperature in Betula
pubescens and Betula tortuosa. Physiol. Plant 54: 275-
282.

Hansteen-Cramer. B. 1922. Zur biochemie und physiologie der
grenzschicten lebender pflanzenzellen. Medl. Norges
Land bruksh. 2:1-160.










Heinrich, G., and H. Lasch. 1987. Photoinhibition of
photosynthesis. Studies on mechanisms of damage and
protection in chloroplasts. p. 19-26. In J. Biggins
(ed.) Progress in photosynthesis research. Vol. 4.
Martinus Nijhoff Publishers, Boston.

Henke, R. R. 1981. Selection of biochemical mutants in plant
cell cultures: Some considerations. Environmental and
Experimental Bot. 21: 347-357.

Herold, A., R. C. Leegood, P. H. McNeil, and S. P. Robinson.
1981. Accumulation of maltose during photosynthesis in
protoplasts isolated from spinach leaves treated with
mannose. Plant Physiol. 67: 85-88.

Hetherington, S. E., R. M. Smillie, A. K. Hardacre, and H.
A. Eagles. 1983. Using chlorophyll fluorescence in vivo
to measure the chilling tolerances of different
populations of maize. Aust. J. Plant Physiol. 10: 247-
256.

Hillard, J. H. 1975. Eragrostis curvula: Influence of low
temperatures on starch accumulation, amylolytic
activity and growth. Crop Sci. 15: 293-294.

Hillard, J. H., and S. H. West. 1970. Starch accumulation
associated with growth reduction at low temperatures in
a tropical plant. Science 168: 494-496.

Ho. T.-H. D., S.-C. Shih, and A. Kleinhots. 1980. Screening
for barley mutants with altered hormone sensitivity in
their aleurone layers. Plant Physiol. 66: 153-157.

Hodges, E. M., G. B. Killinger, J. E. McCaleb, O. C. Ruelke,
R. L. Allen, JR., S. C. Schank, and A. E. Kretschmer,
JR. 1975. Pangola digitgrass. Fla. Agr. Exp. Sta. Bul.
718A.

Hodges, E. M. and D. W. Jones. 1958. Winterkilling of
pangolagrass pastures. Fla. Cattleman and Livestock J.
22: 24.

Horsch, R. B., J. E. Fry, N. L. Hoffman, D. Eicholtz, S. C.
Robergs, and R. J. Fraley. 1985. A simple method for
transferring genes into plants. Science 227: 1229-1231.

Hughes, K. 1983. Selection for herbicide resistance. p. 442-
460. In D. A. Evins, W. R. Sharp, P. V. Ammirato, and
Y. Yamada (eds.) Handbook of plant cell and tissue
culture. Macmillan Publishing Co., New York.









Jacobsen, J. V., and T. J. V. Higgins. 1982.
Characterization of the a-amylases synthesized by
aleurone layers of Himalaya barley in response to
gibberellic acid. Plant Physiol. 70: 1647-1653.

Jacobsen, J. V., and L. R. Beach. 1985. Control of
transcription of a-amylase and rRNA genes in barley
aleurone protoplasts by gibberellin and abscisic acid.
Nature 316: 275-277.

Jensen, W. A. 1962. Carbohydrates and cell wall
constituents. p. 175-209. In W. A. Jensen (ed.)
Botanical histochemistry. W. H. Freeman and Co., San
Francisco.

Jones, C. A. 1985. Temperature. p. 139-161. In C. A. Jones
(ed.) C4 grasses and cereals growth, development, and
stress response. John Wiley and Sons, New York.

Kakefuda, G., and S. H. Duke. 1984. Electrophoretic transfer
as a technique for the detection and identification of
plant amylolytic enzymes in polyacrylamide gels. Plant
Physiol. 75: 278-280.

Kakefuda, G., S. H. Duke, and M. S. Hostak. 1986.
Chloroplast and extrachloroplastic starch-degrading
enzymes in Pisum sativum L. Planta 168: 175-182.

Karbassi, P., L. A. Garrard, and S. H. West. 1971. Reversal
of low temperature effects on a tropical plant by
gibberellic acid. Crop Sci. 11: 755-757.

Karbassi, P., S. H. West, and L. A. Garrard. 1972.
Amylolytic activity in leaves of a tropical and
temperate grass. Crop Sci. 12: 58-60.

Karnok, K. J., and J. B. Beard. 1986. The effects of
chilling temperatures on the chloroplast ultrastructure
of Cynodon dactylon as affected by gibberellic acid.
Acta Agron. Acad. Sci. Hung. 34: 259-266.

Kasperbauer, M. J., and G. C. Eizenga. 1985. Tall fescue
doubled haploids via tissue culture and plant
regeneration. Crop Sci. 25: 1091.

Kleinedorst, A., and R. Brouwer. 1972. The effect of local
cooling on growth and water content of plants. Neth. J.
Agric. Sci. 20: 203-217.









Laemmli, U. K. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature
227:680.

Larkin, P. J. 1987. Somaclonal variation: history, method,
and meaning. Iowa State J. Res. 61: 393-434.

Larkin, P. J., and W. R. Scowcroft. 1981. Somaclonal
variation--A novel source of variability from cell
cultures for plant improvement. Theor. Appl. Genet.,60:
197-214.

Larkin, P. J., and W. R. Scowcroft. 1983. Somaclonal
variation and crop improvement. p. 289-314. In C. P.
Meredith, and A. Hollander (eds.) Genetic engineering
of plants. Plenum, New York.

Larkin, P. J., S. A. Ryan, R. I. S. Bretell, and W. R.
Scowcroft. 1984. Heritable somaclonal variation in
wheat. Theor. Appl. Genet. 67: 443-455.

Larkin, P. J., R. I. S. Brettell, S. A. Ryan, P. A. Davies,
M. A. Pallotta, and W. R. Scowcroft. 1985. Somaclonal
variation: Impact on plant biology and breeding
strategies. p. 83-100. In P. Day, M. Zaitlin, and A.
Hollander (eds.) Biotechnology in plant science.

Lawlor, D. 1987. Photosynthesis by leaves. p. 217-242. In D.
Lawlor (ed.) Photosynthesis: Metabolism, control and
physiology. Singapore Publishers Ltd., Singapore.

Levi, C., and J. Preiss. 1978. Amylopectin degradation in
pea chloroplast extracts. Plant Physiol. 61: 218-220.

Levitt, J. 1972. Freezing resistance-types, measurements,
and changes. p. 75-101. In J. Levitt (ed.) Responses of
plants to environmental stresses. Academic Press, New
York.

Lin, T.-P., S. R. Spilatro, and J. Preiss. 1988. Subcellular
localization and characterization of amylase in
Arabidopsis leaf. Plant Physiol. 86: 251-259.

Long, S. P. 1983. Effects of temperature on photosynthesis.
p. 237-244. In R. Marcelle, H. Clijster, and M. Van
Poucke (eds.) Effects of stress on photosynthesis.
Martinus Nijhoff, The Hague.









Low, P. S., D. R. Ort, W. A. Cramer, J. Whitmarsh, and B.
Martin. 1984. Search for an endotherm in chloroplast
lamellar membranes associated with chilling-inhibition
of photosynthesis. Arch. Biochem. Biophys. 231: 336-
344.

Lyons, J. M., J. K. Raison, and P. L. Steponkus. 1979. The
plant membrane in response to low temperature: An
overview. p. 1-24. In J. M. Lyons, D. Graham, and J. K.
Rasion (eds.) Low temperature stress in crop plants.
Academic Press, New York.

Machado, V. S., J. Shupe, and W. A. Keller. 1984.
Chloroplastic inherited atrazine resistance transmitted
through anther culture in rutabaga. Z. Pflanzenuchtg.
95: 179-184.

Mahfouz, M. N., M. T. deBoucaud, and J. M. Gaultier. 1983.
Caryological analysis of single cell clones of tobacco.
Relation between the ploidy and the intensity of the
caulogenesis. Z. Pflanzenphysiol. 109: 251-257.

Malmberg, R. L. 1979. Temperature-sensitive variants of
Nicotiana tabacum isolated from somatic cell culture.
Genetics 92: 215-221.

Margulies, M. M. 1972. Effect of cold storage of bean leaves
on photosynthetic relations of isolated chloroplasts.
Inability to donate electrons to photosystem II and
relation to manganese content. Biochem. et Biophysica.
Acta 267: 96-103.

Marousky, F. J., and S. H. West. 1988. Shoot development
from ovaries and somatic embryogenesis in pangolagrass.
In vitro cellular and developmental biology. 24: 37A.

Martin, B. 1986. Arrhenius plots and the involvement of
thermotropic phase transitions of the thylakoid
membrane in chilling impairment of photosynthesis in
thermophilic higher plants. Plant, Cell Environment 9:
323-331.

Marx, J. L. 1989. Tracing hormone action in the cell.
Science 245: 1446-1447.

Matthewes, P. S., and I. K. Vasil. 1975. The dynamics of
cell proliferation in haploid and diploid tissue of
Nicotiana tabacum. Z. Pflanzenphysiol. 77: 222-236.









Meins, F., 1974. Mechanisms underlying the persistence of
tumor autonomy in crown-gall disease. p. 233-264. In H.
E. Street (ed.) Tissue culture and plant science.
Academic Press, New York.

Meyer, K. H., and G. C. Gibbons. 1951. The present status of
starch chemistry. Adv. in Enzymol. 12: 341-377.

Mitra, J., and F. C. Steward. 1961. Growth induction in
cultures of Haplopappus reacilis. II. The behavior of
the nucleus. Am. J. Bot. 48: 358-368.

Muller, A. J., and R. Grafe. 1978. Isolation and
characterization of cell lines of Nicotiana tabacum.
Mol. Gen. Genet. 161: 67-76.

Sunday, C. J., and Govindjee. 1969. Light-induced changes in
the fluorescence yield of chlorophyll A in vivo III.
The dip and the peak in the fluorescence transient of
Chlorella pyrenoidosa. Biophys. J. 9: 1-21.

Murashige, T., and F. Skoog. 1962. A revised medium for
rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant. 15: 473-497.

Murata, N., and D. C. Fork. 1975. Temperature dependence of
chlorophyll a fluorescence in relation to the physical
phase of membrane lipids in algae and higher plants.
Plant Physiol. 56: 791-796.

Nolan, R. C., L.-S. Lin, and T.-H. D. Ho. 1987. The effect
of abscisic acid on the differential expression of a-
amylase isozymes in barley aleurone layers. Plant Mol.
Biol. 8: 13-22.

Nolan, R. C., and T.-H. D. Ho. 1988. Hormonal regulation of
a-amylase expression in barley aleurone layers. Plant
physiol. 00: 588-593.

Okita, T. W., and J. Preiss. 1980. Starch degradation in
spinach leaves. Isolation and characterization of the
amylase and R-enzyme of spinach leaves. Plant Physiol.
66: 870-876.

Oono, K. 1981. Trop. Agric. Res. Ser. 11: 109-123.

Oquist, G. 1983. Effects of low temperature on
photosynthesis. Plant, Cell and Environment 6: 281-300.









Oquist, G. 1987. Environmental stress and photosynthesis p.
1-10. In J. Biggins (ed.) Progress in photosynthesis
research. vol. 4. Martinus Nijhoff Publishers, Boston.

Orton, T. J. 1984. Somaclonal variation: Theoretical and
practical considerations. p. 427-468. In P. J.
Gustafson (ed.) Gene manipulation in plant improvement.
Plenum Press, New York.

Palta, J. P., J. Levitt, and E. J. Stadelmann. 1977a.
Freezing injury in onion bulb cells. Plant Physiol. 60:
393-397.

Palta, J. P., J. Levitt, and E. J. Stadelmann. 1977b.
Freezing tolerance of onion bulbs and significance of
freeze-induced tissue infiltration. Cryobiol. 14: 614-
619.

Papageorgiou, G. 1975. Chlorophyll fluorescence: an
intrinsic probe of photosynthesis. p. 319-371. In
Govindjee (ed) Bioenergetics of photosynthesis.
Academic Press, New York.

Patterson, B. D., T. Murata, and D. Graham. 1976.
Electrolyte leakage induced by chilling in Passiflora
species tolerant to different climates. Aust. J. Plant
Physiol. 3: 435-442.

Pearson, C. J., and G. A. Derrick. 1977. Thermal adaptation
of pennisetum: Leaf photosynthesis and photosynthate
translocation. Aust. J. Plant Physiol. 4: 763-769.

Peavey, D. C., M. Steup, and M. Gibbs. 1977.
Characterization of starch breakdown in isolated
spinach chloroplasts. Plant Physiol. 60: 305-308.


Percival, M., M. Bradbury, D. B. Hayden, and N. R. Baker.
1987. Modification of the photochemical apparatus in
maize by photoinhibitory stress at low temperature. p.
47-50. In J. Biggens (ed.) Progress in photosynthesis
research. Vol. 4. Martinus Nijhoff Publishers, Boston.

Preiss, J. 1982. Regulation of the biosynthesis and
degradation of starch. Ann. Rev. Plant Physiol. 33:
431-454.

Preiss, J., and C. Levi. 1979. Metabolism of starch in
leaves. p. 282-312. In M. Gibbs, and E. Latzko (eds.)
Photosynthesis II, Encyclopedia of plant physiology.
Vol. 6. Springer, Berlin.









Preiss, J., C. Levi, and T. W. Okita. 1980. Characterization
of the spinach leaf phosphorylase. Plant Physiol. 66:
864-869.

Preiss, J., M. Bloom, M. Morell, V. L. Knowles, W. C.
Plaxton, T. W. Okita, R. Larson, A. C. Harmon, and C.
Putnam-Evans. 1987. Regulation of starch synthesis:
enzyomatical and genetic studies. p. 133-152. In G.
Bruening, J. Harada, T. Koguge, and A. Hollaender
(eds.) Tailoring genes for crop improvement an
agricultural perspective. Plenum Press, New York.

Qureshi, J. A., H. A. Collin, K. Harwick, and D. A. Thurman.
1981. Metal tolerances in tissue cultures of
Anthoxanthum odoratum. Plant Cell Reports 1: 80-82.

Reisch, B. 1983. Genetic variability in regenerated plants.
p. 748-769. In D. A. Evans, W. R. Sharp, P. V.
Ammirato, and Y. Yamada (eds.) Handbook of plant cell
culture. Macmillian Publishing Company, New York.

Robinson, N. L., and J. Preiss. 1987. Localization of
carbohydrate metabolizing enzyme in guard cells of
Commelina communis. Plant Physiol. 85: 360-364.

Ryan, S. A., and W. R. Scowcroft. 1987. A somaclonal variant
of wheat with additional P-amylase isozymes. Theor.
Appl. Genet. 73: 459-464.

Ryan, S. A., P. J. Larkin, and F. W. Ellison. 1987.
Somaclonal variation in some agronomic and quality
parameters in wheat. Theor. Appl. Genetics 74: 77-82.

Salisburry, F. B. and C. W. Ross. 1985a. Respiration. p.
229-242. In F. B. Salisburry, and C. W. Ross (eds.)
Plant physiology. Wadsworth Publishing Co. Inc.,
California.

Salisburry, F. B. and C. W. Ross. 1985b. Carbon dioxide
fixation and carbohydrate synthesis. p. 136-159. In F.
B. Salisburry, and C. W. Ross (eds.) Plant physiology.
Wadsworth Publishing Co. Inc., California.

Schuler, M. A., and R. E. Zielinski. 1989. Methods in plant
molecular biology. Academic Press, New York.

Scowcroft, W. R. 1985. Somaclonal variation: the myth of
clonal uniformity, p. 217. In B. Hohn and E. S. Dennis
(eds.) Genetic flux in plants. Springer-Verlag, Berlin.









Scowcroft, W. R., and P. J. Larkin. 1988. Somaclonal
variation. p. 21-48. In G. Bock and J. Marsh (eds.)
Applications of plant cell and tissue culture. John
Wiley and Sons, New York.

Secor, G. A., and J. F. Shepard. 1981. Variability of
protoplast-derived potato clones. Crop Sci. 21: 102-
105.

Schank, S. C., E. M. Hodges, G. B. Killinger, and D. E.
McCloud. 1972. Registration of pangola digitgrass. Crop
Sci. 12: 715.

Sheth, A. A. 1955. Sterility problems in Digitaria decumbens
Stent. M. S. diss. University of Florida, Gainesville.
Sheth, A. A., L. Yu, and J. Edwardson 1956. Sterility
in pangolagrass. Agron. J. 48: 505-507.

Shneyour, A., J. D. Rasion, and R. M. Smillie. 1973. The
effect of the rate of photosynthetic electron transfer
in chloroplasts of chilling-sensitive and chilling-
resistant plants. Biochem. Biophys. Acta 292: 152-161.

Skene, K. G. M., and M. Barlass. 1983. Regeneration of
plants from callus culture of Lolium rigidum. Z.
Pflanzenzuchtg. 90: 130-135.

Smillie, R. M., and S. E. Hetherington. 1983. Stress
tolerance and stress induced injury in crop plants
measured by chlorophyll fluorescence in vivo. Plant
Physiol. 72: 1043-1050.

Smith, S. M., and H. E. Street. 1974. The decline of
embryogenic potential as callus and suspension cultures
of carrot (Dacus carota L.) are serially subcultured.
Ann. Bot. 38: 223-241.

Steinback, K. E., and B. A. Mall. 1986. Regulatory and
developmental factors affecting photoinhibition damage
and recovery in chilling sensitive rice. p. 725-730. In
G. Akoyunoglou and H. Senger (eds.) Regulation of
chloroplast differentiation. Alan R. Liss Inc., New
York.

Steponkus, P. L. 1972. Freeze preservation of callus culture
of Chrysanthemum morifolium ramat. HortSci. 7(2): 194.


Steup, M., and C. Schachtele. 1981. Mode of glucan
degradation by purified phosphorylase forms from
spinach leaves. Planta 153: 351-361.









Steup, M., H. Robenek, and M. Melkonian. 1983. In vitro
degradation of starch granules isolated from spinach
chloroplasts. Planta 158: 428-436.

Stitt, M., and T. Rees. 1980. Carbohydrate breakdown by
chloroplasts of Pisum sativum. Biochem. Biophys. Acta
627: 131-143.

Stitt, M., and W. H. Heldt. 1981. Physiological rates of
starch breakdown in isolated intact spinach
chloroplasts. Plant Physiol. 68: 755-761.

Sukumaran, N. P., and C. J. Weiser. 1972. Freezing injury in
potato leaves. Plant Physiol. 50: 564-567.

Tal, M. 1983. Selection for stress tolerance. p. 461-488. In
D. A. Evans, W. R. Sharp, P. V. Ammato, and Y. Yamada
(eds.) Handbook of plant cell culture. Macmallian
Publishing Company, New York.

Templeton-Somers, K. M., W. R. Sharp, and R. M. Pfister.
1981. Selection of cold-resistant cell lines of carrot.
Z. Pflanzenphysiol. 103:139-148.

Torello, W. A., and A. G. Symington. 1984. Regeneration from
perennial ryegrass callus tissue. HortSci. 19: 56-57.

Urata, U., and P. P. Long. 1968. A method of propagating
grasses by culture. p. 24. In Agronomy Abstracts. ASA,
Madison, Wisconsin.

USDA. Plant material introduction by the division of plant
induction, Bureau of Plant Industry. Jan. 1 to March
31, 1931. (Nos. 90837-92336). inventory No. 106: 1-
61. November, 1932.

Vallejos,E. 1983. Enzyme activity staining. p. 469-516. IN
S. D. Tansley and T. J. Oston (eds.) Isozymes in plant
genetics and breeding. Elsevier Science Publishers,
Amsterdam.

Van Harten, A. M., H. Bouter, and C. Broertjes. 1981.In
vitro adventitious bud techniques for vegetative
propagation and mutation breeding of potato (Solanum
tuberosum L.). II. Significance for mutation breeding.
Euphytica 30: 1-8.









Vasil, I. K. 1983. Toward the development of a single cell
system for grasses. p. 131-144. In Cell and tissue
culture techniques for cereal crop improvement. Proc.
Workshop Institute of Genetics, Academia Sinica, and
International Rice Research Institute, Science Press,
Beijing China.

Walker, D. 1987. The use of the oxygen electrode and
fluorescence probes in sample measurements of
photosynthesis. Oxygraphics Limited. West Sussex, UK.

West, S. H. 1970. Biochemical mechanism of photosynthesis
and growth depression in Digitaria decumbens when
exposed to low temperatures. p. 514-517. In Proc. of
the 11th International Grassland Congress, Surfers
Paradise, Australia.

West, S. H., R. H. Biggs, and J. M. Baskin. 1968. Growth and
photosynthesis of pangolagrass, Digitaria decumbens
Stent., in a gradient of temperatures. Soil Crop Sci.
Soc. Fla. Proc. 29: 29-35.

Whitney, A. S., R. E. Green, and 0. R. Younge. 1973. Effects
of gibberellic acid and sublethal levels of four
herbicides on the cool-season regrowth of two tropical
forage grasses. Agron. J. 65: 473-476.

Williams, E. G., and G. Maheswaran. 1986. Somatic
embryogenesis: Factors influencing coordinated behavior
of cells as an embryogenic group. Ann. Bot. 57: 443-
462.

Wiltbank, W. J., and T. W. Oswalt. 1984. Laboratory
determination of the killing temperature of citrus
leaves during the 1981-1982 and 1982-1983 low
temperature periods. The Citrus Industry. Feb., p. 42-
47.

Yung-ling, D., C.-H. Xu, F.-H. Zhao. 1987. Comparative
studies on effects of low temperature on chlorophyll
fluorescence induction kinetics and photochemical
activities of cucumber and winter wheat. p. 99-102. In
J. Biggens (ed.) Progress in photosynthesis research.
Vol. 4. Martinus Nijhoff Publishers, Boston.

Ziegler, P., and E. Beck. 1986. Exoamylase activity in
vacuoles isolated from pea and wheat protoplasts. Plant
Physiol. 82: 1119-1121.













BIOGRAPHICAL SKETCH


Eugene Balliet Ackerman was born on November 4, 1956,

in Bainbridge, Maryland. He completed high school at the

Hill School, Pottstown, Pennsylvania, in 1976. In May, 1981

he received his Bachelor of Science degree in agronomy from

The Pennsylvania State University in College Park,

Pennsylvania. From June 1981 to January 1983, he was granted

an assistantship at the University of Arkansas to work

toward a Master of Science degree in agronomy.

In 1983 he was granted a fellowship to study at the

Marine Biological Laboratory, Woods Hole, Massachusetts. He

conducted research on microbial ecology.

