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11100 cICTRCJDamest-pJA

____——_7

UTILIZATION OF CHLOROPHYLL FLUORESCENCE
IN STORAGE AND VEGETATIVE PROPAGATION
OF
TAXUS
By

Sarah Eleni Bruce

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Horticulture

1 999

ABSTRACT

UTILIZATION OF CHLOROPHYLL FLUORESCENCE
IN STORAGE AND VEGETATIVE PROPAGATION
OF TAXUS
By

Sarah Eleni Bruce

Propagation failures in Taxus are Often attributed to cutting collection from
stock plants of poor quality. If a quick, reliable method of determining potential
rooting of cuttings based on the condition of a specific stock plant was available
for propagators, rooting success could be predicted prior to an investment in
time, labor, and resources. These studies examined chlorophyll fluorescence
(FJF...) as a potential tool for stock plant selection, assessment of storage
conditions, and measurement of stress over the course of propagation. Ten
cultivars of Taxus xmedia (Taxus baccata L. x T. cuspidata Sieb. & Zucc.) were
used: Brownii, Dark Green Pyramidalis, Dark Green Spreader, Densiformis,
Densiforrnis Gem, Hicksii, L.C. Bobbink, Runyan, Tauntoni, and Wardii. Storage
condition treatments consisted of desiccation (low, medium, high), duration (34,
70, 107 days), and temperature (-30, -2.5, O, 2.5, 5, 10, and 20 °C). Cultivars
differed in FJF... initially, as well as over time. Correlations were not found
between initial stock plant FJF... and rooting percentage, root number, root dry
weight, or root length, indicating that NF... is not a reliable indicator of stock plant
propagation potential. Short storage duration at -2.5 to 2.5 °C was found to be
ideal. FJF... could detect substandard storage conditions only at temperature and

desiccation extremes.

ACKNOWLEDGMENTS

I am thankful for the guidance and support of my major advisor, Brad
Rowe. Mike Corbett, Tom Todor, and others at Zelenka Nursery were extremely
helpful sharing their experience in propagation, and generous with their time and
resources. I would also like to thank my committee members, Jim Flore and Don
Dickmann, for their time and helpful criticisms. Lastly, I thank Emily Cloudi for

her friendship, support, and advice.

TABLE OF CONTENTS

LIST OF FIGURES... vii
SYMBOLS AND ABBREVIATIONS... x

LITERATURE REVIEW:

CHLOROPHYLL FLUORESCENCE AND THE PROPAGATION OF
Chlorophyll Fluorescence . 2
Chlorophyll Fluorescence lmplIcetions 4
A Closer Look at Chlorophyll Fluorescence Measurements ...... 5
Chlorophyll Fluorometers... 11
Chlorophyll Fluorescence Use 14
Chlorophyll Fluorescence Potential................................. 19
Taxus HIstory 20
Propagation of Taxus 23
Purpose ofthe Experiments 24
Literature CIted 25

CHAPTER ONE:
CHLOROPHYLL FLUORESCENCE AND VEGETATIVE PROPAGATION OF
AbStract
Introduction
Materials and Methods
Results and Discussmn
Literature Cited
ListofFIgures

QSSSIK‘G’

CHAPTER TWO:

CHLOROPHYLL FLUORESCENCE AND COLD STORAGE OF TAXUS
Abstract... 67
Introduction... 68
Materials and MethOds. 71
Results and DichSSIon 75
Conclusrons 80
Literature Cited 83
ListOfFIgureS 87

LIST OF TABLES

TABLE PAGE

CHAPTER QNE

1997 -1998 Initial chlorophyll fluorescence (Fl/Fm) of ten cultivars of Taxus
xmedia from stock plant material at Zelenka Nursery
50

1998-1999 Initial chlorophyll fluorescence (Fl/Fm) of nine cultivars Of
Taxus xmedia from stock plant material at Zelenka Nursery
51

1998 -1999 Chlorophyll fluorescence (FJFM) of stem cuttings at sticking
(after 32 days of cold storage at 5 °C) Nine cultivars of Taxus xmedia at
Zelenka Nursery.

52

1998 -1999 Chlorophyll fluorescence (FJF...) of stem cuttings at harvest
(after 32 days of cold storage at 5 °C and 100 days in the propagation
bed). Nine cultivars of Taxus xmedia at Zelenka Nursery.

53

1997 -1998 Rooting percentage often cultivars of Taxus xmedia at
Michigan State University.
54

1997 -1998 Mean root dry weights often cultivars of Taxus xmedia at
Michigan State University.
55

1998 -1999 Rooting percentage of nine cultivars of Taxus xmedia at
Zelenka Nursery.
56

1998 -1999 Mean root dry weights Of nine cultivars Of Taxus xmedia at
Zelenka Nursery.
57

1998 -1999 Taxus xmedia chlorophyll fluorescence (FJF...) correlated with

harvest data at Zelenka Nursery at the whole-experiment level. Initial

(FJFm) was taken at cutting collection, sticking (FJFm) after 32 days cold

storage at 5 °C, and harvest (FJFI'II) after 100 days in the propagation bed.
58

TAB LE PAGE

10

1998 ~1999 Taxus xmedia chlorophyll fluorescence (Ev/F...) correlated with
harvest data at Zelenka Nursery at the cultivar level. Initial (FJFm) was
taken at cutting collection, sticking (FJF...) after 32 days cold storage at 5

°C, and harvest (FJF...) after 100 days in the propagation bed.
59

CHAPTER TWQ

1997 - 1998 Taxus xmedia chlorophyll fluorescence (FJF...) and storage
temperature treatments (0, 2.5, 5, 10, and 20 °C) correlated with harvest

data (Pearson correlation coeffecients). Storage duration = 34 days.
85

LIST OF FIGURES

FIGURE PAGE
CHAPTER ONE
1 Chlorophyll fluorescence in Taxus over the course Of propagation at

Zelenka Nursery Oct. 14, 1997 - May 7, 1998. Cuttings were
collected from stock plants in the field and placed in cold storage at
2.5 °C for 37 days and then stuck in rooting beds (100% perlite) at 18 °C
with bottom heat at 21 °C. Chlorophyll Fluorescence readings (FJFM) were
taken periodically until harvest. The first three points per cultivar are each
a mean of 10 readings, while all other points represent means of 24
readings. Vertical bars represent + SE.

62

2 Chlorophyll fluorescence in Taxus over the course of propagation at
Michigan State University Oct. 14, 1997 - May 6, 1998. Cuttings were
collected from stock plants in the field and placed in cold storage at 5 °C
for 39 days and then stuck in rooting beds (100% perlite) at 18-21 °C.
Chlorophyll Fluorescence readings (Ev/Fm) were taken periodically until
harvest. The first three points per cultivar are each a mean of 10 readings,
while all other points represent means of 24 readings. Vertical bars
represent + SE.

63

3 Chlorophyll fluorescence in Taxus over the course of propagation at
Zelenka Nursery Oct. 29, 1998 - March 10, 1999. Cuttings were
collected from stock plants in the field and placed in cold storage at
2.5 °C for 33 days and then stuck in rooting beds (100% perlite) at 18 °C
with bottom heat (21 °C). Chlorophyll Fluorescence readings (FJF...) were
taken periodically until harvest. The first three points per cultivar are each
a mean of 60 readings, while all other points represent means of 24
readings. Vertical bars represent + SE.

64

FIGURE PAGE
CHAPTER TWQ

1 Effect of storage temperature and desiccation on rooting of four cultivars
of Taxus (1997 -— 1998). Storage duration = 34 days. Stem cuttings were
collected from stock plants in the field, placed in cold storage treatments,
and then placed in perlite propagation beds. Rooting percentages were
measured 96 - 99 days after sticking. Points represent means of 24
readings. Vertical bars represent + SE.

89

2 Effect Of storage temperature and desiccation on root dry weight of four
cultivars of Taxus (1997 - 1998). Storage duration = 34 days. Stem
cuttings were collected from stock plants in the field, placed in cold
storage treatments, and then placed in perlite propagation beds. Roots
were harvested 96 - 99 days after sticking, dried at 28 °C for 3 days and
weighted. Points represent means of 24 readings. Vertical bars represent
+ SE.

90

3 Effect of storage duration on rooting of four cultivars Of Taxus (1998 -
1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were collected
from stock plants in the field, placed in cold storage for treatment duration,
and then placed in perlite propagation beds at 18 °C with bottom heat
(21 °C). Rooting percentages were measured 96 - 99 days after sticking.
Points represent means Of 72 readings. Vertical bars represent + SE.