In August 1985 he joined the plant stress unit of the

United States Department of Agriculture-Agricultural

Research Service (USDA-ARS), located at Gainesville,

Florida. He is currently researching plant growth under

stress conditions.

In August 1986 he initiated work towards a Ph.D. degree

in agronomy at the University of Florida. He is currently a

Ph.D. candidate, and upon completion of his graduate work,

he will be employed by Hoffman-LaRoche as an agricultural

specialist in the business development department.







90

In 1989 he was certified as a professional crop

scientist by the American Registry for Certified

Professionals in Agronomy, Crops, and Soils.

He is a member of the American Society of Agronomy and

Crop Science Society of America.

He is married to Amy Lynn and has three children:

Jacob, Rachel, and Taryn.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E84T7G9C1_74PSU8 INGEST_TIME 2017-07-12T20:59:34Z PACKAGE AA00003316_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 2

(1=<0$7,& $1' 3+<6,2/2*,&$/ 678',(6 2) /2: 7(03(5$785( 5(63216( ,1 9(*(7$7,9( $1' 620$&/21$/ 3$1*2/$ %\ (8*(1( %$//,(7 $&.(50$1 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 3

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

PAGE 4

OLJKW RI VXFK ORYH FKDOOHQJHV DUH PHW DQG REVWDFOHV RYHUFRPH 7R P\ ZLIH $P\ DQG RXU FKLOGUHQ -DFRE 5DFKHO DQG 7DU\Q DP WUXO\ JUDWHIXO IRU WKHLU ORYH ZKLFK LV WHPSHUHG ZLWK SHUFHSWLRQ DQG SDWLHQFH LQ

PAGE 5

7$%/( 2) &217(176 3DJH $&.12:/('*(0(176 L L /,67 2) 7$%/(6 YL /,67 2) ),*85(6 YLL $%675$&7 L[ &+$37(56 ,,1752'8&7,21 ,,352'8&7,21 2) 3$1*2/$ 620$&/21(6 $1' 6(/(&7,21 )25 ,1&5($6(' 72/(5$1&( 72 )5((=,1* $1' &+,//,1* 7(03(5$785( 0DWHULDOV DQG 0HWKRGV 5HVXOWV DQG 'LVFXVVLRQ &RQFOXVLRQV ,,,(9$/8$7,21 2) &+,//,1* 7(03(5$785( $1' *,%%(5(//,& $&,' 21 7+( (;35(66,21 2) +<'52/<7,& (1=<0(6 0DWHULDOV DQG 0HWKRGV 5HVXOWV DQG 'LVFXVVLRQ &RQFOXVLRQV ,9(9$/8$7,21 2) +<'52/<7,& (1=<0(6 ,1 )5((=,1* 7(03(5$785(72/(5$17 620$&/21(6 2) 3$1*2/$ 0DWHULDOV DQG 0HWKRGV 5HVXOWV DQG 'LVFXVVLRQ &RQFOXVLRQV 96800$5< $1' &21&/86,216 LY

PAGE 6

3DJH 5()(5(1&(6 %,2*5$3+,&$/ 6.(7&+ 9

PAGE 7

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

PAGE 8

/,67 2) ),*85(6 )LJXUH 3DJH 'HFUHDVH LQ WKH UDWH RI IOXRUHVFHQW HPLVVLRQ )5f IURP SDQJOD FKLOOHG DW r& IRU PLQXWHV 3HDN IOXRUHVFHQFH 3f LV WKH PD[LPXP IOXRUHVFHQW HPLVVLRQ &RQILJXUDWLRQ RI /D&UR\ WUDQVLHQW UHFRUGHU &RQILJXUDWLRQ RI /D&UR\ WUDQVLHQW UHFRUGHU 3DQJOD FDOOXV ZLWK HPEU\RJHQLF Hf DQG QRQHPEU\RJHQLF Qf FDOOXV 2Q 06 PHGLD FRQWDLQLQJ 0 ; 3DQJOD FDOOXV ZLWK HPEU\RJHQLF Hf DQG QRQHPEU\RJHQLF Qf FDOOXV 2Q 06 PHGLD FRQWDLQLQJ 0 ; /RQJLWXGLQDO VHFWLRQ RI SDQJOD FDOOXV ZLWK QRQHPEU\RJHQLF Qf FDOOXV HPEU\RJHQLF Hf FDOOXV DQG \RXQJ VRPDWLF HPEU\R HPf 2Q 06 PHGLD FRQWDLQLQJ L0 ; /RQJLWXGLQDO VHFWLRQ RI SDQJOD FDOOXV ZLWK HPEU\RJHQLF Hf FDOOXV VXSSRUWLQJ D PDWXULQJ VRPDWLF HPEU\R VFXWHOOXP VFf FROHRSWLOH FRf DQG VKRRW DSH[ Vf 2Q 06 PHGLD ZLWKRXW ; 5HJHQHUDWHG SDQJOD SODQW ZLWK SOXPXOH Sf DQG \RXQJ URRW Uf 2Q 06 PHGLD ZLWKRXW ; (IIHFW RI WHPSHUDWXUH DQG *$ RQ H[SUHVVLRQ RI VWDUFKGHJUDGLQJK\GURO\WLF HQ]\PHV LQ SDQJOD :KLWH EDQGV EDQGV f VKRZ HQGRDP\ODVH DFWLYLW\ EOXH EDQG EDQG f LQGLFDWHV GHEUDQFKLQJ HQ]\PH DFWLYLW\ ) UHIHUV WR WKH EURUDRSKHQRO EOXH IURQW PDUNHU 0LFURJUDPV SURWHLQ UHIHUV WR WKH WRWDO SURWHLQ YLL

PAGE 9

)LJXUH 3DJH (IIHFW RI WHPSHUDWXUH DQG *$ RQ SURGXFWLRQ DQG H[SUHVVLRQ RI VWDUFKGHJUDGLQJK\GURO\WLF HQ]\PHV LQ SDQJOD =\PRJUDPV ]f RI K\GURO\WLF DFWLYLW\ DQG FRRPDVVLH SURWHLQ VWDLQV Ff DUH SUHVHQWHG IRU HDFK WUHDWPHQW 0LFURJUDPV SURWHLQ UHIHUV WR WKH WRWDO SURWHLQ =\PRJUDPV RI VWDUFKK\GURO\]LQJ HQ]\PHV LVRODWHG IURP SDQJOD SODQWV WKDW ZHUH H[SRVHG WR r& RU r& :KLWH EDQGV EDQGV f VKRZ HQGRDP\ODVH DFWLYLW\ EOXH EDQG EDQG f LQGLFDWHV GHEUDQFKLQJ HQ]\PH DFWLYLW\ /DQHV DQG UHSUHVHQW WKH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV /DQHV DQG UHSUHVHQW SODQWV UHJHQHUDWHG IURP WLVVXH FXOWXUH ) UHIHUV WR WKH EURPRSKHQRO EOXH IURQW PDUNHU =\PRJUDPV RI VWDUFKK\GURO\]LQJ HQ]\PHV DQG WRWDO SURWHLQ SURILOHV IURP SDQJOD SODQWV H[SRVHG WR r& RU r& /DQHV DQG DUH ]\PRJUDPV :KLWH EDQGV EDQGV f VKRZ HQGRDP\ODVH DFWLYLW\ EOXH EDQG EDQG f LQGLFDWHV GHEUDQFKLQJ HQ]\PH DFWLYLW\ &RRPDVVLH EOXH VWDLQHG SURWHLQV DUH UHSUHVHQWHG LQ ODQH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWVf DQG ODQHV DQG SODQWV UHJHQHUDWHG IURP WLVVXH FXOWXUHf YP

PAGE 10

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH 6FKRRO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ (1=<0$7,& $1' 3+<6,2/2*,&$/ 678',(6 2) /2: 7(03(5$785( 5(63216( ,1 9(*(7$7,9( $1' 620$&/21$/ 3$1*2/$ E\ (XJHQH %DOOLHW $FNHUPDQ 0D\ &KDLUPDQ 'U 6 + :HVW 0DMRU 'HSDUWPHQW $JURQRP\ &RQYHQWLRQDO EUHHGLQJ SURJUDPV DUH XQDEOH WR JHQHUDWH YDULDWLRQ LQ nSDQJODn GLJLWJUDVV 'LJLWDULD GHFXPEHQV 6WHQWf 3DQJOD LV D KLJKO\ VWHULOH FXOWLYDU ZKLFK UHVXOWHG IURP K\EULGL]DWLRQ RI FORVHO\ UHODWHG 'LJLWDULD VSHFLHV 3DQJOD LV D WULSORLG Q f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

PAGE 11

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n K\GURO\]LQJ HQ]\PHV UHVXOWHG LQ WKH LGHQWLILFDWLRQ RI WZR FKLOOVHQVLWLYH HQGRDP\ORO\WLF HQ]\PHV 2QH GHEUDQFKLQJ DQG VL[ HQGRDP\ORO\WLF HQ]\PHV ZHUH XQDIIHFWHG E\ FKLOOLQJ WHPSHUDWXUH 7KH QXPEHU RI K\GURO\WLF HQ]\PHV ZDV XQDIIHFWHG E\ DSSO\LQJ JLEEHUHOOLF DFLG *$f WR WKH SODQW SULRU WR FKLOOLQJ WHPSHUDWXUH H[SRVXUH $OO RI WKH UHJHQHUDWHG SODQWV WKDW ZHUH VHOHFWHG IRU DQ LQFUHDVH LQ IUHH]LQJ WROHUDQFH ZHUH LGHQWLFDO ZLWK UHVSHFW WR WKH QXPEHU RI VWDUFKK\GURO\]LQJ HQ]\PHV UHJDUGOHVV RI WHPSHUDWXUH 6RPDWLF YDULDWLRQ LQ IUHH]LQJ RU FKLOOLQJ WHPSHUDWXUH UHVSRQVH RI SDQJOD ZDV HYLGHQW LQ YLWUR 9DULDQW UHJHQHUDWHG SODQWV WKDW VXVWDLQ OHVV GDPDJH IURP IUHH]LQJ WHPSHUDWXUH FDQ EH LGHQWLILHG E\ D ORZHOHFWURO\WH OHDNDJH [

PAGE 12

&+$37(5 ,1752'8&7,21 3DVWXUHV WKDW DUH HVWDEOLVKHG ZLWK nSDQJODn GLJLWJUDVV 'LTLWDULD GHFXPEHQV 6WHQWf SURGXFH DQ DEXQGDQFH RI OXVK KLJKTXDOLW\ IRUDJH 3DQJOD FDQ EH XVHG DV D SDVWXUH KD\ RU VLODJH FURS +RGJHV HW DO f 'XULQJ WKH HDUO\ V SDQJOD ZDV LQWURGXFHG LQWR WKH 86 *XOI &RDVW DUHDV DQG VRXWKHUQ &DOLIRUQLD 86'$ f 3DQJOD ZDV UHJLVWHUHG E\ WKH &URS 6FLHQFH 6RFLHW\ RI $PHULFD UHJLVWUDWLRQ QXPEHU f LQ 6FKDQN HW DO f 7KH HVWDEOLVKPHQW RI SDQJOD UHVXOWHG LQ SDVWXUHV WKDW VXVWDLQ DEXQGDQW KLJKTXDOLW\ \LHOGV LQ WKH KRW VXPPHU PRQWKV DQG GUDVWLFDOO\ UHGXFHG \LHOGV LQ WKH FRROHU PRQWKV 3DQJOD LV YHU\ IUHH]H VXVFHSWDEOH DV FKDUDFWHUL]HG E\ WKH GHDWK RI JUDVV FURZQV +RGJHV DQG -RQHV f :LQWHUNLOOLQJ UHVXOWV IURP D FRPELQDWLRQ RI IURVW DQG IUHH]LQJ WHPSHUDWXUH 7KH OLYHVWRFN LQGXVWULHV LQ )ORULGD SDUWLFXODUO\ WKH EHHI LQGXVWU\ GHVLUH D SDVWXUH JUDVV WKDW SURYLGHV D \HDU URXQG VXSSO\ RI DEXQGDQW IRUDJH 8QIRUWXQDWHO\ GXULQJ WKH FRROHU PRQWKV SDQJOD SURGXFWLRQ IDOOV VKRUW RI WKH IDUPHUVn QHHGV )DUPHUV DUH IRUFHG WR SXUFKDVH DGGLWLRQDO IHHG VWRFNV DIWHU WKHLU EDQNHG SDQJOD SDVWXUHV KDYH EHHQ

PAGE 13

GHSOHWHG ,QFUHDVLQJ WKH FRVW HIIHFWLYHQHVV RI EHHI SURGXFWLRQ LQ )ORULGD VWURQJO\ GHSHQGV RQ WKH \HDUURXQG SURGXFWLRQ RI DEXQGDQW DQG LQH[SHQVLYH IRUDJH ([WHQGLQJ WKH VXPPHUWLPH JURZWK FKDUDFWHULVWLFV RI SDQJOD LQWR WKH FRROHU PRQWKV ZRXOG VXEVWDQWLDOO\ UHGXFH WKH IDUP DQG UDQFK LQYHVWPHQWV LQ OLYHVWRFN IHHG :KROH SODQW JURZWK KDV EHHQ FKDUDFWHUL]HG E\ %XUJHVV f DV D QDWXUDO SURFHVV WKDW LV UHJXODWHG E\ JHQHWLF ELRFKHPLFDO PRUSKRORJLFDO DQG SK\VLRORJLFDO FKDQJHV LQ HDFK FHOO 0XOWLSOLFDWLRQ DQG GLIIHUHQWLDWLRQ RI SODQW FHOOV UHVXOWV IURP VHOHFWLYH H[SUHVVLRQ RI SDUWV RI WKH JHQRPH 6HOHFWLYH H[SUHVVLRQ RI WKH JHQRPH NHHSV WKH SODQW LQ EDODQFH ZLWK LWV HQYLURQPHQW 'XULQJ WKH ZLQWHU PRQWKV WKH PRVW LPSRUWDQW HQYLURQPHQWDO IDFWRU UHVWULFWLQJ SODQW JURZWK LV FKLOOLQJ r&r&f DQG IUHH]LQJ r& DQG ORZHUf WHPSHUDWXUHV &KULVWLDQVHQ DQG 6W -RKQ f &KLOOLQJ DQG IUHH]LQJ FRQGLWLRQV DUH GHILQHG E\ WKHVH WHPSHUDWXUH UDQJHV $ ZLGH UDQJH RI WROHUDQFH WR ORZWHPSHUDWXUH VWUHVV RIWHQ H[LVWV ZLWKLQ FURS VSHFLHV GXH WR WKH JHQHWLF GLYHUVLW\ JHQHUDWHG E\ JHQH UHFRPELQDWLRQ GXULQJ PHLRVLV -RQHV f 6WHULOH FURSV VXFK DV SDQJOD DUH ODFNLQJ LQ JHQH UHFRPELQDWLRQ DQG WKHUHIRUH UHWDLQ D KLJK GHJUHH RI JHQRW\SLF DQG SKHQRW\SLF XQLIRUPLW\ 7LVVXH FXOWXUH PHWKRGV KDYH EHHQ XVHG WR UHJHQHUDWH SODQWV ZLWK LQFUHDVHG JHQHWLF YDULDWLRQ

PAGE 14

6FRZFURIW DQG /DUNLQ f DQG LQFUHDVHG WROHUDQFH WR XQIDYRUDEOH HQYLURQPHQWV 7DO f 0DQ\ DJURQRPLFDOO\ LPSRUWDQW SODQW VSHFLHV RI WURSLFDO RULJLQ DUH VHQVLWLYH WR FKLOOLQJ WHPSHUDWXUHV LQ WKH UDQJH RI r& GRZQ WR r& %HORZ VRPH FULWLFDO WHPSHUDWXUH JUDVVHV DUH VKDUSO\ UHVWULFWHG LQ JURZWK *XVWD HW DO f 6HYHUDO SODQW IDFWRUV FRQWULEXWH WR UHGXFHG JURZWK XQGHU FKLOOLQJWHPSHUDWXUH FRQGLWLRQV 0HPEUDQH GDPDJH LV D XQLYHUVDO PDQLIHVWDWLRQ RI FKLOOWHPSHUDWXUH GDPDJH LQ ELRORJLFDO V\VWHPV DQG LV FRPPRQO\ UHJDUGHG WR EH WKH SULPDU\ FDXVH RI LQMXU\ /\RQV HW DO f 3KRWRV\QWKHVLV LV RQH RI WKH ILUVW SURFHVVHV DIIHFWHG E\ FKLOOLQJ WHPSHUDWXUHV •TXLVW f $OWHUDWLRQV LQ WKH OLJKW UHDFWLRQV DUH HYLGHQW DW YDULRXV VWDJHV LQ WKH SKRWRV\QWKHWLF HOHFWURQ WUDQVSRUW FKDLQ &KDQJHV LQ WKH GDUN UHDFWLRQV LH FDUERK\GUDWH DQDEROLVPf UHVXOW IURP DOWHUDWLRQV LQ WKH DFWLYLW\ &DUWHU HW DO f DQG V\QWKHVLV -DFREVHQ DQG +LJJLQV f RI DP\ORO\WLF HQ]\PHV 'HVSLWH KDYLQJ GLVWLQFW JHQHWLFDO ELRFKHPLFDO DQG SK\VLRORJLFDO IDFWRUV WKH DFWLRQ DQG LQWHUDFWLRQ RI DOO WKHVH IDFWRUV FRQWULEXWH WR D FRPPRQ UHVXOW UHGXFHG JURZWK 6HYHUDO RI WKH DERYH IDFWRUV ZHUH VWXGLHG DQG UHSRUWHG KHUHLQ 7KH REMHFWLYHV RI WKH VWXG\ UHSRUWHG LQ &KDSWHU ,, ZHUH Df WR JHQHUDWH VRPDWLF FHOO OLQHV RI SDQJOD Ef WR VHOHFW IUHH]LQJ DQG FKLOOLQJ UHVLVWDQW FHOO OLQHV LQ YLWUR Ff WR UHJHQHUDWH ZKROH SODQWV IURP VHOHFWHG FXOWXUHV DQG Gf

PAGE 15

WR WHVW UHJHQHUDWHG SODQWV IRU LQFUHDVHG WROHUDQFH WR IUHH]LQJ DQG FKLOOLQJ WHPSHUDWXUH DW WKH RUJDQHOOH DQG FHOOXODU OHYHO 7KH VWXGLHV UHSRUWHG LQ &KDSWHU ,,, ZHUH FRQGXFWHG ZLWK WKH REMHFWLYHV Df WR HYDOXDWH WKH HIIHFW RI FKLOOLQJ WHPSHUDWXUH RQ WKH H[SUHVVLRQ RI DP\ORO\WLF HQ]\PHV LQ SDQJOD Ef WR LGHQWLI\ DP\ORO\WLF SURWHLQV WKDW PD\ DVVLVW LQ VWDUFK EUHDNGRZQ DIWHU H[SRVXUH WR FKLOOLQJ FRQGLWLRQV DQG Ff WR GHWHUPLQH WKH HIIHFW *$ KDV RQ WKH V\QWKHVLV RI WKHVH SURWHLQV 7KH REMHFWLYHV RI WKH VWXG\ SUHVHQWHG LQ &KDSWHU ,9 ZHUH Df WR HYDOXDWH ORZWHPSHUDWXUHWROHUDQW SDQJOD VRPDFORQHV IRU YDULDWLRQ LQ WKH H[SUHVVLRQ RI DP\ORO\WLF HQ]\PHV DQG Ef WR VHOHFW SODQWV WKDW UHWDLQ DP\ORO\WLF SURWHLQV ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ DIWHU H[SRVXUH WR FKLOOLQJ FRQGLWLRQV

PAGE 16

&+$37(5 ,, 352'8&7,21 2) 3$1*2/$ 620$&/21(6 $1' 6(/(&7,21 )25 ,1&5($6(' 72/(5$1&( 72 )5((=,1* $1' &+,//,1* 7(03(5$785( 7KLV VWXG\ WHVWHG WKH WHQWDWLYH DVVXPSWLRQ WKDW VRPDFORQDO YDULDWLRQ PD\ EH REWDLQHG LQ SDQJOD SODQWV WKDW DUH UHJHQHUDWHG IURP WLVVXH FXOWXUH +\SRWKHWLFDOO\ VRPDWLF YDULDQWV PD\ EH VHOHFWHG IRU LQFUHDVHG ORZWHPSHUDWXUH WROHUDQFH 7R WHVW WKLV K\SRWKHVLV WKH REMHFWLYHV RI WKLV VWXG\ ZHUH Df WR JHQHUDWH VRPDWLF FHOO OLQHV RI SDQJOD Ef WR VHOHFW IUHH]LQJ DQG FKLOOLQJ UHVLVWDQW FHOO OLQHV LQ FXOWXUH Ff WR UHJHQHUDWH SODQWV IURP WKH VHOHFWHG FDOOXV DQG Gf WR WHVW IRU LQFUHDVHG WROHUDQFH WR IUHH]LQJ DQG FKLOOLQJ WHPSHUDWXUH DW WKH ZKROH SODQW OHYHO 3DQJOD HVWDEOLVKHV H[FHOOHQW VXPPHUWLPH SDVWXUHV EXW LV DQ XQSURGXFWLYH IDOO DQG ZLQWHU IRUDJH FURS LQ )ORULGD &KLOOLQJ WHPSHUDWXUHV r&r&f HQFRXQWHUHG E\ SDQJOD GXULQJ WKH FRROHU VHDVRQV UHGXFH JURZWK UDWHV DQG HYHQWXDOO\ VWRS SURGXFWLYH JURZWK :HVW HW DO f 7KH JHQHWLF GLYHUVLW\ WKDW SHUPLWV D IHUWLOH SODQW VSHFLHV WR DGDSW WR WHPSHUDWXUH H[WUHPHV LV QRW SUHVHQW LQ SDQJOD 3DQJOD LV KLJKO\ VWHULOH GXH WR LUUHJXODULWLHV LQ ERWK PLFURVSRURJHQHVLV DQG PDFURVSRURJHQHVLV 6KHWK f

PAGE 17

6KHWK HW DO f DQG 6FKDQN HW DO f UHSRUWHG WKDW SDQJOD SURGXFHG YHU\ IHZ YLDEOH VHHG 7KLV JHQHWLF FURVVLQJ EDUULHU HOLPLQDWHV DQ\ JHQHWLF YDULDWLRQ WKDW LV DFTXLUHG IURP LQGHSHQGHQW JHQH VHJUHJDWLRQ DQG JHQH UHFRPELQDWLRQ $Q LQFUHDVH LQ JHQHWLF YDULDELOLW\ IURP VWHULOH SODQWV PD\ EH REWDLQHG E\ VXEMHFWLQJ WKH WLVVXH WR VRPDWLF FHOO FXOWXUH 5HLVFK f 7KH FRQFHSW RI VRPDFORQDO YDULDWLRQ DV D QHZ VRXUFH RI JHQHWLF YDULDELOLW\ ZDV SUHVHQWHG E\ /DUNLQ DQG 6FRZFURIW f DQG KDV EHHQ UHYLHZHG D QXPEHU RI WLPHV 6FRZFURIW $KORRZDOLD /DUNLQ 5\DQ HW DO f 9DULDWLRQ DULVLQJ DV D FRQVHTXHQFH RI WLVVXH FXOWXUH KDV EHHQ UHSRUWHG LQ WKH IROORZLQJ IRUDJH JUDVVHV U\HJUDVV /ROLXP PXOWLIORUXP /f $KORRZDOLD 7RUHOOR DQG 6\PLQJWRQ 6NHQH DQG %DUODVV f WDOO IHVFXH )HVWXFD DUXQGLQDFHD /f .DVSHUEDXHU DQG (L]HQJD f JXLQHDJUDVV 3DQLFXP PD[LPXP /f %DMDM HW DO f DQG FUDEJUDVV 'LJLWDULD VDQJXLQDOLV /f (OPRUH HW DO f 7KLV VXJJHVWV WKDW VRPDWLF YDULDWLRQ LQ SDQJOD FRXOG EH XVHG WR JHQHUDWH YDULDWLRQ $ IXQGDPHQWDO UHTXLUHPHQW IRU REWDLQLQJ VRPDWLF YDULDWLRQ LV WKH DELOLW\ WR VXSSRUW LQ YLWUR FDOOXV JURZWK DQG SODQW UHJHQHUDWLRQ 8UDWD DQG /RQJ f ZHUH ILUVW WR UHSRUW WKH UHJHQHUDWLRQ RI JUDVV SODQWV IURP WLVVXH FXOWXUH 3DQJRODJUDVV ZDV RQH RI WKH VHYHUDO WURSLFDO JUDVVHV WKDW WKH\ UHJHQHUDWHG WKURXJK RUJDQRJHQHVLV 5HFHQWO\ XVLQJ

PAGE 18

SDQJRODJUDVV 0DURXVN\ DQG :HVW f UHSRUWHG VKRRW GHYHORSPHQW IURP RYDULDQ H[SODQW WLVVXH DQG VRPDWLF HPEU\R IRUPDWLRQ IURP FDOOXV 8QWLO UHFHQWO\ LW ZDV WKRXJKW WKDW WKH RQO\ ZD\ WR REWDLQ XVHIXO QRQFKLPHUDO PXWDQWV ZDV E\ UHJHQHUDWLRQ RI VRPDWLF HPEU\RV *DPERUJ HW DO f %RWK RUJDQRJHQLF VKRRWV +RUVFK HW DO f DQG VRPDWLF HPEU\RV :LOOLDPV DQG 0DKHVZDUDQ f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f VWDWHG WKDW WKLV W\SH RI YDULDWLRQ LV XVXDOO\ EXW QRW QHFHVVDULO\ UHYHUVLEOH 6HFRU DQG 6KHSDUG f UHSRUWHG WKDW SRWDWR 6RODQXP WXEHURVXP /f VRPDFORQHV ZKLFK KDG JRQH WKURXJK D QXPEHU RI YHJHWDWLYH JHQHUDWLRQV LQ WKH ILHOG VWLOO UHWDLQHG VHOHFWHG FKDUDFWHULVWLFV

PAGE 19

0LWUD DQG 6WHZDUG f ZHUH DPRQJ WKH ILUVW WR UHSRUW WKH H[LVWHQFH RI WUXH JHQHWLF FKDQJH LQ VRPDWLF FHOO FXOWXUH 7KH\ REVHUYHG FKURPRVRPDO DEQRUPDOLWLHV SRO\SORLG\ DQG DQHXSORLG\ LQ FDUURW 'DXFXV FDUROD /f FHOO FXOWXUHV 7KHVH JHQHWLF FKDQJHV ZHUH JURVV FKURPRVRPDO PXWDWLRQV DQG DUH QRW VWDEO\ LQKHULWHG RU XVHIXOO $FFRUGLQJ WR 2UWRQ f VWDEO\ LQKHULWHG RU 0HQGHOLDQ YDULDWLRQ DFFRXQWV IRU FKURPRVRPDO FKDQJHV WKDW DUH KHULWDEOH DQG VHJUHJDWH LQ FURVVHV ZLWK LQGLYLGXDOV H[KLELWLQJ D GLVWLQFW SKHQRW\SH 8VLQJ WKLV FULWHULD DQ\ KHULWDEOH SKHQRW\SLF DOWHUDWLRQ FDXVHG E\ D EDVH FKDQJH GHOHWLRQ RU UHDUUDQJHPHQW LQ WKH '1$ LV LQFOXGHG 7KH '1$ PD\ EH ORFDWHG LQ WKH QXFOHXV PLWRFKRQGULD RU FKORURSODVW 7KH VLWH RI PXWDWLRQ PD\ EH DW ORFL WKDW UHVXOWV LQ HLWKHU PDMRU TXDOLWDWLYH HIIHFWV RU VXEWOH TXDQWLWDWLYH HIIHFWV /DUNLQ HW DO f FLWHG UHSRUWV RI GLIIHUHQW VSHFLHV ZKHUH FKURPRVRPDO PRGLILFDWLRQV KDG EHHQ LQGXFHG DQG WKHQ LQKHULWHG LQ D 0HQGHOLDQ PDQQHU 7KH '1$ DOWHUDWLRQV LQFOXGHG SRLQW PXWDWLRQV GHOHWLRQV LQWHUFKDQJHV LQYHUVLRQV DQG DPSOLILFDWLRQV ,Q WZR RI WKH VSHFLHV FRUQ =HD PD\V /f DQG DOIDOID 0HGLFDJR VDWLYD /f LQFUHDVHG WUDQVSRVDEOH HOHPHQW DFWLYLW\ GXULQJ WLVVXH FXOWXUH KDV EHHQ UHSRUWHG E\ %HQ]LRQ HW DO f DQG *URVV DQG %LQJKDP f UHVSHFWLYHO\ 7KH UHDVRQ ZK\ WLVVXH FXOWXUH UHVXOWV LQ DQ LQFUHDVHG IUHTXHQF\ RI FKURPRVRPDO DEHUUDWLRQ LV \HW WR EH H[SODLQHG

PAGE 20

%HQ]RLQ HW DO f K\SRWKHVL]HG WKDW WKH ODWH UHSOLFDWLQJ KHWHURFKURPDWLQ PD\ RFFDVLRQDOO\ UHSOLFDWH VR ODWH WKDW EULGJH IRUPDWLRQ DQG VXEVHTXHQW FKURPRVRPH EUHDNDJH RFFXUV DW DQDSKDVH 7KH UHVXOWLQJ EUHDNDJHIXVLRQEULGJH F\FOH ZRXOG OHDG WR H[FKDQJHV LQWHUFKDQJHV RU WUDQVORFDWLRQV /DUNLQ HW DO f VWXGLHG KHULWDEOH VRPDFORQDO YDULDWLRQ LQ ZKHDW 7ULWLFXP DHVWLYXP /f DQG FRQFOXGHG WKDW WZR PHFKDQLVPV RQH RSHUDWLQJ WR FUHDWH D PXWDQW JHQH DQG D VHFRQG WR PDNH WKH PXWDQW KRPR]\JRXV DFFRXQWHG IRU WKH VRPDFORQDO YDULDWLRQ 7KHLU FRQFOXVLRQV ZHUH EDVHG RQ WKH REVHUYDWLRQV WKDW YDULDWLRQ ZDV HYLGHQW LQ PRUSKRORJLFDO DQG ELRFKHPLFDO WUDLWV WUDLWV XQGHU VLPSOH JHQHWLF FRQWURO JUDLQ FRORUf TXDQWLWDWLYHO\ LQKHULWHG FKDUDFWHULVWLFV KHDGLQJ GDWHf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f PHGLD FRQVWLWXHQWV 9DQ +DUWHQ HW DO +XJKHV f FHOO

PAGE 21

F\FOH GXUDWLRQ 0XOOHU DQG *UDIH %D\OLVV f DQG UHJHQHUDWLRQ +XJKHV 0DKIRX] HW DO f 7KH OHQJWK RI WLPH LQ FXOWXUH LQIOXHQFHV WKH FXOWXUHV DELOLW\ WR UHJHQHUDWH 0DWWKHZV DQG 9DVLO f UHSRUWHG WKDW LQFUHDVLQJ WKH OHQJWK RI WLPH LQ FXOWXUH IDYRUV YDULDQW FHOOV DQG UHVXOWV LQ PRUH SUHFRFLRXV UHJHQHUDWLRQ 7KLV GHFOLQH KDV EHHQ DWWULEXWHG DW OHDVW SDUWO\ WR JHQHWLF PXWDWLRQV 6PLWK DQG 6WUHHW f ,Q SDQJOD 0DURXVN\ DQG :HVW f UHSRUWHG WKDW DIWHU ZHHNV LQ FXOWXUH HPEU\RJHQHVLV RFFXUUHG DW D b IUHTXHQF\ *HQHWLF PXWDWLRQV LQ LQ YLWUR SDQJOD FHOOV PD\ EH IDYRUHG E\ H[WHQGLQJ WKH OHQJWK RI WLPH LQ FXOWXUH EH\RQG ZHHNV ZKLOH QRW WRWDOO\ FRPSURPLVLQJ WKH UHJHQHUDWLYH FDSDFLW\ ,VRODWLRQ RI YDULDQW FHOOV FDQ EH DFKLHYHG E\ SRVLWLYH VHOHFWLRQ LH WKH DSSOLFDWLRQ RI D VWUHVV IROORZHG E\ WKH KDUYHVWLQJ RI VXUYLYLQJ FHOOV 7KH VWUHVV PD\ EH H[HUWHG HLWKHU VWHSZLVH RU JUDGXDOO\ /DUNLQ HW DO f UHSRUWHG WKDW D VWHSZLVH DSSOLFDWLRQ RI SUHVVXUH LV PRUH DSW WR IDYRU QRYHO JHQRW\SHV ZKLFK DULVH IURP HLWKHU JHQH DPSOLILFDWLRQ DQGRU JHQH PXWDWLRQ 9DULDQW SKHQRW\SHV SURGXFHG E\ WKLV SURFHGXUH KDYH UHVXOWHG LQ UHJHQHUDWHG SODQWV WKDW DUH UHVLVWDQW WR GLVHDVH )RURXJKL:HKU HW DO /DUNLQ DQG 6FRZFURIW f PLQHUDO WR[LFLW\ $KORRZDOLD 2RQR f PLQHUDO GHILFLHQF\ 4XUHVKL HW DO f DQG KHUELFLGH GDPDJH &KDOHII DQG %DVFRPE f

PAGE 22

7KH IHZ UHSRUWHG DWWHPSWV WR VHOHFW FKLOOLQJ RU IUHH]LQJWHPSHUDWXUHUHVLVWDQW FHOO OLQHV WKDW PDLQWDLQ UHVLVWDQFH LQ FXOWXUH KDYH KDG PL[HG UHVXOWV 7KH HDUOLHVW DWWHPSWV WR VHOHFW FKLOOLQJ RU IUHH]LQJWHPSHUDWXUH UHVLVWDQW FDOOXV ZDV UHSRUWHG E\ 6WHSRQNXV f 6WHSRQNXV f REWDLQHG FHOO OLQHV RI LY\ +HGHU£ KHOL[ /f ZKLFK VXUYLYHG WKH VHOHFWLRQ SURFHGXUH RI IUHH]LQJ EXW QRQH RI WKH OLQHV PDLQWDLQHG D VWDEOH HQKDQFHG UHVLVWDQFH WR WKH IUHH]LQJ WHPSHUDWXUH 'L[ f %DVHG RQ UHVSLUDWLRQ UDWHV LQ PLWRFKRQGULD 'L[ DQG 6WUHHW f REWDLQHG VRPH FHOO OLQHV RI WREDFFR 1LFRWLDQD VYOYHVWULV /f DQG SHSSHU &DSVLFXP DQQXP /f WKDW PDLQWDLQHG FKLOO UHVLVWDQFH DQG RWKHU OLQHV WKDW ORVW FKLOO UHVLVWDQFH 7HPSOHWRQ6RPHUV HW DO f VHOHFWHG IUHH]LQJWHPSHUDWXUH UHVLVWDQW FDUURW FDOOXV 5HVXEPLVVLRQ RI WKH UHVLVWDQW FDOOXV WR IUHH]LQJ FRQGLWLRQV UHVXOWHG LQ FRQVLGHUDEOH YDULDWLRQ LQ WKH DELOLW\ RI WKH FHOO OLQHV WR WROHUDWH WKH VHOHFWLRQ FRQGLWLRQV &KHQ HW DO f VHOHFWHG FKLOOLQJWROHUDQW VXJDUFDQH 6DFFKDUXP VSSf FHOO OLQHV PRVW RI ZKLFK UHWDLQHG FKLOO WROHUDQFH LQ FXOWXUH ,W KDV QRW EHHQ GHWHUPLQHG LI WKH FKLOOLQJ RU IUHH]LQJ WHPSHUDWXUH UHVLVWDQFH RI WKH VHOHFWHG FDOOXV ZLOO EH H[SUHVVHG LQ UHJHQHUDWHG SODQWV &HOO OLQHV VHOHFWHG IRU FKLOOLQJ RU IUHH]LQJWHPSHUDWXUH WROHUDQFH RIWHQ ORVH WKHLU DELOLW\ WR GLIIHUHQWLDWH DQG UHJHQHUDWH SODQWV 'L[ DQG 6WUHHW &KHQ HW DO f 'L[ f VH[XDOO\

PAGE 23

FURVVHG SODQWV WKDW ZHUH UHJHQHUDWHG IURP FKLOOVHQVLWLYH FDOOXV &DOOXV REWDLQHG IURP SURJHQ\ SODQWV KDG ORVW WKHLU FKLOO VHQVLWLYLW\ +H FRQFOXGHG WKDW WKH UHVLVWDQFH ZDV QRW KHULWDEOH 0DOPEHUJ f VKRZHG WKDW FXOWXULQJ OHDYHV IURP D WREDFFR SODQW WKDW ZDV UHJHQHUDWHG IURP D FKLOOLQJ WHPSHUDWXUHVHQVLWLYH FDOOXV UHVXOWHG LQ FDOOXV WKDW UHWDLQHG FKLOO VHQVLWLYLW\ 7HPSOHWRQ6RPHUV HW DO f UHSRUWHG WKDW FKLOOLQJWHPSHUDWXUH UHVLVWDQFH LQ VHOHFWHG FDOOXV LV QRW DOZD\V H[SUHVVHG GXULQJ HPEU\R GHYHORSPHQW 7KH\ VWDWHG WKDW DOWKRXJK WKHUH LV QR FRQFUHWH HYLGHQFH IRU LQFUHDVHG FKLOO UHVLVWDQFH LQ WKH VRPDWLF HPEU\RV IXUWKHU ZRUN LV QHFHVVDU\ WR GHWHUPLQH WKH H[SUHVVLRQ LQ PRUH PDWXUH SODQWV $ YLVXDO V\PSWRP RI IUHH]LQJWHPSHUDWXUH LQMXU\ LQ PDWXUH SODQW WLVVXH LV WKH LQILOWUDWLRQ RI WLVVXH LQWHUFHOOXODU VSDFH ZLWK ZDWHU &KHQ HW DW f XVHG WKLV VRDNHG DSSHDUDQFH DORQJ ZLWK ORVV RI WXUJRU DV D FULWHULRQ IRU HYDOXDWLQJ IUHH]LQJ LQMXU\ $QRWKHU FRPPRQ UHVXOW RI IUHH]LQJ LQMXU\ LV WKH OHDNDJH RI LRQV IURP FHOOV 8QWLO UHFHQWO\ UHSRUWV VXFK DV /HYLWW f DQG 6XNXUPDQ DQG :HLVHU f DWWULEXWHG WKH HIIOX[ RI LRQV IURP IUR]HQ DQG WKDZHG WLVVXH WR WKH EUHDNGRZQ RI WKH VHPLSHUPHDEOH SURSHUW\ RI WKH FHOO PHPEUDQH 3DOWD HW DO Df KDV VKRZQ WKDW LQ RQLRQ $OOLXP FHSD /f EXOE FHOOV WKH VHPLSHUPHDEOH SURSHUWLHV UHPDLQ LQWDFW GXULQJ WKH SURFHVV RI IUHH]LQJ ZKHUHDV WKH DFWLYH WUDQVSRUW SURSHUWLHV RI WKH FHOO

PAGE 24

PHPEUDQHV DUH GDPDJHG 7KH\ UHSRUWHG WKDW LQDFWLYDWLRQ RI WKH DFWLYH WUDQVSRUW V\VWHP UHVXOWV LQ D ODUJH SDVVLYH HIIOX[ RI LRQV DQG VXJDUV 'XULQJ WKDZLQJ DV LFH PHOWV LQ WKH H[WUDFHOOXODU VSDFH LRQV DQG VXJDUV PRYH GRZQ FRQFHQWUDWLRQ JUDGLHQWV YDFXROH WR H[WUDFHOOXODU VROXWLRQf DV WKH DFWLYH WUDQVSRUW V\VWHP LV XQDEOH WR SXPS WKHP EDFN LQWR WKH YDFXROH $V D UHVXOW RI RVPRWLF HTXLOLEULXP EHWZHHQ WKH RXWVLGH VROXWLRQ DQG WKH FHOO VDS WKH FHOO LV XQDEOH WR DEVRUE ZDWHU 7KH WLVVXHV EHFRPH IODFFLG GXH WR WKH LQILOWUDWLRQ RI WKH WLVVXH ZLWK H[WUDFHOOXODU VROXWLRQ DQG ORVH RI WXUJRU 8VLQJ RQLRQ EXOE FHOOV +DQVWHHQ&UDPHU f ILUVW UHSRUWHG WKDW &D LV QHHGHG IRU WKH VWDELOLW\ RI QDWXUDO PHPEUDQHV 3DOWD HW DO Ef UHSRUWHG D VPDOO EXW LQFUHDVLQJ HIIOX[ RI &D IURP RQLRQ EXOE FHOOV DV D UHVXOW RI IUHH]LQJ LQMXU\ 7KH\ FRQFOXGHG WKDW VXFK UHPRYDO RI &D IURP WKH PHPEUDQHV FDXVHV LQVWDELOLW\ RI WKH PHPEUDQH VWUXFWXUH DQG ILQDOO\ UHVXOWV LQ EUHDNGRZQ RI WKH PHPEUDQH V\VWHP %DVHG RQ WKH OHDNDJH RI LRQV 'H[WHU HW DO f UHSRUWHG WKDW PHDVXUHPHQW RI HOHFWULFDO FRQGXFWLYLW\ RI WKH LRQV FDQ SURYLGH D TXDQWLWDWLYH HYDOXDWLRQ RI IUHH]LQJ LQMXU\ (OHFWURO\WH OHDNDJH KDV EHHQ XVHG E\ 3DWWHUVRQ HW DO f WR PHDVXUH ORZWHPSHUDWXUH VHQVLWLYLW\ LQ VSHFLHV RI SDVVLRQIORZHU 3DVVLIORUDf KDYLQJ D UDQJH RI FOLPDFWLF UHTXLUHPHQWV :LOWEDQN DQG 2VZDOW f UHSRUWHG WKH XVH RI

PAGE 25

HOHFWURO\WH OHDNDJH WR GHWHUPLQH FKDQJHV LQ WKH NLOOLQJ SRLQW WHPSHUDWXUH RI VHYHUDO FLWUXV FXOWLYDUV 8VH RI HOHFWURO\WH OHDNDJH WR PHDVXUH YDULDWLRQ LQ ORZ WHPSHUDWXUH VWUHVV VKRXOG EH DSSOLFDEOH LQ DVVHVVLQJ YDULDWLRQV LQ ORZWHPSHUDWXUH VHQVLWLYLWLHV RI UHJHQHUDWHG SDQJRODJUDVV SODQWV 'DPDJH WR WKH SKRWRV\VWHP DSSDUDWXV RFFXUV DW WHPSHUDWXUHV DERYH WKRVH ZKLFK FDXVH EUHDNGRZQ RI FHOOXODU LQWHJULW\ %HUU\ DQG %MRUNPDQ f 7KLV GDPDJH FDQ EH XVHG DV DQ LQGLFDWLRQ RI WKH DPRXQW RI ORZWHPSHUDWXUH VWUHVV LQ WKH SODQW 'UDNH DQG 6DOLVEXU\ f UHSRUWHG WKDW JURZWK LQKLELWLRQ LQ & VSHFLHV LV SURSRUWLRQDO WR WKH VHYHULW\ RI FKLOOLQJ DQG WR WKH LQWHQVLW\ RI OLJKW UHFHLYHG GXULQJ FKLOOLQJ &KDWWHUWRQ HW DO f DWWULEXWHG UHGXFHG JURZWK LQ SDQJOD GXULQJ FKLOOLQJ WR GDPDJH LQ WKH SKRWRV\QWKHWLF DSSDUDWXV (DUO\ LQGLFDWRUV RI UHGXFHG SKRWRV\QWKHWLF DFWLYLW\ LQ FKLOOVHQVLWLYH SODQWV ZHUH EHOLHYHG WR EH WK\ODNRLG PHPEUDQH SKDVH WUDQVLWLRQV 0XUDWD DQG )RUN 6KQH\RXU HW DO f DQG LQFUHDVHV LQ VWRPDWDO UHVLVWDQFH WR & IOX[ &URRNVWRQ HW DO 'UDNH DQG 6DOLVEXU\ f +RZHYHU ZRUN E\ /RZ HW DO f KDV LQGLFDWHG WKDW ERWK FKLOOLQJVHQVLWLYH DQG FKLOOLQJWROHUDQW SODQWV H[KLELW HTXLYDOHQW WK\ODNRLG SKDVH WUDQVLWLRQV /RZ HW DO f DQG 0DUWLQ f KDYH UHSRUWHG WKDW WKHUH LV LQVXIILFLHQW WK\ODNRLG PHPEUDQH GDPDJH WR DFFRXQW IRU WKH LPSDLUPHQW RI