91

4 Effect Of storage duration on root dry weight of four cultivars Of Taxus
(1998 - 1999). Storage temperature = -2.5 - 2.5 °C. Stern cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in pedite propagation beds at 18 °C with bottom
heat (21 °C). Roots were harvested 96 - 99 days after sticking, dried at 28
°C for 3 days and weighed. Points represent means Of 72 readings.

Vertical bars represent 4- SE.
92

5 Effect of storage duration on root number of four cultivars of Taxus (1998
— 1999). Storage temperature = -2.5 — 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Root numbers were measured 96 - 99 days after sticking.
Points represent means of 72 readings. Vertical bars represent + SE.

93

FIGURE PAGE

6

Effect of storage duration on root length rating of four cultivars of Taxus
(1998 - 1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in pedite propagation beds at 18 °C with bottom
heat (21 °C). Root length ratings were measured 96 - 99 days after
sticking. (0=less than 2.54 cm [1 inch], 1=less than 5.08 cm [2 inch],
2=less than 7.62 cm [3 inch], 3=greater than 7.62 cm) Points represent
means of 72 readings. Vertical bars represent + SE.

94

Effect of storage temperature and desiccation on chlorophyll fluorescence
of cuttings Of Taxus (1997 — 1998). Chlorophyll fluorescence (Fl/Fm) was
measured on the lower surface of individual dark-adapted (15 minutes)
needles with fluorometer (Morgan CF-1000) light level set at 700
umollm’ls. The first point of each line represents a mean of 40 readings,
all other points represent means of 80 readings. Vertical bars represent
+ SE.

95

Effect of storage duration on chlorophyll fluorescence of cuttings Of Taxus
(1998 -—1999). Storage temperature = -2.5 - 2.5 °C. Chlorophyll
fluorescence (FJF...) was measured on the lower surface of individual
dark-adapted (15 minutes) needles with fluorometer (PEA, Hansatech
Instruments Ltd.) light level set at 1200 urnol/mzls. Points represent
means of 20 readings. Vertical bars represent + SE.

96

SYMBOLS AND ABBREVIATIONS

Fo initial dark-adapted chlorophyll fluorescence
F... maximum chlorophyll fluorescence

F. .. stable chlorophyll fluorescence

Fv variable chlorophyll fluorescence (Fm—F0)
PSII photosystem II

PSI photosysteml

IBA IndoIe—3-butyric acid

NAA 1 Naphthalene aceticacid

MSU Michigan State University

LITERATURE REVIEW

Chlorophyll Fluorescence and the Propagation of Taxus

CHLOROPHYLL FLUORESCENCE

Chlorophyll fluorescence is a product of the photosynthetic process. It is
energy that is re-emitted from all green plants. In tracing the path of that energy,
we begin with sunlight, the energy’s origin.

As light strikes a leaf, it travels through cellular layers to the chloroplasts,
cellular organelles which house chlorophyll, the cell’s light-energy absorbing
pigment molecules. Blue (~420 nm) and red (~660 nm) light absorption occurs
across the thylakoid membranes Of a chloroplast where antemae chlorophyll
molecules are arranged in arrays of several hundred each. These anay
chlorophyll molecules funnel energy, via resonance energy transfer, to a
photochemically active chlorophyll molecule in the center of the array. This
central pigment molecule is actually a chlorophyll a dimer situated within a
specialized protein complex. In Photosystem II, the first light absorption
mechanism in photosynthesis, it is called Pm, the “680' referring to the
wavelength of light at or above which it absorbs. Light energy transferred to Pm
raises the molecule’s energy level to an excited state. At this point the collected
energy faces four possible fates: energy transduction, resonance energy transfer,
loss as heat, or loss as light.

Energy transduction is the process that enables the energy in the excited
Pm molecule to be used for the biochemistry of photosynthesis. The excitement
energy collected in Pam is passed down an electron transport chain in the form of
an electron. The first step in this chain is pheophytin, a molecule similar to

chlorophyll a. From pheophytin the electron is passed through a series of at

least two plastoquinones, QA and 03, which are lipid soluble and thus transfer the
electron across the thylakoid membrane to an iron-sulfur complex which releases
the electron into a plastocyanin pool in the thylakoid lumen space (to the inside of
the membrane). The now oxidized pigment, Pam, is quickly reduced with an
electron from the D1 protein which originates in the splitting of water (Krause and
Weis, 1991 ). Photosystem I, the secondary light absorbing step of
photosynthesis, draws its excitement electrons from the plastocyanin pool.
Therefore, PSI uses energy transferred from PSII in addition to light absorbed by
its reaction center pigment, Pm. From PSI, energy travels to the
photophosphorylation processes of photosynthesis and the production of
NADPH, which eventually leads to glucose production.

PSII has been shown to consist of two types, PSIIcl and PSIIp, which differ
in terms of antennae array size and photosynthetic capacity, PSII, being smaller
and having lower activity levels (Holzwarth, 1988). Chlorophyll Fluorescence is
therefore largely a result of PSII“ centers. Melis et al. (1988) suggest that these
forms of PSII may simply reflect a photocenter’s stage of maturity in an assembly
growth process.

About 90% of the energy absorbed by Pam is used for photosynthesis via
the electron transport processes reviewed above (Bjorkman and Demmig, 1987).
The remainder is ‘Iost’ in a number of ways. Some is transferred to non-
photochemically active molecules (resonance energy transfer). Some is lost as
heat. Some is lost as re-emitted light, termed chlorophyll fluorescence. It is this

light, emitted at a slightly longer wavelength than absorbed (~690 nm, red and

far-red light) due to heat dissipation (termed "stokes shift"), that we measure. At
physiological temperatures, almost all fluorescence is emitted from chlorophyll a
molecules associated with PSII Since chlorophyll b molecules transfer their
excitement energy to chlorophyll a molecules (Lichtenthaler, 1988b). The exact
source of re-emittance - Pm, array chlorophylls, accessory pigments, etc.,
remains unknown (Schreiber and Bilger, 1993), although a general consensus
has been formed that it is likely the array chlorophylls which are responsible
(Lichtenthaler, 1 988a).

CHLOROPHYLL FLUORESCENCE IMPLICATIONS

The amount Of chlorophyll fluorescence emitted from a particular sample
of photosynthetically-active tissue is dependent on the light energy utilization
potential of that tissue. At best, chlorophyll fluorescence represents only 3 - 5%
of excitation energy in viva (Coombs et al., 1985; Lichtenthaler, 1988a estimates
it at 0.5 - 3%). In vitro, up to 30% of the energy has been found to fluoresce
(Coombs et al., 1985). Chlorophyll fluorescence an be considered a product of
excess light energy —that light energy that exceeds the absorption potential of the
electron transport process of PSII. This makes chlorophyll fluorescence levels
indicative of the energy processing potential of PSII.

Photosystem II, itself, is an indirect indicator of the condition of the
photosynthetic apparatus components at large (Georgieva and Yordanov, 1993).
Being the primary light absorption process in photosynthesis and lying across

thylakoid membranes, a region sensitive to environmental stresses, PSII electron

transport is believed to play a key role in the response of leaf photosynthesis to
environmental perturbations. Chlorophyll fluorescence measurements have
been have been used extensively in stress physiology studies. Chlorophyll
fluorescence has been found to show a broad inverse relationship to
photosynthetic carbon assimilation (Kautsky and F rank, 1943 in Lichtenthaler,
1990) and reflects the quantum yield of electron transport in PSII (Genty et al.,
1989; Adams et al., 1990).

A CLOSER LOOK AT CHLOROPHYLL FLUORESCENCE MEASUREMENTS

Historically, the chlorophyll fluorescence signal pattern has been difficult to
interpret. In 1977 Lavorell and Etienne described it as being “rich and
ambiguous”. Today we have a better understanding of the signal, achieved
through a very broad base of research. However, many different measurements
are used, a tribute to the difficulty of making point comparisons from fluctuating
signals.