PAGE 26

SKRWRV\QWKHVLV )XUWKHU +DOOJUHQ HW DO f UHSRUWHG WKDW VWRPDWDO FORVXUH GRHV QRW QHFHVVDULO\ LQKLELW & XSWDNH EHFDXVH LQWHUFHOOXODU & SUHVVXUH FDQ UHPDLQ FRQVWDQW GHVSLWH D GHFUHDVHG VWRPDWDO FRQGXFWDQFH &KLOOLQJ FRUQ LQ OLJKW GHFUHDVHG TXDQWXP \LHOG RI & DVVLPLODWLRQ UDWH RI HYROXWLRQ DQG YDULDEOH IOXRUHVFHQW HPLVVLRQ RI SKRWRV\VWHP ,, /RQJ f +LOODUG DQG :HVW f UHSRUWHG WKDW SDQJOD H[KLELWHG GHFUHDVHG UDWHV RI & DVVLPLODWLRQ DIWHU FKLOOLQJ LQ WKH GDUN &KLOOVWUHVVHG SDQJOD SODQWV DOVR KDG D UHGXFWLRQ LQ WKH +LOO UHDFWLRQ :HVW f 7KH H[WHQW RI ORZWHPSHUDWXUH DOWHUDWLRQV LQ YDULDEOH IOXRUHVFHQW HPLVVLRQ RI SKRWRV\VWHP ,, LQ SDQJOD LV XQNQRZQ 6WHLQEDFN DQG 0DOO f UHSRUWHG WKDW WKH ORVV RI SKRWRV\QWKHWLF FRPSHWHQFH LQ ORZ WHPSHUDWXUH LV GXH WR SKRWRLQKLELWLRQ RI SKRWRV\VWHP ,, DFWLYLW\ &KLOOLQGXFHG GDPDJH WR SKRWRV\VWHP ,, KDV EHHQ DVVRFLDWHG ZLWK DOWHUHG HQHUJ\ IORZV LQ WKH HOHFWURQ WUDQVSRUW FKDLQ 3HUFLYDO HW DO +HLQULFK DQG /DVFK f 7KH DFWLYH FHQWHU RI SKRWRV\VWHP ,, LV QRW LPPHGLDWHO\ DIIHFWHG E\ FKLOOLQJ 6PLOOLH SHUVRQDO FRPPXQLFDWLRQf &KLOOLQJWHPSHUDWXUH LQKLELWLRQ RI WKH ZDWHUVSOLWWLQJ VLGH RI SKRWRV\VWHP ,, ZDV UHSRUWHG E\ 0DUJXOLHV f DQG •TXLVW f DV DVVD\HG E\ HOHFWURQ WUDQVIHU DQG IOXRUHVFHQW H[FLWDWLRQ RI FKORURSK\OO D 1R FKLOOLQJ WHPSHUDWXUH HIIHFWV RQ SKRWRV\VWHP ZHUH REVHUYHG E\ •TXLVW f +HWKHULQJWRQ

PAGE 27

HW DO f DQG
PAGE 28

FKLOO VWUHVV UHGXFHG WKH )5 YDOXH LQ FRUQ 7KHLU ZRUN ZLWK FRUQ OHG WR WKH LGHQWLILFDWLRQ RI FKLOOWROHUDQW FRUQ SRSXODWLRQV EDVHG RQ )5 YDOXHV 0DFKDGR HW DO f UDWHG LQFUHDVHG DWUD]LQH UHVLVWDQFH XVLQJ VRPDWLF YDULDQWV UHJHQHUDWHG IURP WLVVXH FXOWXUH E\ WHVWLQJ GLIIHUHQFHV LQ WKH HPLVVLRQ RI FKORURSK\OO IOXRUHVFHQFH 7R GDWH QR UHSRUWV RQ VRPDWLF YDULDWLRQ LQ FKLOOLQJ RU IUHH]LQJWHPSHUDWXUH WROHUDQFH KDYH EHHQ SUHVHQWHG XVLQJ IOXRUHVFHQW HPLVVLRQ PHDVXUHPHQWV 0DWHULDOV DQG 0HWKRGV 3ODQW 0DWHULDO DQG *URZLQJ &RQGLWLRQV $ nSDQJODn GLJLWJUDVV 'LJLWDULD GHFXPEHQV 6WHQWf SODQW ZDV REWDLQHG IURP WKH 8QLYHUVLW\ RI )ORULGDnV $JURQRP\ 6HHG /DERUDWRU\ LQ 6HSWHPEHU RI (OHYHQ YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQWV ZHUH JURZQ LQ D JUHHQKRXVH ZLWK QDWXUDO OLJKWLQJ 7KH DYHUDJH GDLO\ WHPSHUDWXUH UDQJH ZDV r& WR r& 7KH VRXUFH SODQWV ZHUH JURZQ LQ FP SRWV FRQWDLQLQJ 0HWURPL[ JURZLQJ PHGLXP 5DSLG JURZWK ZDV PDLQWDLQHG E\ VXSSO\LQJ VL[ JUDPV RI 2VPRFRWH : 5 *UDFH &DPEULGJH 0$f D VORZUHOHDVLQJ IHUWLOL]HU 1 Sf§.f WR HDFK SRW 7KH SODQWV UHFHLYHG DGHJXDWH ZDWHU RQ D GDLO\ EDVLV DQG ZHUH FOLSSHG WR FP DERYH WKH VRLO VXUIDFH HYHU\ WZR ZHHNV &DUH ZDV WDNHQ WR SUHYHQW URRW ELQGLQJ E\ YHUWLFDOO\ KDOYLQJ WKH HQWLUH SODQW DQG UHSRWWLQJ ZKHQ QHHGHG

PAGE 29

5(/$7,9( )/825(6&(1&( )LJ 'HFUHDVH LQ WKH UDWH RI IOXRUHVFHQW HPLVVLRQ )Uf IURP SDQJRODJUDVV FKLOOHG DW r& IRU PLQXWHV 3HDN IOXRUHVFHQFH 3f LV WKH PD[LPXP IOXRUHVFHQW HPLVVLRQ

PAGE 30

&HOO &XOWXUH ,QLWLDWLRQ 6RXUFH SODQWV ZHUH QR ORQJHU FOLSSHG DIWHU WKH ILUVW RI 0D\ LQ RUGHU WR SURPRWH IORUDO GHYHORSPHQW ,Q HDUO\ -XQH RI LPPDWXUH LQIORUHVFHQFH WR FP LQ OHQJWK ZHUH H[FLVHG IURP FXOPV DW DSSUR[LPDWHO\ $0 (QWLUH LQIORUHVFHQFH ZDV VXUIDFH VWHULOL]HG LQ b YYf VRGLXP K\SRFKORULWH b &ORUR[ LQ ZDWHUf $ GURS RI 7ZHHQ )LVKHU 6FLHQWLILF &RPSDQ\ )DLU /DZQ 1-f ZDV DGGHG SHU P/ RI VROXWLRQ )ORZHU KHDGV ZHUH WRWDOO\ LPPHUVHG LQ WKH VWHULOL]LQJ VROXWLRQ IRU PLQ ZKLOH EHLQJ VKDNHQ DW USP RQ D 1HZ %UXQVZLFN 6FLHQWLILF J\URWRU\ VKDNHU (GLVRQ 1-f 7KH UHPDLQLQJ GHFRQWDPLQDWLRQ SURFHGXUHV DQG WUDQVIHUULQJ WR FXOWXUH PHGLD ZDV GRQH XQGHU DQ (GJHJXDUG ODPLQDU IORZ KRRG 7KH %DNHU &R 6DQIRUG 0(f 7KH IORZHU KHDGV ZHUH ULQVHG IRXU WLPHV ZLWK VWHULOH GHLRQL]HG ZDWHU G+f DQG WKHQ FXW ORQJLWXGLQDOO\ E\ PDNLQJ DQ LQFLVLRQ ZLWK D VFDOSHO WKURXJK WKH OHDI VKHDWKV 7KH LPPDWXUH IORUHWV ZHUH H[SRVHG E\ VSUHDGLQJ WKH WLVVXH DSDUW RQ HLWKHU VLGH RI WKH LQFLVLRQ ZLWK D SDLU RI FXUYHG WLS PLFURIRUFHSV 7KH VSLNHV ZHUH H[FLVHG ZLWK VPRRWK WLS PLFURIRUFHSV DQG FXW LQWR PP VHJPHQWV 7HQ WR VHJPHQWV RU H[SODQWV ZHUH FXOWXUHG LQ HDFK RI ; PP SHWUL GLVKHV FRQWDLQLQJ D VROLG 06 PHGLD 0XUDVKLJH DQG 6NRRJ f 7KH 06 PHGLD FRQWDLQHG SUHPL[HG 06 VDOWV 6LJPD &KHPLFDO &R 6W /RXLV 02f b 7 & DJDU &DUROLQD %LRORJLFDO 6XSSO\ &R

PAGE 31

%XUOLQJWRQ 1&f b VXFURVH DQG GLFKORURSKHQR[\DFHWLF DFLG 'f 7KH SHWUL GLVKHV ZHUH ZUDSSHG LQ SDUDILOP $PHULFDQ &DQ &R *UHHQZLFK &7f DQG SODFHG LQ D 3HUFLYDO JURZWK FKDPEHU %RRQH ,$f DW r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f ZHUH SODFHG LQ HDFK WHPSHUDWXUH WUHDWPHQW 7KH WUHDWPHQWV ZHUH r& r& IRU G r& IRU G r& IRU K r& IRU K r& IRU K $OO FXOWXUHV ZHUH PDLQWDLQHG LQ WKH GDUN 7KH r& WUHDWPHQWV ZHUH SODFHG LQ D 3HUFLYDO JURZWK FKDPEHU ZKHUHDV WKH r& WUHDWPHQWV ZHUH NHSW LQ D :KLUOSRRO IUHH]HU %HQWRQ +DUERU 0,f &DOOXV YLDELOLW\ ZDV HVWLPDWHG E\ WKH DELOLW\ WR UHJURZ RQ IUHVK 06 PHGLD FRQWDLQLQJ 0 DW r& LQ K OLJKWGDUN IRU G

PAGE 32

3ODQW 5HJHQHUDWLRQ $IWHU H[SRVXUH WR WHPSHUDWXUH WUHDWPHQWV WKH FDOOXV ZDV WUDQVIHUUHG WR D 06 PHGLD FRQWDLQLQJ DQG SODFHG DW r& ZLWK D K OLJKWGDUN SHULRG &KLOOLQJ RU IUHH]LQJ WHPSHUDWXUH GDPDJH DQG VXEVHTXHQW GHYHORSPHQWDO PRUSKRORJ\ RI WKH FDOOXV ZHUH REVHUYHG E\ XVLQJ DQ 2O\PSXV 6=7U VWHUR PLFURVFRSH 7RN\R -DSDQf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f : 5 *UDFH DQG &R )RJOHVYLOOH 3$f 7KH SODQWV ZHUH ZDWHUHG DV QHHGHG IRU WKH

PAGE 33

QH[W WKUHH ZHHNV 7KH SODQWV ZHUH WKHQ WUDQVIHUUHG WR FP SRWV DQG PDLQWDLQHG DV SUHYLRXVO\ GHVFULEHG 6FDQQLQJ (OHFWURQ 0LFURVFRSH 0LFURJUDSKV RI HPEU\R GHYHORSPHQW ZHUH REWDLQHG ZLWK D +LWDFKL 6 VFDQQLQJ HOHFWURQ PLFURVFRSH 7RN\R -DSDQf 6HOHFWHG WLVVXH ZDV IL[HG LQ )$$ DQG GULHG WKURXJK DQ DOFRKRO GHK\GUDWLRQ VHULHV 6RPH RI WKH WLVVXH ZDV VHFWLRQHG ZLWK D VLQJOHHGJH UD]RU EODGH 7KH WLVVXH ZDV GULHG LQ D %DO]HU &3' FULWLFDO SRLQW GU\HU )XUVWHQWXP /LHFKWHQVWHLQf DQG FRDWHG ZLWK JROG LQ D 6DPVSXWWHU D DXWRPDWLF FRDWLQJ DSSDUDWXV 7RXVLPLV 5HVHDUFK &RUS 5RFNYLOOH 0'f 3ODQW 6HOHFWLRQ (OHFWURO\WH /HDNDJH $ WRWDO RI UHJHQHUDWHG SODQWV DQG WKUHH YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQWV ZHUH WHVWHG IRU IUHH]LQJ WHPSHUDWXUH GDPDJH XVLQJ PRGLILFDWLRQV LQ WKH HOHFWURO\WH OHDNDJH SURFHGXUH SUHVHQWHG E\ 3DWWHUVRQ HW DO f DQG :LOWEDQN DQG 2VZDOW f 7LVVXH ZDV REWDLQHG IURP SODQWV PDLQWDLQHG LQ WKH JUHHQKRXVH 6L[ \RXQJ IXOO\ H[SDQGHG OHDYHV IURP HDFK SODQW ZHUH FXW IURP WKH SODQW DQG ZDVKHG WKUHH WLPHV LQ GHLRQL]HG ZDWHU /HDI WLSV DQG FP RI OHDI WLVVXH ZHUH LPPHUVHG LQ D P/ WHVW WXEH FRQWDLQLQJ P/ RI GHLRQL]HG ZDWHU &DUH ZDV WDNHQ WR DYRLG VXEPHUJLQJ FXW WLVVXH 7KH WHPSHUDWXUH RI

PAGE 34

WKH OHDI WLVVXH DQG ZDWHU ZDV GHFUHDVHG E\ DSSUR[LPDWHO\ r& SHU PLQ WR D ILQDO WHPSHUDWXUH RI r& IRU PLQ LQ D /DXGD UHIULJHUDWHG FLUFXODWRU %ULQNPDQQ ,QVWUXPHQWV &R :HVWEXUU\ 1
PAGE 35

XVHG IRU GDWD DQDO\VLV 0HDQV ZHUH FDOFXODWHG DQG GDWD ZHUH DQDO\]HG E\ 'XQFDQnV 1HZ 0XOWLSOHUDQJH 7HVW 7KH VDPH SODQWV ZHUH XVHG IRU FKORURSK\OO IOXRUHVFHQFH GHWHUPLQDWLRQV 3ODQW 6HOHFWLRQ &KORURSK\OO )OXRUHVFHQFH ([SHULPHQWV ZHUH FRQGXFWHG GXULQJ 0DUFK 7KH SUHYLRXVO\ VHOHFWHG SODQWV ZHUH UHPRYHG IURP WKH JUHHQKRXVH DW $0 DQG GDUN DGMXVWHG IRU PLQ DW r& EHIRUH REWDLQLQJ LQLWLDO IOXRUHVFHQW HPLVVLRQ PHDVXUHPHQWV 7KH WRS WKLUG RI WKH ILUVW IXOO\ H[WHQGHG OHDI ZDV H[FLVHG IURP WKH SODQW DQG SODFHG LQ D r& +DQVDWHFK /' OHDI FKDPEHU .LQJnV /\QQ 1RUIRON 8.f 7KH OHDI ZDV SRVLWLRQHG DERYH D SDG ZHWWHG ZLWK D 0 VRGLXP ELFDUERQDWH VROXWLRQ 7KH VRGLXP ELFDUERQDWH PDLQWDLQHG DQ DGHTXDWH VXSSO\ RI & IRU SKRWRV\QWKHVLV :DONHU f $ WHPSODWH ZDV FRQVWUXFWHG ZKLFK OLPLWHG WKH OLJKW H[SRVXUH WR PP RI OHDI WLVVXH 7KH WHPSODWH GLG QRW LQWHUIHUH ZLWK JDV IORZ ZLWKLQ WKH FKDPEHU ([FLWDWLRQ HQHUJ\ ZDV DSSOLHG WZR PLQXWHV DIWHU WKH OHDI WLVVXH ZDV SODFHG LQ WKH OHDI FKDPEHU $ TXDQWXP IOX[ GHQVLW\ RI : Pf QPf ZDV VXSSOLHG E\ D +DQVDWHFK /6, OLJKW VRXUFH .LQJnV /\QQ 1RUIRON 8.f IRU SKRWRV\QWKHVLV 7KH /' XQLW ZDV DWWDFKHG WR D /DXGD 50 UHIULJHUDWHG FLUFXODWRU %ULQNPDQQ :HVWEXU\ 1
PAGE 36

LQWHUIHUHQFH ILOWHU 'DWD SRLQWV ZHUH REWDLQHG HYHU\ PV ZLWK D /H&UR\ WUDQVLHQW UHFRUGHU )LJV DQG f 6SULQJ 9DOOH\ 1
PAGE 37

)LJ &RQILJXUDWLRQ RI /H&UR\ WUDQVLHQW UHFRUGHU D $ FUDWH E JDLQ GLIIHUHQWLDO DPSOLILHU LQ FUDWH VORW F $ PHPRU\ LQ FUDWH VORW G ZDYHIRUP DQDO\]HU LQ FUDWH VORW H $ *3,% LQWHUIDFH LQ FUDWH VORW

PAGE 38

)LJ &RQILJXUDWLRQ RI /D&UR\ WUDQVLHQW UHFRUGHU D JDLQ GLIIHUHQWLDO DPSOLILHU FRQWURO ORFN ILOWHU LQ JDLQ PXOWLSO\ LQSXW WR IOXRUHVFHQW GHWHFWRU RXWSXW WR ZDYHIRUP DQDO\]HU E ZDYHIRUP DQDO\]HU SRVW WULJJHU VDPSOHV VDPSOH LQWHUYDO H[WHUQDO FORFN LQSXW FKDQQHOV GLVSOD\ FKDQQHO LQSXW WR

PAGE 39

5HVXOWV DQG 'LVFXVVLRQ 6RPDWLF HPEU\RJHQHVLV LQ SDQJOD IROORZHG D SDWWHUQ RI GHYHORSPHQW VLPLODU WR WKDW UHSRUWHG E\ 0DURXVN\ DQG :HVW f :LWKLQ G RI LQLWLDWLRQ RQ 06 PHGLD FRQWDLQLQJ 0 VRIW IULDEOH FDOOXV IRUPHG IURP WKH SHGLFLOV DQG RYDULDQ WLVVXH 3UROLILF JURZWK ZDV REVHUYHG $IWHU WZR ZHHNV RI FXOWXUH WZR W\SHV RI FDOOXV ZHUH SURGXFHG )LJXUHV f (PEU\RJHQLF FDOOXV ZDV SDOH \HOORZ WR ZKLWH LQ FRORU QRGXODU DQG FRPSDFW LQ DSSHDUDQFH DQG ZDV FRPSRVHG RI VPDOO GHQVHO\ F\WRSODVPLF FHOOV $OWHUQDWLYHO\ QRQ HPEU\RJHQLF FDOOXV ZDV IULDEOH DQG FRQWDLQHG ORRVH FHOOV ZKLFK ZHUH RIWHQ HORQJDWHG DQG KLJKO\ YDFXRODWHG ,VRODWLRQ RI FHOO OLQHV ZLWK DQ LQFUHDVHG WROHUDQFH WR IUHH]LQJ RU FKLOOLQJ WHPSHUDWXUH ZDV DFKLHYHG E\ D VWHSZLVH H[HUWLRQ RI ORZ WHPSHUDWXUH DQG SRVLWLYH VHOHFWLRQ /DUNLQ HW DO f UHSRUWHG WKDW D VWHSZLVH H[HUWLRQ RI VHOHFWLYH SUHVVXUH WHQGV WR VHOHFW WKH QRYHO JHQRW\SHV WKDW DULVH IURP HLWKHU JHQH DPSOLILFDWLRQ DQGRU JHQH PXWDWLRQ )UHH]LQJ DQG FKLOOLQJWHPSHUDWXUH VWUHVV DGYHUVHO\ DIIHFWHG WKH YLDELOLW\ RI HPEU\RJHQLF FDOOXV 7DEOH f &DOOXV YLDELOLW\ GHFUHDVHG ZKHQ WKH WHPSHUDWXUH VHYHULW\ RU GXUDWLRQ RI H[SRVXUH LQFUHDVHG $Q /' ZDV DSSUR[LPDWHG ZKHQ FKLOOLQJ r&f WHPSHUDWXUH ZDV LPSRVHG IRU G RU ZKHQ IUHH]LQJ r&f WHPSHUDWXUH ZDV LPSRVHG IRU K 'DPDJHG DQG QHFURWLF FDOOXV EHFDPH DSSDUHQW VHYHUDO GD\V DIWHU WKH WHPSHUDWXUH

PAGE 40

)LJ 3DQJOD FDOOXV ZLWK HPEU\RJHQLF Hf DQG QRQHPEU\RJHQLF Qf FDOOXV 2Q 06 PHGLD FRQWDLQLQJ [0 ;

PAGE 41

)LJ 3DQJOD FDOOXV ZLWK HPEU\RJHQLF Hf DQG QRQHPEU\RJHQLF Qf FDOOXV 2Q 06 PHGLD FRQWDLQLQJ 0 ;

PAGE 42

7DEOH 3HUFHQWDJH RI SDQJOD FDOOXV VXUYLYLQJ ORZ WHPSHUDWXUHV DQG VXSSRUWLQJ SODQW UHJHQHUDWLRQ 7UHDWPHQW 3DQJOD FDOOXV 7HPSHUD WXUH 'XUDWLRQ 7RWDO 9LDEOH 6XSSRUWLQJ UHJHQHUDWLRQ G RU K b b FRQVWDQW G G K K K 3HULRG RI H[SRVXUH WR WHPSHUDWXUH WUHDWPHQW A3HUFHQWDJH RI YLDEOH FDOOXV IURP ZKLFK SODQWV UHJHQHUDWHG

PAGE 43

)LJ 3DQJOD FDOOXV ZLWK HPEU\RJHQLF Hf DQG QRQHPEU\RJHQLF Qf FDOOXV 2Q 06 PHGLD FRQWDLQLQJ P0 ;

PAGE 44

7DEOH 3HUFHQWDJH RI SDQJOD FDOOXV VXUYLYLQJ ORZ WHPSHUDWXUHV DQG VXSSRUWLQJ SODQW UHJHQHUDWLRQ 7UHDWPHQW 3DQJOD FDOOXV 7HPSHUD WXUH 'XUDWLRQr 7RWDO 9LDEOH 6XSSRUWLQJ UHJHQHUDWLRQ r& G RU K b b FRQVWDQW G G K K K 3HULRG RI H[SRVXUH WR WHPSHUDWXUH WUHDWPHQW A3HUFHQWDJH RI YLDEOH FDOOXV IURP ZKLFK SODQWV UHJHQHUDWHG