The fluorescence induction signal was first described by Kautsky in 1931
and is known as the Kautsky Effect. Depending on degree of resolution, the
signal is referred to as "complex" or "simple" fluorescence kinetics. Complex
fluorescence kinetics involve several minor peaks and dips in the fluorescence
signal that remain unexamined in the simple fluorescence induction curve
described here (for more information see Papageorgiou, 1975). In
measurement, leaves are dark adapted (kept in darkness) for a certain amount of

time (usually under 30 minutes) depending on the species, until the fluorescence

level is brought down to a minimum operating level, F... Then the leaf is brightly
illuminated, sufficiently to saturate PSII, and the level Of chlorophyll fluorescence
immediately rises to a peak level, P... This “fast rise” in chlorophyll fluorescence
reflects the saturation of the initial 0;. electron acceptors in the electron transport
chain of PSII (Krause and Weis, 1991; Coombs et al., 1985). It takes a minimum
of 200 ms for F... to be reached (Krause and Weis, 1991), with times usually
within the 300 - 400 ms range (Lichtenthaler, 1988b). Then as electron transport
begins, the energy is transported to proceeding electron carriers (03.
plastocyanins...) and the initial electron acceptors become available carriers Of
energy from Page again. In this way, energy moves through PSII. Chlorophyll
fluorescence is reduced from F... and, over a period of minutes, reaches a steady
state fluorescence level, F., close to the original F.. level. This reduction of the
chlorophyll fluorescence level by photochemical processes, mainly the re-
oxidation of Q... is termed "photochemical quenching“.

Chlorophyll Fluorescence Quenching is a broad title which refers to a
number of processes that lower the fluorescence level from the maximum, F....
This includes the photochemical quenching mentioned above, but also a number
of non-photochemical quenching means. One of these is energy~dependent
quenching, the major non—photochemical quenching mechanism (Lichtenthaler
1988a). It is a little understood process whereby the pH gradient caused by light-
driven transportation of protons across the thylakoid membrane causes a
reduction in fluorescence (Krause and Weis, 1991). Lichtenthaler (1 988a)

speculates that an increase in the rate constant Of thermal deactivation of PSII

may have occurred, or structural changes that lower PSll’s efficiency. Energy
dissipation switches away from fluorescence and tends to be lost as heat
(Coombs et al., 1985). Another type of quenching is caused by a state transition
in the Pan photocenter due to phosphorylation (Schreiber and Bilger, 1993). This
may serve to balance excitation energy distribution between the two photo
systems since PSII excitation is reduced relative to PSI (Lichtenthaler, 1988a).

Photoinhibition has been the most studied of the non-photochemical
quenching mechanisms. It is the increased deactivation of reaction writers, Pm
and P700, as the result of bright illumination. Two processes are involved: a
process of PSII reaction center deactivation and repair involving the D1 protein,
and avoidance of over-excitation by increased thermal energy dissipation,
probably via the xanthophyll cycle (Long at al., 1994; Krause et al., 1990). The
xanthophyll cycle is a quenching mechanism that acts as the major initial
response to light that exceeds PSII photochemical capacity (reviewed in: Long et
al., 1994). Diepoxide violaxanthin is converted to epoxide-free zeaxanthin, which
distributes excess light energy as heat in antennae chlorophyll molecules. The
conversion is gradually reversed in darkness or low light. Photoinhibition caused
by excessive photon flux densities is a main cause for reduction of FJF... under
natural conditions (Long et al., 1994). Schreiber suggests that energy dependent
quenching may actually be a mechanism to deal with excess light so as to avoid
photoinhibition (Krause and Weis, 1991). However, Huner et al., (1993) argue
that photoinhibition should be viewed as "the capacity of plants to adjust

photosynthetically to the prevailing environmental conditions" rather than an

injury.

Although early research tended to focus on using chlorophyll fluorescence
to study photochemical quenching processes, now it is the non-photochemical
processes that receive the most attention. Lichtenthaler (1990) points out that it
is mostly non-photochemical quenching and not photochemical quenching that is
affected by environmental stress.

The values used to describe chlorophyll fluorescence, and how they are
measured, have varied widely according to researcher, field, and precedent.
This makes comparisons between studies often difficult. An attempt at a
nomenclatural key has been published by van Kooten and Snel (1990), but they
caution that it is too early for rigorous definitions. Confounding issues is the
sensitivity of PSII to varying environmental conditions and their interactions. For
instance, the upper and lower surfaces of a leaf Often show different chlorophyll
fluorescence levels, which may, in part, be caused by a much denser
arrangement of chlorophyll on the upper surface leading to much re-absorption of
emitted fluorescence as compared with the underside of the leaf (Rinderle and
Lichtenthaler, 1 988).

Measurements of the fast rise induction kinetics are perhaps the most
common, probably due to the ease and speed with which data can be taken. F.
and F... have frequently been used, Often in conjunction with ratios derived from
them. F .. tends to be affected by stresses that cause configuration changes in
thylakoid structure, such as heat stress (Hansatech manual). F... is most affected

by non-photochemical quenching mechanisms. The variable fluorescence, F.., is

calculated by subtracting F. from F.... It represents the initial light absorption
ability Of PSII, the number of quinone-type electron acceptors that are available
in a dark-adapted state. Commonly the ratio FJ F... is used. It has been shown
to be a reliable indicator of the quantum yield of PSII (Adams et al., 1990),
provided that the sample is dark-adapted and therefor operating at minimal
fluorescence levels initially, and that the light source is sufficient to saturate PSII.
If PSII is not light saturated, F... values obtained are inaccurate. It is the
measurement most Often used to test the effects of environmental stresses.
Bjorkman and Demmig (1987) found that although there was considerable
fluorescence variation at 692 and 734 nm among different spades, upper and
lower leaf surfaces, and sun and shade leaves, F.,! F... ratios varied little. The 44
species, measured under ideal conditions, had an overall FJ F... average of .832
+I- .004. Conifers averaged .853 +I- .004. Less commonly, the F.,! F. ratio has
been used (Ruter, 1993) and was found helpful in identifying optimal growth
temps in woody plants. It has been shown to be closely linked to leaf water
potential (Hansatech manual).

However, Lichtenthaler (1990) argues that F., F..., F.,, and ratios derived
from them, are inappropriate measurements when dealing with stress and
ecophysiology due to their being measured in the dark-adapted, non-functional
state of photosynthesis. In this state, measurements would not fully reflect the
actual physiological condition of the photosynthetic apparatus and its
functionality. He suggests a ‘vitality index’, Rfd, consisting of the ratio of the

fluorescence signal decrease to steady state fluorescence, or (F...- F.)I F., as a

better indicator of actual photosynthetic functioning. Rfd values have been used
in research involving spruce (Hagg et al., 1992) and Scotch pine (Saarinen and
Liski, 1993). Using a variation of F.,! F... measured under illuminated conditions
(F.,! F...'), Genty et al. (1989) found a clear relationship between it and the
quantum yield of carbon dioxide assimilation in a wide variety Of plant species.

As chlorophyll fluorometers have become more advanced, the possible
measurements have increased. F luorometers are available that can measure
different wavelength ranges and thus chlorophyll fluorescence readings can be
taken for PSII (~690 nm) and PSI (~ 735 nm) individually as well as combined.
The Pm’P‘ns ratio has been used as an indicator of the reciprocal relationship of
in vivo chlorophyll content of leaves and needles (Hagg et al., 1992). It may be
especially suited for measurement of long term stress conditions in plants, and,
due to the speed with which it can be measured, it has potential for aerial forest
surveys (Lichtenthaler, 1988c).

Quenching measurements are Often expressed in terms of ‘quenching
coefficients’ which represent the quenched proportion of F... Quenching origins
have been identified with the study of delayed chlorophyll fluorescence, or
luminescence. The delay time can be as short as 0.3 seconds and luminescence
can last up to several minutes (Schmidt, 1988). Millisecond-long luminescence is
often the result of membrane energization, while luminescence Of ~50
microseconds involves a slow-down of PSII donors (Schreiber and Bilger, 1993).
Schmidt (1988) identifies the origin of luminescence energy as a back-transfer of

charges built-up in the photosynthetic electron transport chain. Luminescence is

10

therefore very closely linked with photosynthetic processes. It also provides high
contrast between damaged and undamaged leaves. However, measurement of
luminescence requires complete darkness due to its weak signal and thus
requires bulky devices (Blaich, 1988).

A calculation of the electron transport rate (ETR) through PSII has also
been used (Brodribb and Hill, 1997). Edwards and Baker (1993) developed the
formula for such which multiplies the photochemical efficiency of PSII (Gentry et
al., 1989) by the incident photosynthetic photon flux density and the average
fluorescence, halved.

Lastly, chlorophyll fluorescence lifetimes are being measured in growing
numbers of experiments. The lifetime of fluorescence is analyzed by measuring
the fluorescence decline after brief exciting flashes of light. This shows the
decline in excitation density of chlorophyll (Krause and Weis, 1991). Chlorophyll

fluorescence lifetimes are good for observing the kinetics of primary

photosynthetic processes.

CHLOROPHYLL FLUOROME‘I'ERS

The technology involved in chlorophyll fluorometers has changed greatly
since their first appearance on the scene in the 1930's.