PAGE 45

WUHDWPHQWV HQGHG 7KH DIIHFWHG FDOOXV DSSHDUHG GDUN EURZQ LQ FRORU VRIWHU LQ FRQVLVWHQF\ DQG GLVFRORUHG WKH VXUURXQGLQJ PHGLD 7KH VXUYLYLQJ HPEU\RJHQLF FDOOL UHWDLQHG WKHLU FKDUDFWHULVWLF DSSHDUDQFH 7KHRUHWLFDOO\ WKH VXUYLYLQJ FHOOV KDG D KLJKHU GHJUHH RI FKLOO WROHUDQFH WKDQ WKH FKLOO VXVFHSWLEOH FHOOV 6HOHFWLRQ SURFHGXUHV UHSRUWHG E\ 'L[ DQG 6WUHHW f %UHLGHQEDFK DQG :DULQJ f 'L[ f 7HPSOHWRQ6RPHUV HW DO f DQG &KHQ HW DO f KDYH VXFFHVVIXOO\ LVRODWHG FHOO OLQHV WKDW WROHUDWHG FKLOOLQJ RU IUHH]LQJ WHPSHUDWXUHV 6RPDWLF HPEU\RV ZHUH REWDLQHG IURP WKH VXUYLYLQJ FDOOL E\ UHGXFLQJ WKH OHYHO RI LQ WKH PHGLXP ZDV UHGXFHG WR 0 )LJXUH f 0DWXULQJ HPEU\RV FRQWDLQHG WKH FKDUDFWHULVWLF RUJDQV RI D JUDVV HPEU\R VFXWHOOXP FROHRSWLOH DQG VKRRW DSH[ )LJXUH f *URVV DEQRUPDOLWLHV LQ HPEU\R GHYHORSPHQW ZHUH DEVHQW 9DULDWLRQ LQ WKH GHJUHH RI W\SLFDO HPEU\R GHYHORSPHQW ZDV QRW DVVHVVHG 9DVLO f UHSRUWHG HPEU\R GHYHORSPHQW UDQJLQJ IURP DW\SLFDO WR W\SLFDO LQ VRPDWLF HPEU\RV RI GLIIHUHQW JUDVVHV (OHYHQ ZHHNV DIWHU FDOOL ZHUH LQLWLDWHG SODQW UHJHQHUDWLRQ IURP VRPDWLF HPEU\RV ZDV DGYDQFHG RQ 06 PHGLD ZLWKRXW )LJXUH f 0DWWKHZHV DQG 9DVLO f UHSRUWHG WKDW LQFUHDVLQJ WKH OHQJWK RI WLPH LQ FXOWXUH IDYRUV YDULDQW FHOOV DQG UHVXOWV LQ PRUH SUHFRFLRXV UHJHQHUDWLRQ 7DEOH VKRZV WKDW DIWHU ZHHNV LQ FXOWXUH

PAGE 46

)LJ /RQJLWXGLQDO VHFWLRQ RI SDQJOD FDOOXV ZLWK QRQHPEU\RJHQLF Qf FDOOXV HPEU\RJHQLF Hf FDOOXV DQG \RXQJ VRPDWLF HPEU\R HPf 2Q 06 PHGLD FRQWDLQLQJ 0 ;

PAGE 47

)LJ /RQJLWXGLQDO VHFWLRQ RI SDQJOD FDOOXV ZLWK HPEU\RJHQLF Hf FDOOXV VXSSRUWLQJ D PDWXULQJ VRPDWLF HPEU\R VFXWHOOXP VFf FROHRSWLOH FRf DQG VKRRW DSH[ Vf 2Q 06 PHGLD ZLWKRXW ;

PAGE 48

)LJ 5HJHQHUDWHG SDQJOD SODQW ZLWK SOXPXOH Sf DQG \RXQJ URRW Uf 2Q 06 PHGLD ZLWKRXW ;

PAGE 49

DW r& b RI WKH YLDEOH HPEU\RJHQLF FDOOL VXSSRUWHG SODQW UHJHQHUDWLRQ 0DURXVN\ DQG :HVW f REVHUYHG HPEU\RJHQHVLV LQ SDQJRODJUDVV FDOOXV DW D SHUFHQW IUHTXHQF\ DIWHU ILYH ZHHNV RI FXOWXUH 7KH SHUFHQWDJH RI YLDEOH HPEU\RJHQLF FDOOXV FDSDEOH RI VXSSRUWLQJ SODQW UHJHQHUDWLRQ GHFUHDVHG ZKHQ WKH WHPSHUDWXUH VHYHULW\ RU GXUDWLRQ RI H[SRVXUH LQFUHDVHG &DOOXV VXUYLYLQJ r& IRU K ZDV XQDEOH WR UHJHQHUDWH SODQWV 'L[ DQG 6WUHHW f DQG &KHQ HW DO f IRXQG WKDW WREDFFR FHOO OLQHV ZKLFK ZHUH VHOHFWHG IRU ORZWHPSHUDWXUH UHVLVWDQFH ZHUH XQDEOH WR VXSSRUW SODQW UHJHQHUDWLRQ $Q REVHUYHG LQFUHDVH LQ WKH FRQFHQWUDWLRQ RI &D LQ WKH HIIOXVDWH RI IUHH]HLQMXUHG OHDI WLVVXH GDWD QRW VKRZQf DJUHHG ZLWK 3DOWD HW DO Df DQG LPSOLHG D ORVV RI SODVPDOHPPD LQWHJULW\ 7KH SHUFHQWDJH RI UHJHQHUDWHG SODQWV H[KLELWLQJ HOHFWURO\WH FRQGXFWLYLW\ YDOXHV OHVV WKDQ WKH YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQWV LV JLYHQ LQ 7DEOH 7KH SHUFHQWDJH RI UHJHQHUDWHG SODQWV UDQJHG IURP D KLJK RI DSSUR[LPDWHO\ b r& r& IRU Gf WR D ORZ RI b r& IRU Kf ,QFUHDVLQJ WKH VHYHULW\ RI WKH LQ YLWUR WHPSHUDWXUH WUHDWPHQW RQ FDOOL UHVXOWHG LQ IHZHU UHJHQHUDWHG SODQWV ZLWK OHVV HOHFWURO\WH OHDNDJH 7DEOH f 6WHSRQNXV f UHSRUWHG D WRWDO ORVV RI FKLOOLQJWHPSHUDWXUH WROHUDQFH LQ FHOOV GXULQJ WLVVXH FXOWXULQJ 2WKHU UHVHDUFKHUV UHSRUWHG D SDUWLDO ORVV RI FKLOOLQJ RU IUHH]LQJ WHPSHUDWXUH WROHUDQFH LQ FHOOV GXULQJ FXOWXULQJ 'L[ DQG

PAGE 50

7DEOH 3HUFHQWDJH RI UHJHQHUDWHG SDQJOD SODQWV ZLWK OHVV HOHFWURO\WH OHDNDJH WKDQ WKH VRXUFH SODQWV &DOOXV WUHDWPHQW 5HJHQHUDWHG SODQWV 7HPSHUDWXUH 'XUDWLRQr 7RWDO (OHFWURO\WH FRQGXFWLYLW\ OHVV WKDQ VRXUFH SODQW G RU K b FRQVWDQW G G K K /HQJWK RI WLPH IRU FDOOXV H[SRVXUH WR WHPSHUDWXUH WUHDWPHQW F 3HUFHQWDJH RI UHJHQHUDWHG SODQWV ZLWK ORZHU HOHFWURO\WH FRQGXFWLYLW\ WKDQ WKH PHDQ HOHFWURO\WH FRQGXFWLYLW\ RI WKUHH VRXUFH SODQWV 6'

PAGE 51

6WUHHW 7HPSOHWRQ6RPHUV HW DO &KHQ HW DO f 7HPSOHWRQ6RPHUV HW DO f UHSRUWHG WKDW FKLOOLQJWHPSHUDWXUH UHVLVWDQFH H[SUHVVHG LQ FDUURW FXOWXUH FHOOV DUH QRW QHFHVVDULO\ H[SUHVVHG LQ WKH VRPDWLF HPEU\R VWDJH RI GHYHORSPHQW 7KHVH REVHUYDWLRQV DUH VXSSRUWHG E\ WKH SUHVHQW VWXG\ ZKHUH IUHH]HWROHUDQW FHOO OLQHV VXSSRUWHG WKH UHJHQHUDWLRQ RI IUHH]HVXVFHSWLEOH SODQWV (OHFWURO\WH FRQGXFWLYLW\ RI WKH VHOHFWHG IUHH]H WROHUDQWUHJHQHUDWHG SODQWV ZDV OHVV WKDQ WKDW IRU WKH VRXUFH SODQWV 7DEOH f 6L[ RI WKH UHJHQHUDWHG SODQWV KDG VLJQLILFDQWO\ UHGXFHG HOHFWURO\WH OHDNDJH ZKHQ FRPSDUHG WR WKH YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQWV 'DWD SUHVHQWHG LQ 7DEOH VXSSRUWHG WKH K\SRWKHVLV WKDW VRPDWLF YDULDWLRQ LQ SDQJRODJUDVV ZDV REWDLQDEOH DQG WKDW YDULDQW UHJHQHUDWHG SODQWV ZHUH DEOH WR KDYH EHHQ VHOHFWHG IRU DQ LQFUHDVH LQ IUHH]H WROHUDQFH 7KH SHUFHQW )5 UHPDLQLQJ LQ UHJHQHUDWHG SODQWV DQG VRXUFH SODQWV DIWHU FKLOOLQJWHPSHUDWXUH H[SRVXUH LV SUHVHQWHG LQ 7DEOH 1R YDULDWLRQ LQ WKH SHUFHQW )5 UHPDLQLQJ DIWHU FKLOOLQJ ZDV GHWHFWHG EHWZHHQ UHJHQHUDWHG SODQWV RU EHWZHHQ UHJHQHUDWHG SODQWV DQG VRXUFH SODQWV 7KHVH UHVXOWV GR QRW VXSSRUW WKH K\SRWKHVLV WKDW VRPDWLF YDULDQWV PD\ EH VHOHFWHG IRU FKLOOLQJ WROHUDQFH XVLQJ FKORURSK\OO IOXRUHVFHQFH +HWKHULQJWRQ HW DO f UHSRUWHG WKDW FKLOO VWUHVV UHGXFHG WKH )5 YDOXH LQ FRUQ D

PAGE 52

7DEOH )UHH]HLQGXFHG HOHFWURO\WH OHDNDJH RI VHOHFWHG UHJHQHUDWHG SDQJOD SODQWV DQG VRXUFH SODQWV 3ODQW 1R 0HDQ FRQGXFWLYLW\ DPSV 6RXUFH SODQW D D E D E F D E F 5HJHQHUDWHG SODQW D E F G E F G H E F G H F G H G H G H G H H H H r f f f 0HDQV QRW IROORZHG E\ VDPH OHWWHU DUH VLJQLILFDQWO\ GLIIHUHQW DW 3 '057f

PAGE 53

7DEOH 3HUFHQW )5 UHPDLQLQJ LQ VHOHFWHG UHJHQHUDWHG SDQJOD SODQWV DQG VRXUFH SODQWV DIWHU H[SRVXUH WR FKLOOLQJ WHPSHUDWXUH 5HJHQHUDWHG SODQWV 6RXUFH SODQWV 3ODQW 1R )U UHPDLQLQJ 3ODQW 1R )5 UHPDLQLQJr b b s s s s s s s s s s s 3HUFHQW )5 UHPDLQLQJ )A r& )5 DIWHU PLQ DW r&f 0HDQ RI VL[ UHSOLFDWLRQV 6'

PAGE 54

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

PAGE 55

&+$37(5 ,,, (9$/8$7,21 2) &+,//,1* 7(03(5$785( $1' *,%%(5(//,& $&,' 21 7+( (;35(66,21 2) +<'52/<7,& (1=<0(6 7KH K\SRWKHVLV WHVWHG LQ WKLV VHFWLRQ ZDV WKDW FKLOOLQJ WHPSHUDWXUHV UHGXFH WKH QXPEHU RI GHEUDQFKLQJ HQ]\PHV HQGRDP\ODVHV DQG H[RDP\ODVHV WKDW EUHDN GRZQ VWDUFK +\SRWKHWLFDOO\ JLEEHUHOOLF DFLG *$f PD\ QXOOLI\ WKH LQIOXHQFH RI ORZ WHPSHUDWXUH RQ WKH UHGXFWLRQ LQ WKH QXPEHU RI VWDUFKGHJUDGLQJ HQ]\PHV 7KH REMHFWLYHV RI WKLV VWXG\ ZHUH Df WR HYDOXDWH WKH LQIOXHQFH RI ORZ WHPSHUDWXUH RQ WKH H[SUHVVLRQ RI VWDUFKn K\GURO\]LQJ HQ]\PHV Ef WR LGHQWLI\ K\GURO\WLF HQ]\PHV ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ XQGHU ORZ WHPSHUDWXUH FRQGLWLRQV Ff WR HYDOXDWH WKH HIIHFW RI *$ RQ WKH H[SUHVVLRQ RI VWDUFKK\GURO\]LQJ HQ]\PHV GXULQJ ORZ WHPSHUDWXUH FRQGLWLRQV DQG Gf WR LGHQWLI\ *$ LQGXFHG LVR]\PLF IRUPV RI K\GURO\WLF HQ]\PHV ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ XQGHU ORZWHPSHUDWXUH FRQGLWLRQV 7KH VHYHUH JURZWK GHSUHVVLRQ RI SDQJOD WKDW UHVXOWV IURP ORZ QLJKWWLPH WHPSHUDWXUHV KDV EHHQ DVVRFLDWHG ZLWK WKH DFFXPXODWLRQ RI VWDUFK JUDQXOHV LQ PHVRSK\OO FHOOV DQG EXQGOH VKHDWK FHOOV +LOODUG DQG :HVW &KDWWHUWRQ HW DO f +LOODUG f UHSRUWHG WKDW FXOWLYDUV RI

PAGE 56

'LJLWDULD ZKRVH JURZWK ZDV OHDVW DIIHFWHG E\ FKLOOLQJ WHPSHUDWXUH ZHUH WKRVH WKDW UHWDLQHG WKH OHDVW DPRXQW RI VWDUFK LQ WKHLU FKORURSODVWV DIWHU FKLOOLQJ QLJKWV 7KH DFFXPXODWLRQ RI ODUJH DVVLPLODWHG VWDUFK JUDLQV LQ WKH FKORURSODVW KDV EHHQ VKRZQ WR UHGXFH JURZWK UDWHV DQG QHW SKRWRV\QWKHVLV YLD SK\VLFDO GDPDJH RI WKH FKORURSODVW PHPEUDQH :HVW f DQG GLVUXSWLRQ RI SKRWRSKRVn SKRU\ODWLRQ DQG HOHFWURQ WUDQVSRUW 3HDUVRQ DQG 'HUULFN f *DUUDUG DQG :HVW f GHWHUPLQHG WKDW ORZ OHDI WHPSHUDWXUH GUDVWLFDOO\ UHGXFHV VWDUFK DQDEROLVP DQG WUDQVORFDWLRQ HYHQ ZKHQ QHDUE\ VLQNV DUH PDLQWDLQHG DW KLJK WHPSHUDWXUHV -RQHV f FRQFXUUHG WKDW WUDQVORFDWLRQ RI SKRWRV\QWKDWH WR WKH JURZLQJ UHJLRQ DQG XWLOL]DWLRQ RI SKRWRV\QWKDWH E\ WKLV UHJLRQ ZDV OLPLWHG E\ FKLOOLQJ WHPSHUDWXUH .OHLQHGRUVW DQG %URXZHU f UHSRUWHG WKDW UHVWULFWLQJ FKLOOLQJ WR WKH OHDYHV KDG OLWWOH HIIHFW RQ PHULVWHPDWLF FHOO GLYLVLRQ 7KH\ REVHUYHG D UHGXFWLRQ LQ FHOO HORQJDWLRQ DQG SRVWXODWHG WKDW WKLV ZDV GXH WR UHGXFHG FDUERK\GUDWH DYDLODELOLW\ IRU FHOO ZDOO IRUPDWLRQ LQ WKH UHJLRQ RI FHOO HORQJDWLRQ 7KH ELRV\QWKHVLV RI FKORURSODVWLF VWDUFK LV ZHOO FKDUDFWHUL]HG DQG KDV EHHQ UHYLHZHG E\ 3UHLVV f 3UHLVV DQG /HYL f DQG 3UHLVV HW DO f 0RVW VWDUFKHV DUH D FRPSRVLWH RI WZR GLIIHUHQW W\SHV RI SRO\VDFFKDULGHV 0H\HU DQG *LEERQV f 2QH LV

PAGE 57

XQEUDQFKHG DQG FRPSRVHG RI ORQJ FKDLQV RI D OLQNHG JOXFRVH XQLWV WKH RWKHU LV EUDQFKHG DQG FRQVLVWV RI VKRUWHU FKDLQV RI D OLQNHG JOXFRVH UHVLGXHV ZKLFK DUH MRLQHG WKURXJK WKH D SRVLWLRQV WR IRUP D ODUJH PROHFXOH 0H\HU FDOOHG WKH OLQHDU FRPSRQHQW DP\ORVH DQG WKH EUDQFKHG RQH DP\ORSHFWLQ 7KH QXPEHU RI JOXFRVH XQLWV LQ YDULRXV DP\ORSHFWLQV UDQJHV IURP WR ZKHUHDV DP\ORVH PD\ FRQWDLQ D IHZ WKRXVDQG JOXFRVH XQLWV 6DOLVEXUU\ DQG 5RVV Ef $VVLPLODWRU\ VWDUFK JUDQXOHV LQ SKRWRV\QWKHWLF WLVVXH DUH ORFDWHG LQ WKH FKORURSODVW 9LHZHG XQGHU D VFDQQLQJ HOHFWURQ PLFURVFRSH VWDUFK JUDQXOHV LQ SHD 3LVXP VDWLYXP /f DUH FRQVLVWHQWO\ VXUURXQGHG E\ WK\ODNRLG PHPEUDQHV %HFN f %HFN f UHSRUWHG WKH DSSHDUDQFH RI WKH JUDQXOHV WR EH GUDPDWLFDOO\ GLIIHUHQW EHWZHHQ LOOXPLQDWHG DQG GDUNHQHG SHD OHDYHV 7KH VWDUFK JUDQXOHV IURP DQ LOOXPLQDWHG OHDI XQOLNH D GDUNHQHG OHDI DSSHDUHG SDVW\ DQG KDG D ODFN RI VKDUS FRQWRXUV 7KH SDVW\ VWDUFK PDQWOH VXUURXQGHG D FU\VWDOOLQH VWDUFK FRUH %HFN 6WHXS HW DO f %HFN f LQYHVWLJDWHG WKH ZDWHU FRQWHQW RI WKH VWDUFK JUDQXOH DQG UHSRUWHG WKDW WKH PDQWOH KDG D KLJKHU ZDWHU FRQWHQW WKDQ WKH FRUH $FFRPSDQ\LQJ WKH KLJKHU ZDWHU FRQWHQW WKH PDQWOH DOVR FRQWDLQHG PRUH D EUDQFKLQJ WKDQ WKH FRUH

PAGE 58

&RQIXVLRQ LQ WKH OLWHUDWXUH FRQFHUQLQJ WKH PRGH RI VWDUFK GHJUDGDWLRQ UHIOHFWV WKH IDFW WKDW WKH SUHGRPLQDQW SDWKZD\ RI VWDUFK GHJUDGDWLRQ ZLWKLQ WKH FKORURSODVW LV XQUHVROYHG 8QWLO UHFHQWO\ PDQ\ UHVHDUFKHUV EHOLHYHG WKDW VWDUFK SKRVSKRURO\VLV E\ SKRVSKRU\ODVH 6WLWW DQG +HOGW /HYL DQG 3UHLVV 6WLWW DQG 5HHV f ZDV WKH SUHGRPLQDQW SDWKZD\ RI VWDUFK GHJUDGDWLRQ 2WKHU UHVHDUFKHUV EHOLHYHG WKDW VWDUFK K\GURO\VLV E\ HQGRDP\ODVH H[RDP\ODVH 5HQ]\PH GHEUDQFKLQJ DFWLYLW\f DQG 'HQ]\PH WUDQVJO\FRV\ODVHf +HUROG HW DO 3HDY\ HW DO &KDQJ f ZDV WKH SUHGRPLQDQW SDWKZD\ RI VWDUFK GHJUDGDWLRQ $VVLPLODWHG VWDUFK LV QRZ EHOLHYHG WR EH GHJUDGHG E\ WKH FRPELQHG DFWLRQ RI K\GURO\VLV DQG SKRVSKRURO\VLV /LQ HW DO .DNHIXGD HW DO 5RELQVRQ DQG 3UHLVV (FKHYHUULD DQG %R\HU f 6WDUFK SKRVSKRU\ODVH FDWDO\VHV WKH SKRVSKRURO\VLV RI D JOXFRV\O FKDLQV \LHOGLQJ JOXFRVHOSKRVSKDWH D DP\ODVH DWWDFNV D OLQNDJHV RI DP\ORVH RU DP\ORSHFWLQ \LHOGLQJ JOXFRVH DQG VPDOO DPRXQWV RI PDOWRVH 6DOLVEXUU\ DQG 5RVV Df MDP\ODVH DWWDFNV RQO\ WKH QRQUHGXFLQJ HQGV RI FKDLQV VSOLWWLQJ RII SDLUV RI JOXFRV\O XQLWV DV PDOWRVH 6DOLVEXUU\ DQG 5RVV Df 5HQ]\PH DWWDFNV WKH EUDQFKLQJ SRLQWV RI DP\ORSHFWLQ PDNLQJ WKLV FRPSRQHQW RI VWDUFK VXVFHSWLEOH WR IXUWKHU DWWDFN E\ K\GURO\VLV RU SKRVSKRURO\VLV 6DOLVEXUU\ DQG 5RVV Df 'HQ]\PH FDQ WUDQVIHU JURXSV RI JOXFRV\O XQLWV EHWZHHQ VKRUW FKDLQ

PAGE 59

GH[WULQV SURGXFLQJ D PL[WXUH RI ORQJHU DQG VKRUWHU FKDLQ GH[WULQV DQG JOXFRVH 6DOLVEXUU\ DQG 5RVV Df 'H[WULQV ZHUH GHVFULEHG E\ 0H\HU DQG *LEERQV f DV VWDUFK EUHDNGRZQ SURGXFWV WKDW ZHUH FRPSRVHG RI PL[WXUHV RI FKDLQ IUDJPHQWV RI ORZ EXW YDU\LQJ PROHFXODU ZHLJKWV 'H[WULQV DUH URXWLQHO\ DQDO\]HG XVLQJ D SRWDVVLXPLRGLGH LRGLQH UHDFWLRQ -HQVHQ f .DNHIXGD DQG 'XNH f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f %HFN f QRWHG WKDW GXULQJ WKH QRFWXUQDO EUHDNGRZQ RI WKH VWDUFK JUDQXOH WKH PDQWOH ZDV GHJUDGHG PRUH UDSLGO\ WKDQ WKH FRUH (QGRJHQRXV K\GURO\WLF HQ]\PHV DFFRXQWHG IRU WKH GHJUDGDWLRQ RI WKH SDVW\ PDQWOH %HFN HW DO f DQG 6WHXS DQG 6FKDFKWHOH f UHSRUWHG WKDW DVVLPLODWRU\ VWDUFK LV D YHU\ SRRU VXEVWUDWH IRU WKH FKORURSODVWLF SKRVSKRU\ODVH LQ VSLQDFK 6SLQDFLD ROHU£FHDf 7KH\ IRXQG WKDW DP\ORO\VLV RI DVVLPLODWRU\ VWDUFK UHVXOWHG LQ GHJUDGDWLRQ SURGXFWV WKDW ZHUH DFWHG RQ E\ SKRVSKRU\ODVH

PAGE 60

WKUHH WR IRXU WLPHV PRUH UDSLGO\ WKDQ VWDUWLQJ PDWHULDOV 7KHVH UHVXOWV GHPRQVWUDWH WKH FRRSHUDWLYLW\ DQG QRW D FRPSHWLWLRQ EHWZHHQ WKH DP\ORO\WLF V\VWHP DQG SKRVSKRU\ODVH LQ DVVLPLODWRU\ VWDUFK EUHDNGRZQ %HFN f VWDWHG WKDW DOWKRXJK DP\ODVHV FRRSHUDWH ZLWK SKRVSKRU\ODVHV LQ WKH GHJUDGDWLRQ RI DVVLPLODWRU\ VWDUFK WKH RYHUDOO FHOOXODU ORFDWLRQ DQG SK\VLRORJLFDO UROH RI WKHVH HQ]\PHV LQ OHDYHV LV IDU IURP FOHDU )RU H[DPSOH VSLQDFK OHDI FKORURSODVWV FRQWDLQ HQGRDP\ODVH GHEUDQFKLQJ HQ]\PH SKRVSKRU\ODVH DQG 'HQ]\PH 2NLWD DQG 3UHLVV 3UHLVV HW DO f ,Q FRQWUDVW SHD OHDI FKORURSODVWV FRQWDLQ SKRVSKRU\ODVH 5HQ]\PH 'HQ]\PH .DNHIXGD HW DO f DQG D VPDOO DPRXQW RI MDP\ODVH /HYL DQG 3UHLVV f 7KH DDP\ODVH LV HLWKHU DEVHQW RU LQ YHU\ ORZ DFWLYLW\ LQ WKH FKORURSODVW -DFREVHQ DQG %HDFK HW DO .DNHIXGD HW DO f %HHUV DQG 'XNH f UHSRUWHG WKDW WKH ORFDWLRQ RI H[WUDFKORURSODVWLF DDP\ODVH LV SULPDULO\ DSRSODVWLF $OWKRXJK DP\ODVH DQG SKRVSKRU\ODVH DFWLYLWLHV FRQVWLWXWH WKH PDMRU GHJUDGDWLYH DFWLYLW\ LQ WKH FKORURSODVW RQO\ HQGRDP\ODVH KDV EHHQ GHPRQVWUDWHG WR DWWDFN DVVLPLODWHG VWDUFK JUDQXOHV 6WHXS HW DO f 7KH H[WUDFKORURSODVWLF ORFDWLRQ RI DDP\ODVH SUHVHQWV D ORJLVWLF SUREOHP 1R H[WUDFKORURSODVWLF KRPRJOXFDQV ZKLFK FRXOG DFW DV VXEVWUDWHV IRU VXFK DP\ODVHV DUH NQRZQ DQG QR

PAGE 61

H[SODQDWLRQV KDYH EHHQ SUHVHQWHG WR DFFRXQW IRU WKHVH HQ]\PHV RXWVLGH WKH FKORURSODVW ,Q FRUQ (FKHYHUULD DQG %R\HU f UHSRUWHG VWDUFK DFFXPXODWLRQ LQ WKH FKORURSODVWV RI EXQGOH VKHDWK FHOOV 6WDUFK ZDV DEVHQW IURP WKH FKORURSODVWV RI PHVRSK\OO FHOOV $QDO\VLV RI VWDUFKGHJUDGDWLYH HQ]\PHV VKRZHG WKDW b RI WKH GHJUDGDWLYH HQ]\PHV ZHUH ORFDWHG LQ PHVRSK\OO FHOOV ZKHUH QR VWDUFK DFFXPXODWHG 7KH UHPDLQLQJ b RI VWDUFK GHJUDGDWLYH HQ]\PHV ZHUH ORFDWHG LQ WKH EXQGOH VKHDWK FHOOV ,Q EXQGOH VKHDWK FHOOV b RI WKH DFWLYLW\ RI WKHVH HQ]\PHV ZHUH FKORURSODVWLF DQG b ZHUH ORFDWHG LQ WKH F\WRVRO 6WDUFK SKRVSKRU\ODVH ZDV OLPLWHG WR EXQGOH VKHDWK FHOOV ZLWK RQO\ b RI SKRVSKRU\ODVH DFWLYLW\ EHLQJ FKORURSODVWLF ,Q SDQJOD ORZ WHPSHUDWXUH FDXVHV DQ H[FHVVLYH DFFXPXODWLRQ RI DVVLPLODWHG VWDUFK DQG D GHFUHDVH LQ WKH DFWLYLW\ RI VWDUFKGHJUDGLQJ HQ]\PHV &DUWHU HW DO f +LOODUG f UHSRUWHG WKDW FXOWLYDUV RI ZHHSLQJ ORYHJUDVV (UDTURVWLV FXUXYOD /f DQG GLJLWJUDVV ZKRVH JURZWK ZDV OHDVW DIIHFWHG E\ ORZ WHPSHUDWXUH UHWDLQHG WKH OHDVW VWDUFK LQ WKHLU FKORURSODVWV DIWHU FKLOOLQJ QLJKWV DQG KDG WKH KLJKHVW DP\RO\WLF HQ]\PH DFWLYLWLHV DW FKLOOLQJ WHPSHUDWXUHV .DUEDVVL HW DO f UHSRUWHG D UHGXFWLRQ LQ VWDUFK GHJUDGLQJ DFWLYLW\ ZKHQ SDQJRODJUDVV SODQWV ZHUH SUHWUHDWHG DW FKLOOLQJ QLJKW WHPSHUDWXUHV RU ZKHQ WKH HQ]\PH DVVD\ ZDV FRQGXFWHG XQGHU FKLOOLQJ FRQGLWLRQV

PAGE 62

&DUWHU HW DO f UHSRUWHG WKH ORFDOL]DWLRQ RI WZR EDQGV RI DP\RO\WLF HQ]\PHV E\ SRO\DFU\ODPLGH JHO HOHFWURSKRUHVLV XVLQJ DP\ORVH FRQWDLQLQJ JHOV 7KH UHODWLYH DFWLYLW\ RI ERWK EDQGV ZHUH GHFUHDVHG ZKHQ H[SRVHG WR r& LQFXEDWLRQ WHPSHUDWXUHV ZLWK WKH EDQG QHDUHVW WKH RULJLQ GHFUHDVLQJ WKH OHDVW XQGHU ORZ WHPSHUDWXUH &DUWHU HW DO f PRGLILHG WKH HOHFWURSKRUHWLF SURFHGXUH WR VHSDUDWH WKH DP\RO\WLF HQ]\PHV LQWR VHYHQ EDQGV 7KH EDQGV VHSDUDWHG LQWR D IDVW PLJUDWLQJ SDLU EDQGV DQG f DQG D VORZ PLJUDWLQJ JURXS EDQGV WR f &KLOOLQJ WHPSHUDWXUH GHFUHDVHG WKH DFWLYLW\ RI EDQG WKH PRVW ZKLOH EDQG ZDV GHFUHDVHG WKH OHDVW 1R FKDQJH LQ WKH QXPEHU RI EDQGV ZDV GHWHFWHG XQGHU WKH GLIIHUHQW WHPSHUDWXUH WUHDWPHQWV 3URGXFWLRQ RI DDP\ODVH LQ WKH EDUOH\ +RUGHXP YXORDUH /f DOHXURQH OD\HUV KDV EHHQ VKRZQ WR UHVSRQG WR *$ -DFREVHQ DQG +LJJLQV f $GGLWLRQ RI WKH KRUPRQH WR WKH GHHPEU\RQDWHG VHHG RU LVRODWHG DOHXURQH OD\HUV UHVXOWV LQ D ODUJH LQFUHDVH LQ DDP\ODVH DFWLYLW\ $W]RUQ DQG :HLOHU f DQG GHEUDQFKLQJ HQ]\PH DFWLYLW\ %HZOH\ DQG %ODFN f *$M DFWV DW WKH WUDQVFULSWLRQDO OHYHO LQFUHDVLQJ D DP\ODVH P51$V%HZOH\ DQG %ODFN f 7KDW LQFUHDVH LQ D DP\ODVH DFWLYLW\ LV WKH UHVXOW RI SURGXFWLRQ RI PXOWLSOH LVR]\PH IRUPV &DOOLV DQG +R f 1RODQ DQG +R f DQG 1RODQ HW DO f UHSRUWHG WKDW *$ GLIIHUHQWLDOO\ FRQWUROV WKH H[SUHVVLRQ RI WZR DDP\ODVH JHQHV RU D JURXS RI

PAGE 63

JHQHV JLYLQJ ULVH WR WZR JURXSV RI DDP\ODVH LVR]\PHV ZLWK GLIIHUHQW SURSHUWLHV $WNLQ HW DO f UHSRUWHG WKDW FKLOOLQJWHPSHUDWXUH H[SRVXUH RI FRUQ SODQWV UHGXFHV WKH DPRXQW RI JLEEHUHOOLQ LQ WKH [\OHP H[XGDWH 7KH\ VXJJHVWHG WKDW VORZHU JURZWK UDWHV GXULQJ FKLOOLQJWHPSHUDWXUH H[SRVXUH PD\ EH UHODWHG WR D GHFUHDVH LQ WKH JLEEHUHOOLQ FRQFHQWUDWLRQ LQ WKH OHDI WLVVXH )ROLDU DSSOLFDWLRQ RI *$ WR n7LIGZDUIn D EHUPXGDJUDVV &YQRGRQ GDFWYORQ /f WKDW LV GDPDJHG E\ FKLOOLQJ WHPSHUDWXUH VWLPXODWHV JURZWK GXULQJ FKLOOLQJ WHPSHUDWXUHV 'XGHFN DQG 3HDFRFN f *$ KDV EHHQ VKRZQ WR LPSURYH JURZWK UDWHV RI WURSLFDO IRUDJH JUDVV ZKHQ DSSOLHG KRXUV SULRU WR FKLOOLQJ .DUEDVVL HW DO :KLWQH\ HW DO f )ROLDU DSSOLFDWLRQ RI *$ RQ SDQJOD LQFUHDVHV WKH DP\ORO\WLF DFWLYLW\ LQ WKH SODQWV DW HLWKHU DPELHQW RU FKLOOLQJ WHPSHUDWXUHV &DUWHU HW DO .DUEDVVL HW DO f .DUQRN DQG %HDUG f VKRZHG WKDW *$ DSSOLFDWLRQ RQ FKLOOVWUHVVHG EHUPXGDJUDVV UHGXFHG WKH QXPEHU RI VWDUFK JUDQXOHV LQ WKH EXQGOH VKHDWK FKORURSODVW &DUWHU HW DO f UHSRUWHG WKDW WKH DSSOLFDWLRQ RI *$ LQFUHDVHG WKH UHODWLYH DFWLYLW\ RI WKH VHYHQ DP\ORO\WLF HQ]\PHV WKDW ZHUH VHSDUDWHG E\ JHO HOHFWURSKRUHVLV 7KH DFWLYLW\ RI EDQG ZDV LQFUHDVHG WKH PRVW ZKLOHWKH DFWLYLW\ RI EDQG ZDV OHDVW LQIOXHQFHG 2I WKH JURXS WKH HQ]\PH RI EDQG ZDV PRUH UHVSRQVLYH DQG WKRVH RI EDQGV DQG

PAGE 64

ZHUH WKH OHVV HQKDQFHG E\ *$ *$ DSSOLFDWLRQ GLG QRW FDXVH WKH SURGXFWLRQ RI DGGLWLRQDO DP\ORO\WLF LVR]\PHV 7KLV UHVSRQVH LV XQOLNH WKDW LQ WKH DOHXURQH OD\HUV RI EDUOH\ VHHG DQG PD\ EH XQLTXH WR FKORURSODVWV 0DWHULDOV DQG 0HWKRGV 3ODQW 0DWHULDO DQG 7UHDWPHQW $SSOLFDWLRQ ([SHULPHQWV ZHUH FRQGXFWHG GXULQJ WKH PRQWKV RI -XO\ DQG $XJXVW DW ZKLFK WLPH WKH SKRWRSHULRG GHFUHDVHG IURP K WR K DQG WKH TXDQWXP IOX[ GHQVLW\ DYHUDJHG WR HLQVWHLQV Pn Vf QPf DW PLGGD\ $YHUDJH GDLO\ WHPSHUDWXUH LQ WKH JUHHQKRXVH ZDV PDLQWDLQHG EHWZHHQ D UDQJH RI r& WR r& 9HJHWDWLYHO\ SURSDJDWHG VRXUFH SODQWV ZHUH JURZQ LQ WKH JUHHQKRXVH DV GHVFULEHG LQ FKDSWHU ,, *LEEHUHOOLF DFLG ZDV DSSOLHG WR D YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQW IROORZLQJ WKH SURFHGXUHV RXWOLQHG E\ .DUEDVVL HW DO f ZLWK WKH IROORZLQJ PRGLILFDWLRQV $ X0 *$ VROXWLRQ DGMXVWHG WR S+ ZLWK .2+ ZDV VSUD\HG WR WKH GULS SRLQW RQ DOO YHJHWDWLYH SDUWV RI WKH SODQW K EHIRUH WKH r& WHPSHUDWXUH WUHDWPHQW ZDV LPSRVHG 7HPSHUDWXUH WUHDWPHQWV ZHUH LQLWLDWHG DW WKH HQG RI WKH SKRWRSHULRG 2QH YHJHWDWLYHO\ SURSDJDWHG VRXUFH SODQW ZDV SODFHG LQ D GDUNHQHG r& 3HUFLYDO JURZWK FKDPEHU $ VHFRQG VRXUFH SODQW DQG WKH *$ WUHDWHG VRXUFH SODQW ZHUH SODFHG LQ DQ LGHQWLFDO JURZWK FKDPEHU DW r& 3ODQWV UHPDLQHG LQ WKH JURZWK FKDPEHUV IRU K XQWLO VDPSOLQJ

PAGE 65

7UHDWPHQW DSSOLFDWLRQ DQG VXEVHTXHQW DQDO\VLV RI VWDUFKn K\GURO\]LQJ HQ]\PHV ZHUH UHSOLFDWHG WLPHV XVLQJ GLIIHUHQW SODQWV :KROH /HDI ([WUDFW 3UHSDUDWLRQ 2QH JUDP IUHVK ZHLJKW RI WKH ILUVW IXOO\ H[WHQGHG OHDI ZDV REWDLQHG DW WKH HQG RI WKH WHPSHUDWXUH FRQWUROHG K GDUN SHULRG 7KH WLVVXH ZDV ZDVKHG ZLWK GHLRQL]HG ZDWHU DQG FXW LQWR WR PP VHJPHQWV ZLWK D VWUDLJKW HGJH UD]RU EODGH 6HJPHQWV ZHUH JURXQG IRU PLQ LQ D FKLOOHG PRUWDU FRQWDLQLQJ P/ RI LFH FROG H[WUDFWLRQ EXIIHU 7DEOH f PHUFDSWRHWKDQRO DQG SRO\YLQ\OSRO\S\UROLGRQH ZHUH DGGHG WR WKH H[WUDFWLRQ EXIIHU MXVW SULRU WR JULQGLQJ LQ D SRUFHODLQ PRUWDU &RRUV VL]H 1R KDYLQJ DQ XQJOD]HG JULQGLQJ VXUIDFH *URXQG JODVV ZDV DGGHG WR IDFLOLWDWH D ILQH JULQG 7KH VOXUU\ ZDV PDLQWDLQHG DW r& GXULQJ FHQWULIXJDWLRQ DW J IRU PLQ 7KH VXSHUQDWDQW ZDV PDLQWDLQHG DW r& DQG XVHG DV VDPSOHV IRU HOHFWURSKRUHVLV 3URWHLQ FRQWUROV ZHUH SUHSDUHG IRU HOHFWURSKRUHVLV E\ GLOXWLQJ DDP\ODVH IURP EDFLOOXV 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f DP\ODVH IURP VZHHW SRWDWR 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f DQG SXOODQDVH D GHEUDQFKLQJ HQ]\PH IURP (QWHUREDFWHU DHURJHQHV 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02f ZLWK H[WUDFWLRQ EXIIHU

PAGE 66

7DEOH 5HDJHQWV DQG JHO SUHSDUDWLRQ IRU VODE JHO HOHFWURSKRUHVLV QDWLYH VWDUFK 3$*( ([WUDFWLRQ EXIIHU 0 7ULV S+ 0 *O\FHURO b 0HUFDSWRHWKDQRO R nR SRO\YLQ\OSRO\S\UROLGRQH PJPJ IZ SODQW (OHFWURGH EXIIHU 7UL]PD EDVH P0 7ULV>K\GUR[\PHWK\O@DPLQRPHWKDQHf *O\FLQH P0 S+ 6HSDUDWLQJ JHO b JHO 0 7ULV G+ P/ 6ROXEOH VWDUFK PJ 0 7ULV S+ P/ $FU\ODPLGHELV b7 b&f P/ $PPRQLXP SHUVXOIDWH bf + / 7(0(' L / 111n1n7HWUDPHWK\OHWK\OHQHGLDPLQH 6WDFNLQJ JHO b JHO 0 7ULV G+ P/ 0 7ULV S+ P/ $FU\ODPLGHELV b7 b&f P/ $PPRQLXP SHUVXOIDWH bf ILK 7(0(' + / 0DUNHU EXIIHU 0 7ULV S+ 0 *O\FHURO b %0HUFDSWRHWKDQRO b %URPRSKHQRO EOXH b 7KH S+ RI SUHSDUDWLRQV ZHUH DGMXVWHG ZLWK +&O DW r&

PAGE 67

6ODE (OHFWURSKRUHVLV 5HVHUYRLU VWDFNLQJ DQG VHSDUDWLQJ JHO EXIIHUV IRU 6'6 3$*( JHOV DV GHVFULEHG E\ /DHPPOL f ZHUH PRGLILHG IRU QDWLYH 3$*( JHO SUHSDUDWLRQV 7DEOH f 1DWLYH SURWHLQV DUH VHSDUDWHG EDVHG RQ WKHLU FKDUJH PROHFXODU ZHLJKW DQG FRQILJXUDWLRQ 7KH VHSDUDWLQJ JHO ZDV DPHQGHG E\ VXEVWLWXWLQJ WKH ZDWHU SKDVH ZLWK D VROXEOH VWDUFK VROXWLRQ $ 6( YHUWLFDO VODE JHO HOHFWURSKRUHVLV XQLW +RHIIHU 6FLHQWLILF ,QVWUXPHQWV 6DQ )UDQFLVFR &$f ZDV RSHUDWHG ZLWK D GLVFRQWLQXRXV EXIIHU V\VWHP XVLQJ WZR PP ORQJ PP ZLGH DQG PP WKLFN JHOV 7KH b DFU\ODPLGH VHSDUDWLQJ JHO 7DEOH f ZDV SUHSDUHG E\ ERLOLQJ PJ RI VROXEOH VWDUFK LQ P/ RI G+ IRU WZR PLQ DQG DOORZHG WR FRRO 7KHQ P/ RI 0 75,6 S+ f DQG P/ RI DFU\ODPLGHELV b7 b&f ZHUH DGGHG 7KH VROXWLRQ ZDV GHJDVVHG IRU PLQ EHIRUH DGGLQJ M/ RI DPPRQLXP SHUVXOIDWH bf DQG c-/ RI 7(0(' 7ZHQW\ WZR DQG D KDOI PLOOLOLWHUV RI WKH DFU\ODPLGH VROXWLRQ ZHUH LPPHGLDWHO\ WUDQVIHUUHG WR EHWZHHQ WKH JODVV VDQGZLFK ZLWK D SLSHWWH DQG RYHUOD\HG ZLWK D WKLQ OD\HU RI EXWDQRO $IWHU PLQ D VKDUS JHOEXWDQRO LQWHUSKDVH ZDV YLVLEOH 7KH EXWDQRO ZDV SRXUHG RXW RI WKH JODVV VDQGZLFK DQG WKH UHPDLQLQJ EXWDQRO ZDV ULQVHG ZLWK G+ EHIRUH GU\LQJ WKH JODVV 7KH VWDFNLQJ JHO VROXWLRQ 7DEOH f ZDV SUHSDUHG E\ DGGLQJ P/ G+ DQG P/ RI 0 75,6 S+ f WR

PAGE 68

P/ RI DFU\ODPLGHELV 7KH VROXWLRQ ZDV GHJDVVHG IRU PLQ EHIRUH DGGLQJ / RI DPPRQLXP SHUVXOIDWH bf DQG / 7(0(' 7KH VWDFNLQJ JHO VROXWLRQ ZDV WKHQ SLSHWWHG LQWR WKH UHPDLQLQJ VSDFH EHWZHHQ WKH JODVV VDQGZLFK XQWLO PP IURP WKH WRS RI WKH JODVV 7KH FRPE ZDV LQVHUWHG LQWR WKH VWDFNLQJ JHO VROXWLRQ DQG UHPRYHG DIWHU SRO\PHUL]DWLRQ :HOOV IRUPHG E\ WKH FRPE ZHUH HDFK ORDGHG ZLWK [/ RI DQ DSSURSULDWHO\ GLOXWH HQ]\PH SUHSDUDWLRQ )RUW\ PLFUROLWHUV [/ DQG [/ RI HQ]\PH SUHSDUDWLRQ ZHUH DGMXVWHG WR [/ ZLWK WKH PDUNHU EXIIHU 7DEOH f EHIRUH ORDGLQJ LQWR VHSDUDWH ZHOOV $IWHU ORDGLQJ WKH HOHFWURSKRUHVLV ZDV FRQGXFWHG DW YROWV IRU KRXUV DW r& (DFK VDPSOH ZDV UHSOLFDWHG WZLFH RQ HDFK JHO VR WKDW DIWHU HOHFWURSKRUHVLV KDOI WKH JHO FRXOG EH XVHG IRU SURWHLQ VWDLQLQJ DQG KDOI IRU HQ]\PH DFWLYLW\ VWDLQLQJ 3URWHLQ DQG (Q]\PH $FWLYLW\ 6WDLQLQJ 7KH TXDQWLW\ RI SURWHLQ ORDGHG LQWR HDFK ZHOO IRU HOHFWURSKRUHVLV ZDV GHWHUPLQHG VSHFWURSKRWRPHWULFDOO\ XVLQJ %LR5DGnV SURWHLQ DVVD\ %LR5DG 5LFKPRQG &$f $ SURWHLQ VWDQGDUG FXUYH ZDV FRQVWUXFWHG UDQJLQJ IURP [J SURWHLQ XVLQJ ERYLQH VHUXP DOEXPLQ 8QLWHG 6WDWHV %LRFKHPLFDO &RUSRUDWLRQ &OHYHODQG 2+f $EVRUEDQFH DW QP ZDV PHDVXUHG ZLWK D +HZOHWW 3DFNDUG $ GLRGH DUUD\ VSHFWURSKRWRPHWHU 8QLWHG 6WDWHVf 7ZHQW\ PLFUROLWHUV 0/ DQG MX/ RI HQ]\PH SUHSDUDWLRQ ZHUH DGMXVWHG WR [/