Modern fluorometers vary with the uses they were designed for.
Lightweight portable versions are the most common. These usually consist of a
microprocessor, with some type of display and control panel, attached to a fiber

Optic tube. There may be some type of actinic light source with a shutter in the

11

control box or a light source such as light emitting diodes may be attached to the
opposite and of the fiber optic. One new model uses an HeINe-laser (Daley,
1995: Lichtenthaler, 1990). A variety of plastic clip designs exist which are
clamped onto the portion of the sample to be measured. These allow for dark
adaptation of the sample in a lighted lab or out of doors, as well as hold the
photodiode, located at the end of the fiber optic, in place for the chlorophyll
fluorescence measurement. Photodiodes measure photons by the electric
current triggered when photons hitting the photodiode cause photochemical
reactions, causing the excitation of an electron and leaving an empty, positively
charged 'blank' where the electron used to be. In order to measure only
fluoresced light and not reflected actinic light (actinic light is light meant for use in
photosynthesis), optical filters are used to sort out the different wavelengths, or,
more rarely, only lightwaves of less than 620 nm are used for the actinic light
source (Bolhar-Nordenkampf at al., 1989). After F. is measured, the sample is
brightly illuminated by the light source and fluorescence measurements are
taken, often as quickly as 100,000 readings per second for the fast rise and then
gradually slowing to somewhere around 10 readings per second as the signal
stabilizes to F. (Hansatech and Morgan manuals). The quantity of fluorescence
is normally measured in arbitrary units set by the manufacturer. These are often
referred to as "relative units” in the literature.

As mentioned, some chlorophyll fluorometers are being designed to
measure the fluorescence individually at two different wavelengths. These

wavelengths correspond to the two chlorophyll fluorescence spectra peaks, 690

12

and 735 nm, emitted from PSII and PSI, respectively. This gives the researcher
fluorescence information from both photocenters, as well as making an estimate
of chlorophyll content possible (Hagg at al., 1992; Lichtenthaler, 1990). Although
stress detection is possible (Lichtenthaler, 1988b), at physiological temperatures,
changes in chlorophyll fluorescence due to stress are largely dominated by
emissions from PSII (Bolar-Nordenkampf et al., 1989).

A relatively new modification is the Pulse Amplitude Fluorometer, PAM,
also sometimes called a Pulse Modulation Fluorometer. After F., F..., and F.
measurements are taken, an actinic light is turned on and the sample is
subjected to additional saturating light pulses every 10 seconds or so. This
allows the measurement Of photochemical and non-photochemical quenching
coefficients, non-photochemical quenching being that which doesn't change
during a saturation pulse Of light (Schreiber and Bilger, 1993). These pulse-
modulation measurements can be taken in daylight conditions, an important
development for photoinhibition experiments and field studies.

Also new is the advent of fluorescence video imaging. Daley (1995)
describes a portable fluorescence imaging system which records video images
and than is able to use digitized versions of the images for analytical purposes.
Mapping of the spatial distribution of photosynthetic activity becomes possible.

Aerial sensing Of large-scale vegetation fluorescence is under study
(Lichtenthaler, 1990). Instead of focusing on individual leaf chlorophyll
fluorescence, this instead deals with the overall reflection signature of a large

group of plants at once. Stressed or dying trees tend to have lower chlorophyll

l3

contents and this affects their reflection signal in two ways. First, visible light
(800-900nm) reflection is increased because less photosynthesis is going on
(less photochemical quenching). Second, the amount of infrared light reflected is
decreased due to structural changes in the leaf cell arrangement. Combining
these two, we get what is termed a 'blue shift' in the reflected light from stressed
plants. However, this blue shift is little understood in terms of what determines

when it will occur and how different stresses are involved.

CHLOROPHYLL FLUORESCENCE USE

Much of early chlorophyll fluorescence research dealt with understanding
the mechanism behind fluorescence itself and getting a better understanding of
what it implied. It was used as a "probe of photosynthesis" (Papageorgiou, 1975)
and was involved mainly in basic photosynthesis research. By the 1980's
however, numerous experiments were being done comparing indicators of
photosynthesis rates and various chlorophyll fluorescence measurements.
Bjorkman and Demmig (1987) showed that the quantum yield of PSII, as
determined by oxygen evolution, correlates linearly to F.,! F... measurements
under various stress conditions in a wide variety of plants. Genty et al. (1989)
found chlorophyll fluorescence measurements representative of the
photoeffeciency of PSII, as measured by carbon dioxide assimilation. Krause at
al., (1990) found PSII electron transport capacity to be linearly related to F.,! F...
in spinach leaves subjected to photoinhibitory treatments. Edwards and Baker

(1993) found that chlorophyll fluorescence parameters can be used under a wide

14

range Of conditions to accurately predict carbon dioxide assimilation rates in
maize. From studies like these (for review see Lichtenthaler, 1990) the
precedent was set for the use of chlorophyll fluorescence as a stress detection
and quantification tool.

Numerous studies have been done involving heat stress and its effect on
photosynthesis. Georgieva and Yordanov (1993) used FJ F... values to study
heat tolerance in pea. The effects of heat stress on cation-induced chlorophyll
fluorescence rises in pea have been studied by Velitchkova and lvanov (1993).
Bilger et al. (1984) found heat-induced fluorescence increase to be a suitable
indicator of heat dosage accumulation to lethal levels in a variety of vascular
plants. Havaux (1992) used chlorophyll fluorescence to study the combined and
separate effects of heat and water stress and found the combination of stresses
to have less damaging effects on PSII than heat streSs alone. Ruter has shown
F.,! F... to be an effective measurement of heat stress in various holly cultivars
during heat tolerance experiments (Ranney and Ruter, 1997; Ruter, 1993).
Likewise, Ranney and Peat (1994) used chlorophyll fluorescence to examine
heat tolerance in five birch taxa.

Chlorophyll fluorescence studies have also dealt with cold stresses,
including tolerance, frost effects, acclimation, and photoinhibition interactions.
One possible way of measuring freezing tolerance, in terms of photosynthetic
functionality, is the reduction of fluorescence parameters after short duration
exposure to freezing temperatures. Frost tolerance in different birch subspecies

has been examined using chlorophyll fluorescence (Hallgren at al., 1982) and

15

Smillie and Hetherington (1983) used fluorescence values to examine chilling
tolerance in wheats and citrus crops. During at al. (1990) found F.,! F...
measurement of frost injury in grapevine buds to be comparable to visual
estimation of injury. Wrth an acclimation period, readings remained at a much
healthier level and injury was reduced. In plants adjusted to low temperatures, it
seems to be the proportion of PSII reaction centers operating that is reduced
rather than a change in the reaction centers themselves (Huner at al., 1993).
Gray at al. (1997) found that, in cold acclimated plants, the increased excitation
pressure on PSII itself was enough to influence the expression of a nuclear gene
involved in cold acclimation. During acclimation of rape to 2.5 °C , F... and F.
values were found to drop dramatically in a two-day period, and than level off and
recover when removed to 20 °C for 2-3 days (Maciejewska and Bauer, 1992).

F isker et al. (1995) found chlorophyll fluorescence to be an accurate estimate of
needle freezing damage and seedling survival in Douglas fir and could detect
non-visible damage, however, fluorescence measurements were unable to
predict cold hardiness prior to the temperature treatments. Likewise, Welander
at al. (1994) were unable to use chlorophyll fluorescence to predict growth
response after a night of frost followed by high irradiation. Lindgren and Hallgren
(1993) found chlorophyll fluorescence to be an effective method for the detection
of freezing injury and ranking of cold acclimation in lodgepole pine and Scotch
pine, and could be done one week earlier than a visual assessment. Cold
storage has been studied in white spruce (VIdaver at al., 1989) and low

temperature effects on Scotch pine (Hallgren at al, 1990; reviewed in Lindgren

16

and Hallgren, 1993).

Photoinhibition studies frequently overlap with cold stress studies. A
number of studies found decreasing temperature to lead to a decrease in amount
of light needed to saturate photosynthesis (F.,) (Hallgren et al., 1982). F.,! F...
values were found to decrease (increasing fluorescence) in Scotch pine (Hallgren
et al., 1990) and spruce (Welander et al., 1994; Westin et al., 1995) the day afler
a night frost. Cold temperatures have been found to specifically predispose a
plant to photoinhibition and Greer's studies with combinations of dark and light
cold treatments suggest that photoinhibition may be the actual mechanism of
cold stress effects on photosynthesis (Greer, 1990). Bolhar-Nordenkampf and
Lechner (1988) identify photoinhibition, along with membrane disintegration at
temperatures below -4 °C, as the two actual mechanisms of cold damage to
photosynthesis. Cold acclimation was found to increase resistance to
photoinhibition in spinach, limiting photoinhibition to high light intensities
(Somersalo and Krause, 1988).

Although normally associated with cold stress, photoinhibition can occur in
otherwise unstressed plants (i.e. Salix in this study) on sunny summer days
(Ogren and Oquist, 1988). Bilger et al. (1995) used chlorophyll fluorescence-
based calculations of the quantum efficiency of PSII and electron transport rate
to study the daily response of beach and cucumber leaves to changing photon
flux densities. Photoinhibition due to continuous illumination combined with
defoliation in sour cheny was examined by Layne and Flora (1993). Despite the

reduction in photosynthetic processes, recovery potential of photoinhibition-

l7

impaired photosynthesis has been demonstrated in numerous studies
(Somersalo and Krause, 1988; Bilger et al., 1995; . . .).

Study of the seasonal changes in chlorophyll fluorescence can help
identify acclimation and dormancy periods. F.,! F... values have been used to
assess the dormancy of Douglas fir (reviewed in Lindgren and Hallgren, 1993).
Scotch and Lodgepole pines were found to have FvIF m values which decreased
from .83 in August, to .77 in September, and .75 in October, and finally
descended to .21 in December (Lindgren and Hallgren, 1993). In a study by
Westin at al. (1995), Nonlvay spruce F., I F... values were found to remain high in
eariy fall, decline from November to April to about .35 - .5, experience a sharp
drop in April presumably due to increasing photoinhibition, and then quickly rise
to high values in May. WInter values were found to fluctuate with local
temperature changes.

Many other environmental stresses have been studied with chlorophyll
fluorescence to a lesser degree than the temperature and light effects described
above. Spruce under limited mineral nutrition showed no affect on Rfd values
over a ten-month study period (Hagg et al., 1992). Saarinen (1993) found low
FvIFm values to be an indicator of pollution in Scots pine in sites with heavy
vehicular traffic and trees located near oil refineries (Saarinen and Liski, 1993).
However, no change in F.,! F... was found in Norway spruce with elevated CO:
and 03 levels, and potassium deficiency (Barnes et al., 1995). Clorophyll
fluorescence has been used to study forest decline in Nonrvay spruce

(Lichtenthaler and Rinderle, 1988) and Bukhov at al. (1990) used it to examine

18

leaf dehydration.

There has been some study of low temperature injury to fruit using
chlorophyll fluorescence. Studies include banana, mango, cucumber, eggplant,
and green bell peppers. F.,! F... measurements were found ineffective at
predicting apple scald susceptibility (Mir et al., 1998a; Mir et al., 1998b).

Little studied is the relationship between chlorophyll fluorescence and
propagation. Van Huylenbroech and Debergh (1992) used fluorescence as a tool
to examine stress levels and stages during the acclimatization period of
micropropagated Transvaal daisy (Gerbera jamesonii Bol. ex Adlam). I have

found no research involving chlorophyll fluorescence and stem propagation.

CHLOROPHYLL FLUORESCENCE POTENTIAL

Chlorophyll fluorescence is developing as valuable tool in the estimation
and measurement Of photosynthetic health. Its non-destructive, rapid, portable,
and objective nature make it useful in scientific and commercial spheres.
Scientific interest has shifted from interest in primary reactions and induction
kinetics to overall electron transport efficiency and steady state reactions
(Schreiber and Bilger, 1993). Its role in stress physiology measurements will
continue to grow. Schreiber and Bilger (1993) emphasize its potential for remote
sensing of photosynthetic health.

During at al., (1990) suggest chlorophyll fluorescence measurements as
an objective tool for estimating frost injury of buds in grapevine.

F iskar at al., (1995) propose chlorophyll fluorescence as a tool for rapidly

19

identifying cold-damaged seedlings in nurseries.

Edwards and Baker (1993) point to the time and labor that could be saved
by estimating carbon dioxide assimilation using chlorophyll fluorescence values.
The technique would also have advantages of measuring the plants in their
normal environment instead of a gas chamber.

Daley (1994) describes a recently completed chlorophyll fluorescence
video system. Using a camcorder and LED illumination, fluorescence can be
visualized over a small leaf area. This allows for the detection of virus lesions
before they are visible to the eye, as well as low virulence strains which
otherwise may never be seen.

Chlorophyll fluorescence may have some use as a fruit-grading tool.
Beaudry at al. (1997 in Mir at al., 1998a) suggest its use as a tool for quality
measurement of stored apples. Mir at al. (1998b) find a potential use in the
sorting of fruit having superficial defects.

TAXUS HISTORY

The genus Taxus has a worldwide distribution in the Northern
Hemisphere. Three to four species are recognized, but the distinctions between
such are more geographical than morphological. English yews belong to 7'.
baccafa L., Japanese yews to T. cuspidata Siebold and Zuccarini, and North
American yews to T. canadensis Marshall (Chadwick and Keen, 1976). A
western North American species is also referred to, T. brevifolia (Heinstein and
Chang, 1 994).

20

Morphologically, these conifers are usually small trees (20 to 40 It.) or
shmbs. Extremely slow growing and long-lived, their average lifespan in the wild
is around 500 years, but many live to be well over 1000 years old (TIttensor,
1980). Needles tend to be a dark and glossy green (except in some cultivated
varieties) and bark is fibrous and reddish. Most plants are dioecious (Chadwick
and Keen, 1976). Pollen cones open in early spring. The arils ripen in early
winter, single-seeded berries with fleshy, scarlet seed coats for bird-dispersal.
Taxus may take up to 70 years to reach sexual maturity (Hulme, 1996). The
needles and seeds are known to be toxic to humans (Chadwick and Keen, 1976),
and have caused problems in cattle (Hulme, 1996).

In medieval Britain, yews were originally used for archery bows, due to
their strong and flexible wood (Chadwick and Keen, 1976), and as land markers
on property lines due to their longevity (Tittensor, 1980). The first mention of a
cultivated yew comes from an English garden description in 1686 (Chadwick and
Keen, 1976). Since then, yews have become a mainstay of gardens everywhere,
with upright, globe, and spreading varieties.

Taxus nomenclature in America is notoriously confused. Cultivated Taxus
is usually of T. baccafa or T. cuspidata origin, or belongs to a hybrid species of
the two, Taxus xmedia Rehder. Nomenclatural confusion can be traced back to
the enactment of Quarantine 37 by the US. government in 1918. These laws
prohibited importation of nursery stock, and growers suddenly faced with a
scarcity of stock or profitable cultivars, resorted to taking cuttings from less that

desirable material and often applied salable cultivar names to such (Chadwick

21

and Keen, 1976). The confusion created still exists today. The Living Herbarium
of Taxus, at the Secrest Arboretum (Ohio Agricultural Research Experiment
Station) was set up in 1942 to try to sort out some of the misnomers, and by
1976 had 141 accessions (Chadwick and Keen, 1976).

One of the studies conducted using material from Secrest, attempted to
use isozyme electrophoresis to distinguish cultivar differences (Greer et al.,
1993). Looking at 51 plants belonging to 21 cultivars, results Often showed
different electrophoretic fingerprints for members of the same cultivar and
identical fingerprints for different cultivars. Extensive nomenclatural problems
exist.

Aside from its traditional value as an ornamental, Taxus was discovered to
harbor great pharmaceutical value in the 1980’s. Taxol, a diterpene extraction
from Taxus bark, needles, twigs, and roots, has been identified as one of the
most promising anti-cancer drugs from a plant source (Heinstein and Chang,
1994). It has been shown to have anti—cancer activity in ovarian, breast, lung,
head, and neck cancers. A complicated chemical structure has stumped the
development of any synthesis process, however, semi-synthesis of taxol can be
performed using another, more abundant Taxus extract, baccatin ll. Due to the
slow growth Of Taxus cell cultures, and rarity of T. brevifolia, the Taxus bark with
highest taxol content, Heinstein and Chang (1994) identify commercial
ornamental nursery stock as the most economical source of taxol and baccatin II

at this time.

22

PROPAGATION OF TAXUS

Propagation of Taxus from seed is possible but slow and would not
conserve the cultivar traits we value today. For seed propagation, Chadwick and
Keen (1976) advise stratifying cleaned seeds at 35 - 50 °F (2 - 10 °C) until the
following October, or planting directly in protected beds for spring germination.