PAGE 69

ZLWK H[WUDFWLRQ EXIIHU DQG VXEMHFWHG WR WKH VWDQGDUG DVVD\ SURFHGXUH 7KH TXDQWLW\ RI SURWHLQ LQ WKH H[WUDFWV ZDV GHWHUPLQHG E\ FRPSDULVRQ WR WKH SURWHLQ VWDQGDUG FXUYH *HOV ZHUH VWDLQHG IRU SURWHLQ XVLQJ WKH PHWKRG RI 6FKXOHU DQG =LHOLQVNL f 7KH JHO ZDV SODFHG LQ P/ RI D IL[DWLYH FRQWDLQLQJ b PHWKDQRO b JODFLDO DFHWLF DFLG DQG b FRRPDVVLH EULOOLDQW EOXH IRU PLQ DQG JHQWO\ VKDNHQ 7KH JHO ZDV WKHQ GHVWDLQHG LQ P/ RI b PHWKDQRO DQG b JODFLDO DFHWLF DFLG 7KH GHVWDLQLQJ VROXWLRQ ZDV FKDQJHG WZLFH RYHU D WKUHH KRXU SHULRG $IWHU FOHDULQJ WKH EDFNJURXQG VWDLQ WKH JHOV ZHUH SKRWRJUDSKHG DV TXLFNO\ DV SRVVLEOH DQG GULHG RQ D VODE JHO GU\HU +RHIIHU 6FLHQWLILF ,QVWUXPHQWV 6( f 6WDLQLQJ RI WKH JHO IRU VWDUFKGHJUDGLQJ HQ]\PHV IROORZHG WKH SURFHGXUH RXWOLQHG E\ 9DOOHMRV f 7KH JHO ZDV LQFXEDWHG LQ D VROXWLRQ FRQVLVWLQJ RI P0 VRGLXP DFHWDWH DQG 0 FDOFLXP FKORULGH DGMXVWHG WR D S+ RI ZLWK +& DW r& IRU PLQ $IWHU LQFXEDWLRQ WKH JHO ZDV ULQVHG WKRURXJKO\ ZLWK G+ DQG VWDLQHG LQ P0 LRGLQH DQG P0 SRWDVVLXP LRGLGH :KHQ WKH ]\PRJUDP GHYHORSHG D GDUN SXUSOH EDFNJURXQG LW ZDV ULQVHG LQ G+ DQG SKRWRJUDSKHG TXLFNO\ EHIRUH EHLQJ GULHG RQ D VODE JHO GU\HU +RHIIHU 6FLHQWLILF ,QVWUXPHQWV 6( f

PAGE 70

5HVXOWV DQG 'LVFXVVLRQ 1DWLYH VWDUFK 3$*( JHOV VWDLQHG GLIIHUHQWLDOO\ DFFRUGLQJ WR WKH W\SH RI K\GURO\WLF HQ]\PH GDWD QRW VKRZQf D$P\ODVH DFWLYLW\ UHVXOWHG LQ D FOHDU EDQG DP\ODVH DFWLYLW\ UHVXOWHG LQ D UHG EDQG DQG GHEUDQFKLQJ DFWLYLW\ SURGXFHG D EOXH EDQG 7KHVH UHVXOWV VXSSRUW SUHYLRXV UHSRUWV =LHJOHU DQG %HFN f DQG .DNHIXGD DQG 'XNH f UHSRUWHG WKH LGHQWLILFDWLRQ RI WKH W\SH RI K\GURO\WLF HQ]\PH DFWLYLW\ EDVHG RQ WKH FRORU RI EDQGV DIWHU LRGLQH VWDLQLQJ RI VWDUFK LPSUHJQDWHG QDWLYH JHOV )ROORZLQJ HOHFWURSKRUHVLV LQFXEDWLRQ DQG VWDLQLQJ RI JHOV FRQWDLQLQJ SDQJOD OHDI H[WUDFWV GLVWLQFW EDQGLQJ FRORUV DQG EDQGLQJ SDWWHUQV ZHUH HYLGHQW ,QWHUSUHWDWLRQ RI WKH W\SH RI HQ]\PH DFWLYLWLHV ZHUH EDVHG RQ WKH EDQG FRORU DV UHODWHG WR WKH NQRZQ DDP\ODVH DP\ODVH DQG SXOODQDVH VDPSOHV DQG =LHJOHU DQG %HFN f DQG .DNHIXGD DQG 'XNH f UHSRUWV )RU UHIHUHQFH SXUSRVHV ]\PRJUDPV ZLOO EH LGHQWLILHG E\ SODQW WUHDWPHQW ZLWK UHJDUGV WR QLJKW WHPSHUDWXUH DQG *$ DSSOLFDWLRQ DV IROORZV r&FRQWUROf r& r&*$ 5HG EDQGV UHVXOWLQJ IURP H[RDP\ORO\WLF HQ]\PH DFWLYLW\ ZHUH QRW GHWHFWHG UHJDUGOHVV RI WHPSHUDWXUH RU *$ WUHDWPHQW )LJXUH f (QRDP\ORO\WLF HQ]\PH DFWLYLW\ ZDV HYLGHQW LQ WKH r& WUHDWPHQW E\ WKH DSSHDUDQFH RI HLJKW FOHDU EDQGV 7KH EDQGV VHSDUDWHG LQWR D IDVW PLJUDWLQJ JURXS EDQGV f D VORZHU PLJUDWLQJ SDLU EDQGV DQG f DQG D VLQJOH VORZO\

PAGE 71

PLJUDWLQJ EDQG EDQG f )LJXUH f &DUWHU HW HO f SUHYLRXVO\ IRXQG RQH IDVW PLJUDWLQJ EDQG DQG D VORZHU PLJUDWLQJ SDLU RI EDQGV LQ SDQJOD %\ DOWHULQJ WKH HOHFWURSKRUHWLF FRQGLWLRQV WKH\ IXUWKHU VHSDUDWHG HQGRDP\ORO\WLF HQ]\PHV LQWR D IDVW PLJUDWLQJ SDLU RI EDQGV DQG D VORZHU PLJUDWLQJ JURXS RI ILYH EDQGV &DUWHU HW DO f 7KH UHVXOWV IURP WKLV VWXG\ GLIIHU IURP WKH SUHYLRXVO\ FLWHG OLWHUDWXUH LQ WKDW DQ DGGLWLRQDO HQGRDP\ORO\WLF HQ]\PH ZDV UHVROYHG DQG EDQGV RQ WKH JHOV GLG QRW IRUP VLPLODU PLJUDWRU\ SDWWHUQV 7UHDWPHQW DW r& UHGXFHG WKH QXPEHU RI YLVDEOH HQGRDP\ORO\WLF EDQGV %DQGV DQG ZHUH QRW YLVDEOH ZKHQ FRPSDUHG WR WKH r& WUHDWPHQW )LJXUH f %DVHG RQ GHQVLWRPHWHU WUDFLQJV &DUWHU HW DO f DQG &DUWHU HW DO f UHSRUWHG D UHGXFWLRQ LQ WKH HQ]\PH DFWLYLW\ ZLWKLQ WKH EDQGV DQG QRW D UHGXFWLRQ LQ WKH QXPEHU RI EDQGV $SSUR[LPDWHO\ GRXEOLQJ WKH WRWDO SURWHLQ ORDG LQ WKH r& WUHDWPHQW UHODWLYH WR WKH r& WUHDWPHQW ILT WR >LT UHVSHFWLYHO\ GLG QRW UHVXOW LQ WKH DSSHDUDQFH RI EDQG RU 7KLV LQGLFDWHV WKH SUHVHQFH RI WZR FKLOOVHQVLWLYH HQGRDP\ORO\WLF LVR]\PHV 'DWD SUHVHQWHG LQ )LJXUH VXSSRUWHG WKH K\SRWKHVLV WKDW FKLOOLQJ WHPSHUDWXUH UHGXFHG WKH QXPEHU RI VWDUFKn GHJUDGLQJ HQ]\PHV VSHFLILFDOO\ HQGRDP\ORO\WLF LVR]\PHV 7KH UHPDLQLQJ HQGRDP\ORO\WLF LVR]\PHV PD\ QRW EH DEOH WR VDWLVIDFWRULO\ K\GURO\]H VWDUFK SDUWLFXODUO\ LI WKH

PAGE 72

7UHDWPHQW r& r& r&*$ 3URWHLQ 0J )LJ (IIHFW RI WHPSHUDWXUH DQG *$ RQ H[SUHVVLRQ RI VWDUFKGHJUDGLQJK\GURO\WLF HQ]\PHV LQ SDQJOD $UHDV RI HQGRDP\ODVH DFWLYLW\ VKRZ XS DV ZKLWH EDQGV f WKDW RI GHEUDQFKLQJ HQ]\PH DFWLYLW\ EOXH EDQG f DJDLQVW WKH GDUN EDFNJURXQG RI WKH DP\ORVHDP\ORSHFWLQ FRQWDLQLQJ JHO ) UHIHUV WR WKH EURPRSKHQRO EOXH IURQW PDUNHU 0LFURJUDPV SURWHLQ UHIHUV WR WKH WRWDO SURWHLQ

PAGE 73

UHPDLQLQJ HQGRDP\ODVHV KDYH D ORZHU DFWLYLW\ DV UHSRUWHG E\ &DUWHU HW DO f DQG &DUWHU HW DO f 7KH FRQVHTXHQFH RI LV D VWDUFK EXLOGXS &RRPDVVLH VWDLQ IRU WRWDO SURWHLQ GLG QRW UHYHDO D VSHFLILF SURWHLQ EDQG DW HQGRDP\ODVH HQ]\PH SRVLWLRQ RU UHVSHFWLYH RI SURWHLQ ORDG RU WUHDWPHQW )LJXUH f 7KH PHFKDQLVP RI VHQVLWLYLW\ UHPDLQV XQFOHDU LH FRQWLQXHG SURWHLQ V\QWKHVLV ZLWK UHGXFHG HQ]\PH DFWLYLW\ RU WHUPLQDWLRQ RI SURWHLQ V\QWKHVLV 7KH QXPEHU RI K\GURO\WLF HQ]\PH EDQGV LQ WKH r& WUHDWPHQW ]\PRJUDP ZHUH QRW DOWHUHG DV D UHVXOW RI WUHDWPHQW ZLWK *$ )LJXUH f $SSUR[LPDWHO\ GRXEOLQJ WKH WRWDO SURWHLQ ORDG LQ WKH r&*$ WUHDWPHQW UHODWLYH WR WKH r& WUHDWPHQW GLG QRW LQFUHDVH WKH QXPEHU RI GHWHFWDEOH EDQGV )LJXUH f &DUWHU HW DO f DQG .DUDEDVVL HW DO f UHSRUWHG WKDW UHODWLYH DFWLYLWLHV RI DP\ORO\WLF HQ]\PHV DQG QRW WKH QXPEHU RI LVR]\PHV ZHUH LQFUHDVHG E\ DSSO\LQJ *$ ,Q ERWK VWXGLHV 0 *$ ZDV DSSOLHG WR SDQJRODJUDVV KRXUV SULRU WR ORZWHPSHUDWXUH WUHDWPHQW 7KH GDWD SUHVHQWHG LQ )LJXUH GLG QRW VXSSRUW WKH K\SRWKHVLV WKDW *$ QXOOLILHV WKH HIIHFW RI ORZ WHPSHUDWXUH RQ WKH UHGXFWLRQ LQ QXPEHU RI VWDUFKGHJUDGLQJ HQ]\PHV $ VORZ PRYLQJ EOXH EDQG EDQG f LQGLFDWLYH RI GHEUDQFKLQJ HQ]\PH DFWLYLW\ ZDV HYLGHQW LQ WKH r& WUHDWPHQW .DNHIXGD HW DO f LGHQWLILHG D GHEUDQFKLQJ

PAGE 74

3URWHLQ 0J )LJ (IIHFW RI WHPSHUDWXUH DQG *$ RQ SURGXFWLRQ DQG H[SUHVVLRQ RI VWDUFKGHJUDGLQJK\GURO\WLF HQ]\PHV LQ SDQJOD =\PRJUDPV ]f RI K\GURO\WLF DFWLYLW\ DQG FRRPDVVLH SURWHLQ VWDLQV Ff DUH SUHVHQWHG IRU HDFK WUHDWPHQW 0LFURJUDPV SURWHLQ UHIHUV WR WKH WRWDO SURWHLQ

PAGE 75

HQ]\PH LQ FUXGH SHD OHDI H[WUDFW XVLQJ HOHFWURSKRUHVLV DV GHVFULEHG E\ .DNHIXGD DQG 'XNH f 7KH VORZ PRYLQJ EOXH EDQG UHPDLQHG XQFKDQJHG UHJDUGOHVV RI WHPSHUDWXUH RU *$ WUHDWPHQW )LJXUH f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

PAGE 76

&+$37(5 ,9 (9$/8$7,21 2) +<'52/<7,& (1=<0(6 ,1 )5((=,1*7(03(5$785( 72/(5$17 620$&/21(6 2) 3$1*2/$ 7KLV VWXG\ WHVWHG WKH WHQWDWLYH DVVXPSWLRQ WKDW FKLOO WROHUDQWHQGRDP\ORO\WLF LVR]\PHV PD\ EH SUHVHQW LQ ORZ WHPSHUDWXUHWROHUDQW VRPDFORQHV RI SDQJRODJUDVV 7KH REMHFWLYHV RI WKLV VWXG\ ZHUH Df WR HYDOXDWH ORZ WHPSHUDWXUHWROHUDQWSDQJROD VRPDFORQHV IRU YDULDWLRQ LQ WKH H[SUHVVLRQ RI K\GURO\WLF HQ]\PHV DQG Ef WR VHOHFW SODQWV WKDW UHWDLQ K\GURO\WLF HQ]\PH DFWLYLW\ ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ DIWHU H[SRVXUH WR ORZ WHPSHUDWXUHV 7KH UDSLGLW\ RI VFUHHQLQJ DQG VLPSOH LQKHULWDQFH PDNH LVR]\PHV D SRZHUIXO WRRO LQ WKH DQDO\VLV RI YDULDWLRQ 9DULDWLRQ LQ LVR]\PH SDWWHUQV RI VWDUFKK\GURO\]LQJ HQ]\PHV KDV EHHQ VWXGLHG H[WHQVLYHO\ GXULQJ WKH JHUPLQDWLRQ SURFHVV LQ VHHGV %HZOH\ DQG %ODFN -DFREVHQ DQG %HDFK $W]RUQ DQG :HLOHU 1RODQ DQG +R f 6WDUFKK\GURO\]LQJ LVR]\PH SDWWHUQV KDYH EHHQ XVHG WR VKRZ LQGXFHG YDULDWLRQ ZLWKLQ WKH VDPH VHHG W\SH +HQNH f XVHG D KDOIVHHG DDP\ODVH DVVD\ FDSDEOH RI VFUHHQLQJ WKRXVDQGV RI KDOIVHHGV LQ D VKRUW WLPH $IWHU VFUHHQLQJ PXWDJHQL]HG EDUOH\ VHHGV IRU WKH DELOLW\ WR H[SUHVV DDP\ODVH LQ WKH DEVHQFH RI *$ RU LQ WKH SUHVHQFH RI *$

PAGE 77

SOXV DQ LQKLELWRU KH IRXQG b RI WKH VHHGV WR EH YDULDQW (PEU\RV RI WKH YDULDQW KDOIVHHGV ZHUH JURZQ WR PDWXULW\ DQG VHOIHG 7KH UHVXOWLQJ VHHG SURYLGHG HYLGHQFH IRU LQKHULWDQFH RI D PXWDQW WUDLW +R HW DOf XVHG D FRPSDUDEOH DSSURDFK DQG REWDLQHG VLPLODU UHVXOWV )HZ VWXGLHV XVHG VWDUFKK\GURO\]LQJ HQ]\PHV IRU WKH LGHQWLILFDWLRQ RI VRPDWLF YDULDQWV $ VRPDFORQDO YDULDQW ZKLFK HIIHFWV WKH LVR]\PH SDWWHUQ RI RQH RI WKH VHHG SURWHLQ FRPSOH[HV RI ZKHDW QDPHO\ DDP\ODVH ZDV UHSRUWHG E\ /DUNLQ HW DO f 6HHGV IURP VRPDFORQHV ZHUH DVVD\HG WR LGHQWLI\ HLJKW VRPDWLF YDULDQWV 2I WKH YDULDQW SODQWV IRXU ZHUH *$LQVHQVLWLYH RQH ZDV *$VXSHUVHQVLWLYH DQG IRXU ZHUH $%$LQVHQVLWLYH 7KH FKDUDFWHULVWLFV ZHUH KHULWDEOH WKURXJK WZR VHHG JHQHUDWLRQV 5\DQ DQG 6FRZFURIW f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

PAGE 78

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r& 3HUFLYDO JURZWK FKDPEHU $Q LGHQWLFDO FRPELQDWLRQ RI SODQWV ZDV SODFHG LQ D GDUNHQHG r& 3HUFLYDO JURZWK FKDPEHU 3ODQWV UHPDLQHG LQ WKH JURZWK FKDPEHUV IRU KRXUV XQWLO VDPSOLQJ

PAGE 79

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n GHJUDGLQJ HQ]\PH DFWLYLW\ ZDV IROORZHG DV GHVFULEHG LQ &KDSWHU ,,, 5HVXOWV DQG 'LVFXVVLRQ =\PRJUDPV RI WKH UHJHQHUDWHG SODQWV IURP &KDSWHU ,, FRQWDLQHG WKH FKDUDFWHULVWLF K\GURO\WLF EDQGLQJ SDWWHUQ IRXQG LQ WKH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV )LJXUH VKRZV WKH FRPSDULVRQ RI D W\SLFDO ]\PRJUDP IURP WKH UHJHQHUDWHG SODQWV WR WKDW RI D YHJHWDWLYHO\ SURSDJDWHG SODQW 1R YDULDWLRQ LQ WKH LVR]\PH EDQGLQJ SDWWHUQ ZDV

PAGE 80

7UHDWPHQW )LJ r& r& =\PRJUDPV RI VWDUFKK\GURO\]LQJ HQ]\PHV LVRODWHG IURP SDQJOD SODQWV WKDW ZHUH H[SRVHG WR r& RU r& :KLWH EDQGV EDQGV f VKRZ HQGRDP\ODVH DFWLYLW\ EOXH EDQG EDQG f LQGLFDWHV GHEUDQFKLQJ HQ]\PH DFWLYLW\ /DQHV DQG UHSUHVHQW WKH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV /DQHV DQG UHSUHVHQW SODQWV UHJHQHUDWHG IURP WLVVXH FXOWXUH ) UHIHUV WR WKH EURPRSKHQRO EOXH IURQW PDUNHU

PAGE 81

REVHUYHG EHWZHHQ WKH UHJHQHUDWHG SODQWV RU EHWZHHQ UHJHQHUDWHG SODQWV DQG YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV UHVSHFWLYH RI WHPSHUDWXUH WUHDWPHQW 1LQH K\GURO\WLF EDQGV ZHUH HYLGHQW LQ ]\PRJUDPV IURP WKH r& WUHDWPHQW )LJXUH f ([RDP\ORO\WLF DFWLYLW\ ZDV QRW GHWHFWHG (QGRDP\ORO\WLF DFWLYLW\ UHVXOWHG LQ HLJKW FOHDU EDQGV ZKLFK VHSDUDWHG LQWR D IDVW PLJUDWLQJ JURXS EDQGV f D VORZHU PLJUDWLQJ SDLU EDQG DQG f DQG D VLQJOH VORZO\ PLJUDWLQJ EDQG EDQG f 'HEUDQFKLQJ HQ]\PH DFWLYLW\ UHVXOWHG LQ D VLQJOH H[WUHPHO\ VORZ PLJUDWLQJ EOXH EDQG 7KH r& WUHDWPHQW UHVXOWHG LQ D EDQGLQJ SDWWHUQ LGHQWLFDO WR WKH r& WUHDWPHQW H[FHSW IRU WKH HOLPLQDWLRQ RI EDQGV VL[ DQG HLJKW )LJXUH f 'DWD GR QRW VXSSRUW WKH K\SRWKHVLV WKDW VRPDWLF YDULDWLRQ LQ ORZWHPSHUDWXUH WROHUDQW VRPDFORQHV FDQ EH XVHG WR REWDLQ FKLOOWROHUDQW HQGRDP\ORO\WLF LVR]\PHV $PSOLI\LQJ WKH QXPEHU RI WHVW SODQWV ZRXOG LQFUHDVH WKH SUREDELOLW\ RI GHWHFWLQJ D GHVLUHG YDULDWLRQ &RRPDVVLH VWDLQLQJ RI WKH SURWHLQ GLG QRW VKRZ VSHFLILF EDQGLQJ IRU WKH HQGRDP\ORO\WLF SURWHLQ DW EDQG QXPEHU VL[ RU HLJKW IRU UHJHQHUDWHG SODQWV RU YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV LQ HLWKHU WHPSHUDWXUH WUHDWPHQW )LJXUH f (YLGHQWO\ FRRPDVVLH VWDLQ ZDV QRW VHQVLWLYH HQRXJK WR GHWHFW WKH JXDQWLW\ RI SURWHLQ DW EDQG VL[ RU HLJKW SRVLWLRQ 6LOYHU VWDLQ ZKLFK GHWHFWV QJ RI SURWHLQ DQG LV