Today Taxus is commercially propagated via stem cuttings. Chadwick
and Keen (1976) suggest four to eight inch cuttings taken in August and placed
in cold storage or cuttings taken in March. Gerald Verkade (1976) of Verkade
Nurseries, New London, Conn, describes eight inch cuttings taken in November
or December, striped of needles on the bottom 2.5 inches for an auxin dip, and
placed in a 1:2 perlite! coarse sand medium. He reports 90 - 95% rooting.
Joseph P. Von Komya (1976) of Bobbink Nurseries, Freehold, NJ, reports using
six inch cuttings for spreading yews and eight inch cuttings for upright yews.
These cuttings are taken after two to four killing frosts and placed in 100% perlite
at 65-68 °F. He stresses the importance of bottom heat.

The difficulty of rooting certain cultivars has been a persistent problem. A
1964 study attempted to link ease of rooting differences in cultivars to sex
differences between cultivars without success (Davidson, 1964). The highest
Taxus rooting percentages in an Italian study (Eccher, 1988) were achieved with
apical cuttings at 20 °C and no bottom heat. Rooting media did not affect rooting
percentages, however, sand/peat mixtures led to thinner, more fibrous roots than
agriperlite. IBA increased rooting speed in most cultivars but did little to raise

long-tenn rooting percentages. Long propagation times were key.

23

PURPOSE OF THE EXPERIMENTS

If a quick, reliable method of determining potential rooting of cuttings
V based on the condition of a specific stock plant was available for propagators,
then rooting success could be predicted prior to an investment in time, labor, and
resources. A reduction in production costs could be realized.

Chlorophyll fluorescence measurement is a tool in plant stress detection
with high potential for commercial application. It has been shown effective in the
detection of environmental and other stresses on a broad range of plant species,
including many conifers.

Two different experiments were performed. The first was a cultivar study
designed to look at the differences in chlorophyll fluorescence among ten
cultivars of Taxus, to study the changes in chlorophyll fluorescence over the
course of propagation, and to correlate chlorophyll fluorescence and rooting
among cultivars.

The second study examined different storage conditions, quantifying
stress in relation to subsequent rooting by using chlorophyll fluorescence
measurements. Seven different temperatures, three desiccation levels, and

three time durations were studied.

24

LITERATURE CITED

Adams, W.W. III, B. Demmig-Adams, K Winter, and U. Schreiber. 1990. The
ratio of variable to maximum fluorescence from photosystem II, measured in
leaves at ambient temperature and at 77K as an indicator of the photon yield of
photosynthesis. Planta 1 80: 166-1 74.

Barnes, J.D., T. Pfinmann , K Steiner, C. Lutz , U. Busch , H. Kuchenhoff , and
H.D. Payer. 1995. Effects of elevated 002 and potassium deficiency in Norway
Spruce (Picea abies (L.)Karst.): Seasonal changes in photosynthesis and non-
structural carbohydrate content. Plant, Cell, and Environ. 18:1 345-1357.

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

Bilger, W., U. Schreiber, and M. Bock. 1995. Determination of the quantum
efficiency of photosystem II and of non-photochemical quenching of chlorophyll
fluorescence in the field. Oecologia 102:425-432.

Bilger, H.W., U. Schreiber, and O. Lange. 1984. Determination of leaf heat
resistance: comparative investigation of chlorophyll fluorescence changes and
tissue necrosis methods. Oecologia 63:256-262.

Bjorkman, O. and B. Demmig. 1987. Photon yield of 02 evolution and chlorophyll
fluorescence characteristics at 77K among vascular plants of diverse origins.
Planta 170:489-504.

Blaich, R. 1988. Early detection of damage conditions in plants by delayed
chlorophyll fluorescence, p. 223-228. In: H.K Lichtenthaler (ed.). Applications of
Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht, The Netherlands.

Bolhar-Nordenkampf, HR. and E. Lechner. 1988. WInter stress and chlorophyll
fluorescence in Norway spruce, p. 173-180. In: H.K Lichtenthaler (ed.).
Applications of Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht,
The Netherlands.

Bolhar-Nordenkampf,H.R., S.P. Long, N.R. Baker, G. Oquist, U. Schreibers, and
E.G. Lechner. 1989. Chlorophyll fluorescence as a probe of the photosynfl'Ietic
competence of leaves in the field: a review of current instrumentation. Functional
Ecol. 3:491-514.

Brodribb, TM. and RS. Hill. 1997. Light response characteristics of a
morphologically diverse group of Southern Hemisphere conifers as measured by
chlorophyll fluorescence. Oecologia 1 10: 1 0-1 7.

25

 

Bukhov, N.G., P. Mohanty, and SS. Sabat. 1990. Effect of leaf rehydration on
kinetics of variable chlorophyll fluorescence. Doklady Adademii Nauk SSR Sept
1989, 308(1):251-255. In: Plant Physiology, Plenum Press, p. 73—76.

Chadwick, LC. and RA Keen. 1976. A Study of the genus Taxus. Ohio Agr.
Res. Dev. Ctr. Mar. Bul. 1086.

Coombs, J., D.O. Hall, S.P. Long, and J.M.O. Scurlock (eds). 1985. Techniques
in Bioproductivity and Photosynthesis. 2nd ed., Pergamon Press, N.Y.

Daley, PF. 1995. Chlorophyll Fluorescence analysis and imaging in plant stress
and disease. Can. J. Plant Pathol. 17: 1 67-1 73.

Davidson, H. and A Olney. 1964. Clonal and sexual differences in the
propagation of Taxus, p. 156-161. In: lntemational Plant Propagator's Society
Combined Proceedings, vol. 14.

During, H., T.V. Ortoidze, and B. Bushnell. 1990. Effects of subzero
temperatures on chlorophyll fluorescence of grapevine buds. J. Plant Physiol.
1 36:758-760.

Eccher, T. 1988. Response of cuttings of 16 Taxus cultivars to rooting
treatments. Acta Horticulturae 227:251-253.

Edwards, GE. and NR. Baker. 1993. Can CO2 assimilation in maize leaves be

predicted accurately from chlorophyll fluorescence analysis? Photosyn. Res.
37:89-102.

Fisker, S., R. Rose, and D.L. Haase. 1995. Chlorophyll fluorescence as a
measure of cold hardiness and freezing stress in 1+1 Douglas-Fir seedlings.
Forest Sci. 41 (3):564-575.

Garrett, RH. and CM. Grisham. 1995. Biochemistry. Sounders College Publ.,
Fort Worth, T.X.

Genty, 8., J.M. Briantais, and NR. Baker. 1989. The relationship between the
quantum yield of photosynthetic electron transport and quenching of chlorophyll
fluorescence. Biochim. Biophy. Acta 990:87-92.

Georgieva, K and l. Yordanov. 1993. Temperature dependence of chlorophyll
fluorescence parameters of pea seedlings. J. Plant Physiol. 142:151-155.

Gray, G.R.; LP. Chauvin, F. Sarhan, and N.P.A. Huner. 1997. Cold acclimation
and freezing tolerance: A complex interaction of light and temperature. Plant
Physiol. 114:467-474.

 

Greer, C.E., R.E. Schutzki, A Fernandez, and J.F. Hancock. 1993.
Electrophoretic characterization of Taxus cultivars. HortTechnol. 3(4):430-433.

Greer, DH. 1990. The combined effects of chilling and light stress on
photoinhibition of photosynthesis and its subsequent recovery. Plant Physiol.
Biochem. 28(4):447455.

H899. C., F. Stober, and H.K Lichtenthaler. 1992. Pigment content, chlorophyll
fluorescence and photosynthetic activity of spruce clones under normal and
limited mineral nutrition. Photosynthetica 27(3):385-400.

Hallgren, J.E., T. Lundmark, and M. Strand. 1990. Photosynthesis of Scots pine
in the field after night frosts during summer. Plant Physiol. Biochem. 28(4):437-
445.

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

Hansatech manual. 1996. Hansatech Instruments Ltd., Norfolk, England, UK

Havaux, M. 1992. Stress tolerance of photosystem II in vivo: Antagonistic effects
of water, heat, and photoinhibition stresses. Plant Physiol. 100:424-432.

Heinstein, PF. and OJ. Chang. 1994. Taxol. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 45:663-674.

Holzwarth, AR. 1988 Time resolved chlorophyll fluorescence, p. 21 -31. In: H.K
Lichtenthaler (ed.). Applications of Chlorophyll Fluorescence. Kluwer Academic
Publ., Dordrecht, The Netherlands.

Hulme, PE. 1996. Natural regeneration of yew (Taxus baccata L.): microsite,
seed, or herbivore limitation? J. Ecol. 84:853-861.

Huner, N.P., G. Oquist, V.M. Hurry, M. Krol, S. Falk, and M. Griffith. 1993.
Photosynthesis, photoinhibition and low temperature acclimation in cold tolerant
plants. Photosyn. Res. 37:19-39.