PAGE 82

7UHDWPHQW r& r& )LJ =\PRJUDPV RI VWDUFKK\GURO\]LQJ HQ]\PHV DQG WRWDO SURWHLQ SURILOHV IURP SDQJOD SODQWV H[SRVHG WR r& RU r& /DQHV DQG DUH ]\PRJUDPV :KLWH EDQGV EDQGV f VKRZ HQGRDP\ODVH DFWLYLW\ EOXH EDQG EDQG f LQGLFDWHV GHEUDQFKLQJ HQ]\PH DFWLYLW\ &RRPDVVLH EOXH VWDLQHG SURWHLQV DUH SUHVHQWHG LQ ODQH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWVf DQG ODQHV DQG SODQWV UHJHQHUDWHG IURP WLVVXH FXOWXUHf

PAGE 83

DSSUR[LPDWHO\ WLPHV PRUH VHQVLWLYH WKDQ FRRPDVVLH VWDLQ PD\ GHWHFW SURWHLQ DW EDQG VL[ RU HLJKW SRVLWLRQ &RQFOXVLRQV $OO RI WKH UHJHQHUDWHG SODQWV WKDW ZHUH VHOHFWHG LQ FKDSWHU ,, IRU IUHH]LQJWHPSHUDWXUH WROHUDQFH ZHUH VLPLODU LQ WKDW WKH\ VKRZHG QR VRPDFORQDO YDULDWLRQ LQ VWDUFKn K\GURO\]LQJ HQ]\PHV RU WRWDO SURWHLQ EDQGLQJ SDWWHUQV 7KH FKDUDFWHULVWLF EDQGLQJ SDWWHUQV RI K\GURO\WLF HQ]\PHV LQ WKH r& WUHDWPHQW HLJKW HQGRDP\ORO\WLF DQG RQH GHEUDQFKLQJ HQ]\PHf DQG WKH r& WUHDWPHQW VL[ HQGRDP\ORO\WLF DQG RQH GHEUDQFKLQJ HQ]\PHf ZHUH FRQVLVWHQW EHWZHHQ UHJHQHUDWHG SODQWV DQG EHWZHHQ UHJHQHUDWHG SODQWV DQG YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV 7KH UHJHQHUDWHG SODQWV DQG WKH YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQW KDG WZR VLPLODU WHPSHUDWXUHVHQVLWLYHHQGRDP\ORO\WLF HQ]\PHV

PAGE 84

&+$37(5 9 6800$5< $1' &21&/86,216 $ ODUJH QXPEHU RI IDFWRUV FRQWULEXWH WR WKH UHGXFWLRQ RI JURZWK RI SDQJOD XQGHU IUHH]LQJ DQG FKLOOLQJWHPSHUDWXUH FRQGLWLRQV 6HYHUDO RI WKH PDMRU IDFWRUV DUH ORVV RI SODVPDOHPPD LQWHJULW\ UHGXFWLRQ LQ SKRWRV\QWKHWLF HOHFWURQ WUDQVSRUW DQG GHFUHDVHG DFWLYLW\ RI DVVLPLODWHG VWDUFKn K\GURO\]LQJ HQ]\PHV ,UUHVSHFWLYH RI EHLQJ GLVWLQFW IDFWRUV WKH DFWLRQ DQG LQWHUDFWLRQ RI DOO WKHVH IDFWRUV FRQWULEXWH WR D FRPPRQ UHVXOW UHGXFHG JURZWK UHVXOWLQJ IURP IUHH]LQJ RU FKLOOLQJWHPSHUDWXUH H[SRVXUH 6HYHUDO H[SHULPHQWV ZHUH FRQGXFWHG WR LQGXFH SKHQRW\SLF YDULDWLRQ DQG VHOHFW YDULDQW SODQWV IRU D UHGXFWLRQ LQ ORZ WHPSHUDWXUH GDPDJH 7KH VWXGLHV UHSRUWHG LQ &KDSWHU ,, ZHUH FRQGXFWHG ZLWK WKH REMHFWLYHV Df WR JHQHUDWH FDOOXV OLQHV RI SDQJOD Ef WR VHOHFW IUHH]LQJ RU FKLOOLQJWHPSHUDWXUH UHVLVWDQW FHOO OLQHV LQ YLWUR Ff WR UHJHQHUDWH ZKROH SODQWV IURP VHOHFWHG FXOWXUHV DQG Gf WR WHVW UHJHQHUDWHG SODQWV IRU LQFUHDVHG WROHUDQFH WR IUHH]LQJ DQG FKLOOLQJ WHPSHUDWXUH DW WKH RUJDQHOOH DQG FHOOXODU OHYHO 6RPDWLF HPEU\RJHQHVLV LQ SDQJOD IROORZHG D SDWWHUQ RI GHYHORSPHQW VLPLODU WR WKRVH GHVFULEHG IRU RWKHU JUDVVHV ,QFUHDVLQJ WKH LQ YLWUR

PAGE 85

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f WR HYDOXDWH WKH LQIOXHQFH RI FKLOOLQJ WHPSHUDWXUH RQ WKH H[SUHVVLRQ RI VWDUFKK\GURO\]LQJ HQ]\PHV Ef WR LGHQWLI\ K\GURO\WLF HQ]\PHV ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ DIWHU FKLOOLQJWHPSHUDWXUH H[SRVXUH DQG Ff WR HYDOXDWH WKH HIIHFW RI *$M RQ WKH H[SUHVVLRQ RI VWDUFKK\GURO\]LQJ HQ]\PHV GXULQJ FKLOOLQJWHPSHUDWXUH FRQGLWLRQV &XUUHQW VWXGLHV LQGLFDWH WKDW DQDEROLVP RI DVVLPLODWHG VWDUFK LQ FKORURSODVWV LV DFFRPSOLVKHG E\ WKH FRRSHUDWLRQ RI K\GURO\WLF DQG SKRVSKRURO\WLF HQ]\PHV 7KH FHOOXODU ORFDWLRQ DQG SK\VLRORJLFDO UROH RI WKHVH HQ]\PHV UHPDLQ XQFOHDU DQG QHHG WR EH LGHQWLILHG IRU WKH W\SH RI SODQW LQ TXHVWLRQ

PAGE 86

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f WR HYDOXDWH WKH FKLOOWHPSHUDWXUHWROHUDQW SDQJOD VRPDFORQHV IRU YDULDWLRQ LQ WKH H[SUHVVLRQ RI K\GURO\WLF HQ]\PHV DQG Ef WR VHOHFW SODQWV WKDW UHWDLQ K\GURO\WLF HQ]\PH DFWLYLW\ ZKLFK PD\ DVVLVW LQ VWDUFK EUHDNGRZQ DIWHU H[SRVXUH WR FKLOOLQJ WHPSHUDWXUH $OO RI WKH UHJHQHUDWHG SODQWV WKDW ZHUH VHOHFWHG IRU )UHH]LQJ WHPSHUDWXUH WROHUDQFH LQ &KDSWHU ,, VKRZHG QR VRPDFORQDO YDULDWLRQ ZLWK UHVSHFW WR WKH QXPEHU RI VWDUFKK\GURO\]LQJ HQ]\PHV RU WRWDO SURWHLQ EDQGLQJ SDWWHUQV 7KH FKDUDFWHULVWLF EDQGLQJ SDWWHUQV RI K\GURO\WLF HQ]\PHV LQ WKH r WUHDWPHQW HLJKW HQGRDP\ORO\WLF HQ]\PHV DQG RQH GHEUDQFKLQJ HQ]\PHf DQG WKH r& WUHDWPHQW VL[ HQGRDP\ORO\WLF HQ]\PHV DQG RQH GHEUDQFKLQJ HQ]\PHf ZHUH FRQVLVWHQW EHWZHHQ UHJHQHUDWHG SODQWV DQG EHWZHHQ UHJHQHUDWHG SODQWV DQG YHJHWDWLYHO\ SURSDJDWHG FRQWURO SODQWV 7KH UHJHQHUDWHG SODQWV DQG WKH YHJHWDWLYHO\

PAGE 87

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f VKRXOG EH SURGXFHG DQG VFUHHQHG IRU IUHH]LQJ DQG FKLOOLQJWHPSHUDWXUH WROHUDQFH 7KLV PD\ OHDG WR WKH LGHQWLILFDWLRQ RI LQGLYLGXDOV ZLWK LQFUHDVHG WROHUDQFH LQ DOO WKUHH VHOHFWLRQ IDFWRUV

PAGE 88

5()(5(1&(6 $KORRZDOLD % 6 3ODQW UHJHQHUDWLRQ IURP FDOOXV FXOWXUH LQ ZKHDW &URS 6FL $KORRZDOLD % 6 6SHFWUXP RI YDULDWLRQ LQ VRPDFORQHV RI WULSORLG U\HJUDVV &URS 6FL $KORRZDOLD % 6 /LPLWDWLRQ WR WKH XVH RI VRPDFORQDO YDULDWLRQ LQ FURS LPSURYHPHQW S ,Q 6HPDO HGf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f 5HJXODWLRQ RI FDUERQ SDUWLWLRQLQJ LQ SKRWRV\QWKHWLF WLVVXH $PHULFDQ 6RF 3ODQW 3K\VLRORJLVWV 5RFNYLOOH 0' %HFN ( 3 3RQJUDW] DQG 5HXWHU 7KH DP\ORO\WLF V\VWHP RI LVRODWHG FKORURSODVWV DQG LWV UROH LQ WKH EUHDNGRZQ RI DVVLPLODWRU\ VWDUFK S ,BQ $NR\XQRJORX HGf 3KRWRV\QWKHVLV 3URF WK ,QW &RQJ 3KRWRV\QWK %DODEDQ ,QW 6FLHQFH 6HUYLFH 3KLODGHOSKLD

PAGE 89

%HHUV ( 3 DQG 6 + 'XNH /RFDWLRQ RI DDP\ODVH LQ WKH DSRSODVW RI SHD 3LVXP VDWLYXPf VWHPV 3ODQW 3K\VLRO %HQ]LRQ 5 / 3KLOOLSV DQG + : 5LHV &DVH KLVWRULHV RI JHQHWLF YDULDELOLW\ LQ YLWUR RDWV DQG PDL]H S ,Q ,. 9DVLO HGf &HOO FXOWXUH DQG VRPDWLF FHOO JHQHWLFV RI SODQWV $FDGHPLF 3UHVV 1HZ
PAGE 90

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f 3ODQW WLVVXH FXOWXUH 3URF WK ,QWHUQDWLRQDO &RQJUHVV RI 3ODQW 7LVVXH DQG &HOO &XOWXUH /DNH
PAGE 91

'L[ 3 DQG + ( 6WUHHW 6HOHFWLRQ RI SODQW FHOO OLQHV ZLWK HQKDQFHG FKLOOLQJ UHVLVWDQFH $QQ %RW 'UDNH % DQG ) % 6DOLVEXU\ $IWHUHIIHFWV RI ORZ DQG KLJK WHPSHUDWXUH SUHWUHDWPHQW RQ OHDI UHVLVWDQFH LQ ;DQWKLXP 3ODQW 3K\VLRO 'XGHFN $ ( DQG & + 3HDFRFN n7LIGZDUIn EHUPXGDJUDVV JURZWK UHVSRQVH WR FDUER[LQ DQG *$ GXULQJ VXERSWLPXP WHPSHUDWXUHV +RUW6FLHQFH (FKHYHUULD ( DQG & %R\HU /RFDOL]DWLRQ RI VWDUFK ELRV\QWKHWLF DQG GHJUDGDWLYH HQ]\PHV LQ PDL]H OHDYHV $PHU %RW (OPRUH $ & )UDQNOLQ DQG 5 $ *RQ]DOHV (FRQRPLFDOO\ LPSRUWDQW VRPDFORQDO YDULDQWV RI D IRUDJH JUDVV 'LJLWDULD VXQJXQDOLV /f ,Q YLWUR FHOOXODU DQG GHYHORSPHQWDO ELRORJ\ $ )RURXJKL:HKU % : )ULHGW 5 6FKXFKPDQQ ) .RKOHU DQG :HQ]HO ,Q YLWUR VHOHFWLRQ IRU UHVLVWDQFH S ,Q 6HPDO HGf 6RPDFORQDO YDULDWLRQV DQG FURS LPSURYHPHQW 0DUWLQXV 1LMKRII 3XEOLVKHUV %RVWRQ *DPERUJ / ) &RQVWDEOH DQG 5 $ 0LOOHU (PEU\RJHQHVLV DQG SODQW SURGXFWLRQ RI DOELQR SODQWV IURP FHOO FXOWXUHV RI %URPXV LQHUPLV 3ODQWD *DUUDUG / $ DQG 6 + :HVW 6XERSWLPDO WHPSHUDWXUH DQG DVVLPLODWH DFFXPXODWLRQ LQ OHDYHV RI n3DQJOD GLJLWJUDVV 'LJLWDULD GHFXPEHQV 6WHQWf &URS 6FL *URVV 5 : DQG ( 7 %LQJKDP $Q XQVWDEOH DQWKRF\DQLQ PXWDQW UHFRYHUHG IURP WLVVXH FXOWXUH RI DOIDOID 0HGLFDJR VDWLYDf 3ODQW &HOO 5HS *XVWD / 9 %XWOHU & 5DMDVKHNDU DQG 0 %XUNH )UHH]LQJ UHVLVWDQFH RI SHUHQQLDO WXUIJUDVV +RUW6FL +DOOJUHQ ( ( 6XQERP DQG 0 6WUDQG 3KRWRV\QWKHWLF UHVSRQVHV WR ORZ WHPSHUDWXUH LQ %HWXOD SXEHVFHQV DQG %HWXOD WRUWXRVD 3K\VLRO 3ODQW +DQVWHHQ&UDPHU % =XU ELRFKHPLH XQG SK\VLRORJLH GHU JUHQ]VFKLFWHQ OHEHQGHU SIODQ]HQ]HOOHQ 0HGO 1RUJHV /DQG EUXNVK

PAGE 92

+HLQULFK DQG + /DVFK 3KRWRLQKLELWLRQ RI SKRWRV\QWKHVLV 6WXGLHV RQ PHFKDQLVPV RI GDPDJH DQG SURWHFWLRQ LQ FKORURSODVWV S ,Q %LJJLQV HGf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
PAGE 93

-DFREVHQ 9 DQG 7 9 +LJJLQV &KDUDFWHUL]DWLRQ RI WKH DDP\ODVHV V\QWKHVL]HG E\ DOHXURQH OD\HUV RI +LPDOD\D EDUOH\ LQ UHVSRQVH WR JLEEHUHOOLF DFLG 3ODQW 3K\VLRO -DFREVHQ 9 DQG / 5 %HDFK &RQWURO RI WUDQVFULSWLRQ RI DDP\ODVH DQG U51$ JHQHV LQ EDUOH\ DOHXURQH SURWRSODVWV E\ JLEEHUHOOLQ DQG DEVFLVLF DFLG 1DWXUH -HQVHQ : $ &DUERK\GUDWHV DQG FHOO ZDOO FRQVWLWXHQWV S ,Q : $ -HQVHQ HGf %RWDQLFDO KLVWRFKHPLVWU\ : + )UHHPDQ DQG &R 6DQ )UDQFLVFR -RQHV & $ 7HPSHUDWXUH S ,BQ & $ -RQHV HGf & JUDVVHV DQG FHUHDOV JURZWK GHYHORSPHQW DQG VWUHVV UHVSRQVH -RKQ :LOH\ DQG 6RQV 1HZ
PAGE 94

/DHPPOL 8 &OHDYDJH RI VWUXFWXUDO SURWHLQV GXULQJ WKH DVVHPEO\ RI WKH KHDG RI EDFWHULRSKDJH 7 1DWXUH /DUNLQ 3 6RPDFORQDO YDULDWLRQ KLVWRU\ PHWKRG DQG PHDQLQJ ,RZD 6WDWH 5HV /DUNLQ 3 DQG : 5 6FRZFURIW 6RPDFORQDO YDULDWLRQf§$ QRYHO VRXUFH RI YDULDELOLW\ IURP FHOO FXOWXUHV IRU SODQW LPSURYHPHQW 7KHRU $SSO *HQHW /DUNLQ 3 DQG : 5 6FRZFURIW 6RPDFORQDO YDULDWLRQ DQG FURS LPSURYHPHQW S ,Q & 3 0HUHGLWK DQG $ +ROODQGHU HGVf *HQHWLF HQJLQHHULQJ RI SODQWV 3OHQXP 1HZ
PAGE 95

/RZ 3 6 5 2UW : $ &UDPHU :KLWPDUVK DQG % 0DUWLQ 6HDUFK IRU DQ HQGRWKHUP LQ FKORURSODVW ODPHOODU PHPEUDQHV DVVRFLDWHG ZLWK FKLOOLQJLQKLELWLRQ RI SKRWRV\QWKHVLV $UFK %LRFKHP %LRSK\V /\RQV 0 5DLVRQ DQG 3 / 6WHSRQNXV 7KH SODQW PHPEUDQH LQ UHVSRQVH WR ORZ WHPSHUDWXUH $Q RYHUYLHZ S ,Q 0 /\RQV *UDKDP DQG 5DVLQ HGVf /RZ WHPSHUDWXUH VWUHVV LQ FURS SODQWV $FDGHPLF 3UHVV 1HZ
PAGE 96

0HLQV ) 0HFKDQLVPV XQGHUO\LQJ WKH SHUVLVWHQFH RI WXPRU DXWRQRP\ LQ FURZQJDOO GLVHDVH S ,Q + ( 6WUHHW HGf 7LVVXH FXOWXUH DQG SODQW VFLHQFH $FDGHPLF 3UHVV 1HZ
PAGE 97

•TXLVW (QYLURQPHQWDO VWUHVV DQG SKRWRV\QWKHVLV S ,Q %LJJLQV HGf 3URJUHVV LQ SKRWRV\QWKHVLV UHVHDUFK YRO 0DUWLQXV 1LMKRII 3XEOLVKHUV %RVWRQ 2UWRQ 7 6RPDFORQDO YDULDWLRQ 7KHRUHWLFDO DQG SUDFWLFDO FRQVLGHUDWLRQV S ,Q 3 *XVWDIVRQ HGf *HQH PDQLSXODWLRQ LQ SODQW LPSURYHPHQW 3OHQXP 3UHVV 1HZ
PAGE 98

3UHLVV & /HYL DQG 7 : 2NLWD &KDUDFWHUL]DWLRQ RI WKH VSLQDFK OHDI SKRVSKRU\ODVH 3ODQW 3K\VLRO 3UHLVV 0 %ORRP 0 0RUHOO 9 / .QRZOHV : & 3OD[WRQ 7 : 2NLWD 5 /DUVRQ $ & +DUPRQ DQG & 3XWQDP(YDQV 5HJXODWLRQ RI VWDUFK V\QWKHVLV HQ]\RPDWLFDO DQG JHQHWLF VWXGLHV S ,Q %UXHQLQJ +DUDGD 7 .RJXJH DQG $ +ROODHQGHU HGVf 7DLORULQJ JHQHV IRU FURS LPSURYHPHQW DQ DJULFXOWXUDO SHUVSHFWLYH 3OHQXP 3UHVV 1HZ
PAGE 99

6FRZFURIW : 5 DQG 3 /DUNLQ 6RPDFORQDO YDULDWLRQ S ,Q %RFN DQG 0DUVK HGVf $SSOLFDWLRQV RI SODQW FHOO DQG WLVVXH FXOWXUH -RKQ :LOH\ DQG 6RQV 1HZ
PAGE 100

6WHXS 0 + 5REHQHN DQG 0 0HONRQLDQ ,Q YLWUR GHJUDGDWLRQ RI VWDUFK JUDQXOHV LVRODWHG IURP VSLQDFK FKORURSODVWV 3ODQWD 6WLWW 0 DQG 7 5HHV &DUERK\GUDWH EUHDNGRZQ E\ FKORURSODVWV RI 3LVXP VDWLYXP %LRFKHP %LRSK\V $FWD 6WLWW 0 DQG : + +HOGW 3K\VLRORJLFDO UDWHV RI VWDUFK EUHDNGRZQ LQ LVRODWHG LQWDFW VSLQDFK FKORURSODVWV 3ODQW 3K\VLRO 6XNXPDUDQ 1 3 DQG & :HLVHU )UHH]LQJ LQMXU\ LQ SRWDWR OHDYHV 3ODQW 3K\VLRO 7DO 0 6HOHFWLRQ IRU VWUHVV WROHUDQFH S ,Q $ (YDQV : 5 6KDUS 3 9 $PPDWR DQG <
PAGE 101

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
PAGE 102

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f ORFDWHG DW *DLQHVYLOOH )ORULGD +H LV FXUUHQWO\ UHVHDUFKLQJ SODQW JURZWK XQGHU VWUHVV FRQGLWLRQV ,Q $XJXVW KH LQLWLDWHG ZRUN WRZDUGV D 3K' GHJUHH LQ DJURQRP\ DW WKH 8QLYHUVLW\ RI )ORULGD +H LV FXUUHQWO\ D 3K' FDQGLGDWH DQG XSRQ FRPSOHWLRQ RI KLV JUDGXDWH ZRUN KH ZLOO EH HPSOR\HG E\ +RIIPDQ/D5RFKH DV DQ DJULFXOWXUDO VSHFLDOLVW LQ WKH EXVLQHVV GHYHORSPHQW GHSDUWPHQW

PAGE 103

,Q KH ZDV FHUWLILHG DV D SURIHVVLRQDO FURS VFLHQWLVW E\ WKH $PHULFDQ 5HJLVWU\ IRU &HUWLILHG 3URIHVVLRQDOV LQ $JURQRP\ &URSV DQG 6RLOV +H LV D PHPEHU RI WKH $PHULFDQ 6RFLHW\ RI $JURQRP\ DQG &URS 6FLHQFH 6RFLHW\ RI $PHULFD +H LV PDUULHG WR $P\ /\QQ DQG KDV WKUHH FKLOGUHQ -DFRE 5DFKHO DQG 7DU\Q

PAGE 104

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nVn 3URIHVVRU RI +RUWLFXOXUDO 6FLHQFH DV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 'U *UD\ $VVRFLDWH 3URIHVVRU RI +RUWLFXOWXUDO 6FLHQFH

PAGE 105

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHTUHH RI 'RFWRU RI 3KLORVRSK\ GM/ 'U 5 / 6PLWK 3URIHVVRU RI $TURQRP\ 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI $JULFXOWXUH DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 0D\ 'HDQ n&ROOHJH RI $ FXOWXUH 'HDQ *UDGXDWH 6FKRRO

PAGE 106

81,9(56,7< 2) )/25,'$ P LOO LQQ LQQ mL L}