Krause, G.H., S. Somersalo, E. Zumbusch, B. Weyers, and H. Laasch. 1990. On
the mechanism of photoinhibition in chloroplasts: Relationship between changes
in fluorescence and activity of photosystem II. J. Plant Physiol. 136:472-479.

Krause, G.H. and E. Weis. 1991. Chlorophyll fluorescence and photosynthesis:
The basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:313-349.

Lavorell, J. and AL. Ettiene. 1977. In vivo chlorophyll fluorescence, p. 203-268.

27

In: J. Barber (ed.). Primary Processes in photosynthesis. Elsevierl North Holland
Biomedical Press, Amsterdam, The Netherlands.

Layne, DR. and J.A. Flore. 1993. Physiological responses of Prunus oerasus to
whole-plant source manipulation, leaf gas exchange, chlorophyll fluorescence,
water relations, and carbohydrate concentrations. Physiol. Plant. 88:44-51.

Lichtenthaler, H.K (Ed.) 1988a. Applications Of Chlorophyll Fluorescence, p. 1-
12. Kluwer Academic Publ., Dordrecht, The Netherlands.

Lichtenthaler, H.K 1988b. In vivo chlorophyll fluorescence as a tool for stress
detection in plants, p. 129-142. In: H.K Lichtenthaler (ed.). Applications of
Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht, The Netherlands.

Lichtenthaler, H.K 1988c. Remote sensing of chlorophyll fluorescence in
oceanography and in terrestrial vegetation: an introduction, p. 287-297. In: H.K
Lichtenthaler (ed.). Applications of Chlorophyll Fluorescence. Kluwer Academic
Publ., Dordrecht, The Netherlands.

Lichtenthaler, H.K 1990. Applications of chlorophyll fluorescence in stress
physiology and remote sensing, p. 278-305. In: M.O. Steven and J.A. Clark
(eds.). Applications of Remote Sensing in Agriculture. Buttenlvorths, London.

Lichtenthaler, H.K and U. Rinderle. 1988. Chlorophyll fluorescence signatures as
vitality indicator in forest decline research, p. 143-149. In: H.K Lichtenthaler
(ed.). Applications of Chlorophyll Fluorescence. Kluwer Academic Publ.,
Dordrecht, The Netherlands.

Lindgren, K and J.E. Hallgren. 1993. Cold acclimation of Pinus contorta and
Pinus sylvestris assessed by chlorophyll fluorescence. Tree Physiol. 13:97-106.

Long, S.P., S. Humphries, and PG. Falkowski. 1994. Photoinhibition of
photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:633-662.

Maciejewska, U. and H. Bauer. 1992. Effects of cold acclimation on chlorophyll
fluorescence in winter rape leaves. Photosynthetica 27(4):559-562.

Melis, A, G.E. Guenther, P.J. Morrissey, and ML. Ghirardi. 1988. Photosystem ll
heterogeneity in chloroplasts, p. 33-43. In: H.K Lichtenthaler (ed.). Applications
of Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht, The
Netherlands.

Mir, N., M. Wendorf, R. Perez, and RM. Beaudry. 1998a. Chlorophyll

fluorescence in relation to superficial smld development in apple. Amer. Soc.
Hort Sci. 123(5):887-892.

28

Mir, N., M. Wendorf, R. Perez, and RM. Beaudry. 1998b. Chlorophyll
fluorescence as affected by some superficial defects in stored apples. J. Hort
Sci. Biotech. 73(6):846-850.

Morgan CF-1000 manual. 1992. P.K Morgan Instruments, Inc., Andover, MA,
USA

Ogren, E. and G. Oquist. 1988. Screening for photoinhibition of photosynthesis in
the field using a portable fluorometer, p. 165-172. In: H.K Lichtenthaler (ed.).
Applications of Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht,
The Netherlands.

Papageorgiou, G. 1975. Chlorophyll fluorescence: An intrinsic prove of
photosynthesis, p. 320-366. In: Govindjee (ed.). Bioenergetics of Photosynthesis.
Academic Press, N.Y. ‘

Ranney, T.G. and MM. Peat. 1994. Heat tolerance of five taxa of birch (Betula):
Physiological responses to supraoptimal leaf temperatures. J. Amer. Soc. Hort.
Sci. 1 19(2):243-248.

Ranney, T.G. and J.M. Ruter. 1997. Foliar heat tolerance of three holly species
(Ilex spp.): Responses of chlorophyll fluorescence and leaf gas exchange in
supraoptimal leaf temperatures. J. Amer. Soc. Hort. Sci. 122(4):499-503.

Rinderle, U., and H.K Lichtenthaler. 1988. The chlorophyll fluorescence ratio
Fag/F735 as a possible stress indicator, p. 189-196. In: H.K Lichtenthaler (ed.).
Applications of Chlorophyll Fluorescence. Kluwer Academic Publ., Dordrecht,
The Netherlands.

Ruter, J.M. 1993. Foliar heat tolerance of two hybrid hollies. HortSci. 28(6):650-
652.

Saarinen, T. 1993. Chlorophyll fluorescence, and nitrogen and pigment content
Of Scots pine (Pinus sylvestris) needles in polluted urban habitats. Ann. Bot.
Fennici 30:1-7.

Saarinen, T. and J. Liski. 1993. The effect of industrial air pollution On chlorophyll
fluorescence and pigment contents of Scots pine (Pinus sylvestris) needles.
Euro. J. For. Pathol. 23:353-361.

Schmidt, W. 1988. Long term delayed luminescence in green organisms, p. 217-
22.1. In: H.K Lichtenthaler (ed.). Applications of Chlorophyll Fluorescence.
Kluwer Academic Publ., Dordrecht, The Netherlands.

Schreiber, U. and W. Bilger. 1993. Progress in chlorophyll fluorescence research:

Major developments during the past years in retrospect, p. 151-169. In: Progress

29

in Botany, vol. 54. Springer-Verlag, Berlin.

Smillie, RM. and SE. Hetherington. 1983. Stress tolerance and stress-induced
injury in crop plants measured by chlorophyll fluorescence in. viva: Chilling,
freezing, ice cover, heat, and high light. Plant Physiol. 72:1043-1050.

Somersalo, S., and G.H. Krause. 1988. Changes in chlorophyll fluorescence
related to photoinhibition of photosynthesis and cold acclimation in green plants,
p. 157-164. In: H.K Lichtenthaler (ed.). Applications of Chlorophyll Fluorescence.
Kluwer Academic Publ., Dordrecht, The Netherlands.

Tittensor, R. 1980. Ecological history of yew, Taxus baccafa L., in southern
England. Biol. Conserv. 17:243-265.

Van Huylenbroech, J. and P. Degergh. 1992. Acclimation of micropropagated
Gerbera jamesonii, Use of chlorophyll fluorescence. Forum for Applied
Biotechnology. Brugge (Belgium). 24-25 Sept. Mededelingen-van-de-Faculteit-
Landbouwwetenschappen-Rijksuniversiteit-Gent (Belgium). 57(4a):1595-1579.

Van Kooten, O. and J.F.H. Snel. 1990. The use of chlorophyll fluorescence
nomenclature in plant stress physiology. Photosyn. Res. 25:147-150.

Velitchkova, MY. and AG. lvanov. 1993. Effects of short-time heat stress on the
parameters of cation induced increase in chlorophyll fluorescence in pea
thylakoid membranes. J. Plant Physiol. 142:144-150.

Verkade, G. 1976. Propagation Of Taxus by cuttings, p. 177. In: lntemational
Plant Propagator's Society Combined Proceedings, vol 26.

Vidaver, E., W. Binder, R. Brooke, G. Lister, and P. Toivonen. 1989. Assessment
of photosynthetic activity of nursery grown Picea glauca seedlings using an
integrating fluorometer to monitor variable chlorophyll fluorescence. Can. J. For.
Res. 19:1478-1482.

Von Komya, J.P. 1976. Propagation of Taxus cuttings, pp. 178-179. In:
lntemational Plant Propagator’s Society Combined Proceedings, vol. 26.

Welander, N.T., P. Gemmel, O. Hallgren, and B. Ottosson. 1994. The
consequences of freezing temperatures followed by high irradiance on in vivo
chlorophyll fluorescence and growth in Picea abies. Physiol. Plant. 91 :121-127.

Westin, J., L.G. Sundblad, and J.E. Hallgren. 1995. Seasonal variation in

photochemical activity and hardiness in clones of Norway spruce (Picea abies).
Tree Physiol. 15:685-689. _

30

Chapter One
Chlorophyll Fluorescence. and Vegetative Propagation Of Taxus

(Formatted according to publication guidelines of the American Society of
Horticultural Science)

31

Chlorophyll Fluorescence and Vegetative Propagation of Taxus

S. E. Bruce‘ and D. B. Rowe2
Department of Horticulture, Michigan State University, East Lansing, MI 48824

Received for publication . Accepted for publication . This paper is a
portion of a MS. thesis submitted by S. E. Bmce. '

Acknowledgement: This experiment was a Michigan State University study
funded by: Zelenka Nursery in Grand Haven, MI, International Plant Propagator's
Society, Michigan Nursery and Landscape Association, and Michigan Agricultural
Experiment Station.

‘Graduate research assistant
2Assistant professor

32

 

Propagation and Tissue Culture

Chlorophyll Fluorescence and Vegetative Propagation of Taxus

Additional index words. Yew, stem cuttings

Abstract. Chlorophyll fluorescence (FJF...) over the course of stem cutting
propagation was examined in ten cultivars of Taxus xmedia (Taxus baccata L. x
7'. cuspidata Sieb. 8 Zucc.) including Brownii, Dark Green Pyramidalis, Dark
Green Spreader, Densiformis, Densifonnis Gem, Hicksii, L.C. Bobbink, Runyan,
Tauntoni, and Wardii. The study‘s objective was to examine chlorophyll
fluorescence as a method for stock plant selection and monitoring of propagation
in the various cultivars. Differences were found in FJF... among cultivars, initially
and over time, however, there was significant overlapping between some
cultivars. FJF... was found to decrease dramatically during cold storage, but
usually returned to original levels after several weeks in the propagation beds.
This seemed to be more a reflection of ambient temperatures than actual rooting.
Correlations were not found between initial stock plant F.,/F... and rooting
percentage, root number, root dry weight, or root length, indicating that FJF... is

not a reliable indicator Of stock plant propagation potential.

33

 

Yew (Taxus sp. L.) is a mainstay of ornamental gardens with its dark,
evergreen needles and red winter arils. In the mid 80’s great pharmaceutical
value was added to its list of virtues with the discovery of taxol, a diterpene found
in its bark, needles, twigs, and roots, and identified as one of the most promising
anti-cancer dmgs from a. plant source (Heinstein and Chang, 1994). The nursery
industry supplies Taxus for both ornamental and pharmaceutical industries and
growers face great propagation pressures. Propagation is usually done via stem
cuttings, taken from stock plants in earty fall, and kept in cold storage until placed
in propagation beds in mid-winter. Adventitious rooting can be unpredictable, with
some cultivars persistently difficult to root. Davidson (1964) unsuccessfully
attempted to link ease of rooting differences in cultivars to sex differences (Taxus
spp. are largely dioecious). Eccher (1988) reported long propagation times to be
key to rooting of Taxus. Since nursery propagation failures are often related to
stock plant material, interest arose in a method for evaluating stock plant quality
prior to collection of cuttings.

Chlorophyll fluorescence measurements may be a potential method for
evaluating stock plants. Chlorophyll fluorescence is created when light energy,
absorbed by chlorophyll a, exceeds the photochemical processing capacity of
photosystem II (PSII). One way this ‘extra’ energy is dissipated is by being re-
emitted as light, which we call chlorophyll fluorescence. The fluorescence
measured at physiological temperatures is largely a product of chlorophyll a
molecules involved in PSII, although other light capturing pigments do fluoresce,

and PSI fluorescence can be measured. Since chlorophyll fluorescence levels

34

are tied to the amount of light energy not used for photosynthetic processes, they
are inversely related to the amount of energy that is used for photosynthesis, and
serve as indicators of plant photosynthetic potential.

Chlorophyll fluorescence measurement is increasingly used as a
quantitative measure of photosynthetic health. The emitted light signal follows a
general intensity pattern known as the Kautsky Effect. Pre-darkened samples,
with a minimum fluorescence level (F.), show a "fast rise’ in fluorescence to a
maximum value (F...) upon exposure to a light source. As photochemical
processing of the light energy increases, fluorescence values are gradually
reduced to a steady state (F.), somewhere between F. and F.... A common
parameter used in stress studies is FJF... F., being the variable fluorescence,
calculated by subtracting F. from F.... Numerous studies have shown FJF..., and
other chlorophyll fluorescence parameters, to be effective measurements of the
photoefficiency of PSII. Bjorkman and Demmig (1987) linearty correlated FJF...
with the quantum yield of PSII, as determined by oxygen evolution, in a variety of
stressed plants.

Studies utilizing chlorophyll fluorescence to quantify plant stress have
dealt with heat and cold stress, especially tolerance studies, photoinhibition,
mineral nutrition, pollution, and water stress. Studies on conifers have found
effects of vehicle and oil refinery pollution on Scotch pine (Pinus sylvestris L.)
(Saarinen, 1993; Saarinen and Liski, 1993), photoinhibition effects in Scotch pine
(Hallgren at al., 1990), and forest decline and photoinhibition effects in Norway
spruce (Picea abies (L.) Karst.)(Lichtenthaler and Rinderle, 1988; Welander et

35

al., 1994). Seasonal changes in F.,/F... values have been used to assess
dormancy in Douglas fir (Pseudotsuga menziesii (Mirbel) Franco) (Hawkins and
Lister, 1985), Scotch and lodgepole pines (Pinus contorta Dougl. ex Loud.)
(Lindgren and Hallgren, 1993), and Norway spruce (Westin et al., 1995). Fisker
at al. (1995) found chlorophyll fluorescence to be an accurate estimate of freeze
damage in needles and seedling survival in Douglas fir, and could detect non-
visible damage. However, fluorescence measurements were unable to predict
cold hardiness prior to the temperature treatments. Likewise, Welander et al.

(1994) were unable to use chlorophyll fluorescence to predict growth response

 

after a night frost followed by high irradiation. 'I
The only fluorescence study involving propagation examines stress levels

during the acclimatization of micropropagated Transvaal daisy (Gerbera

jamesonii Bol. ex Adlam) (Van Huylenbroech and Debergh, 1992). To our

knowledge, no studies of Taxus or stem cutting propagation have involved

chlorophyll fluorescence. Therefore, the Objective of this study was to examine

the differences in chlorophyll fluorescence among ten cultivars of Taxus xmedia

(Taxus beccata L. x 7'. cuspidata Sieb. & Zucc.), to study changes in chlorophyll

fluorescence over the course of propagation in these cultivars, and to correlate

initial chlorophyll fluorescence values with rooting.
MATERIALS AND METHODS

Ten cultivars of Taxus xmedia were selected for this two year study:

Brownii, Dark Green Pyramidalis (first year only), Dark Green Spreader,

36

Densiformis, Densifonnis Gem, Hicksii, L.C. Bobbink, Runyan, Tauntoni, and
Wardii. Trials were performed at two locations, Michigan State University, East
Lansing, MI (MSU) and Zelenka Nursery, Grand Haven, MI, for the first year of
the study, and solely at Zelenka Nursery the second year.

Propagation procedures were similar for all trials. In early fall, 15 - 20 cm
cuttings were taken from field grown plants at Zelenka Nursery. Cuttings were
bagged in plastic and placed in cold storage at various temperatures. Needles
were taken for initial chlorophyll fluorescence readings prior to placement in cold
storage. After 4 - 5 weeks in cold storage, cuttings were cut to 11.4 cm (apical
and basal portions removed), treated with Woods Rooting Hormone (IBA 1.03%;
NAA 0.66%) at 2800 ppm (1 :5 ratio), and placed into 100% perlite. Cuttings were
placed in rows 3.8 cm apart with 1.3 cm between individual cuttings within each
row. Cuttings were watered as needed until harvest in the spring, at which time
they were gently uprooted, and root lengths rated (0=less than 2.54 cm [1 inch],
1=less than 5.08 cm [2 inch], 2=less than 7.62 cm [3 inch], 3=greater than 7.62
cm), and root numbers recorded. Roots were dried at 28 °C for five days and
weighed (Explorer Balance, Ohaus Corp., Florham Park, NJ). Chlorophyll
fluorescence measurements were recorded periodically throughout the
propagation process.

Greenhouse conditions differed between the two locations. Zelenka
propagation benches were 15 cm deep and received bottom heat. MSU growth

trays were 7.6 cm deep and received no bottom heat. Greenhouse temperatures

at Zelenka were maintained at 18 °C, while MSU temperatures were difficult to

37

control, often exceeding 21 °C and sometimes reaching 32 °C during a sunny
day.

The 1997-1998 trial at MSU consisted of a randomized complete block
design of six blocks and ten cultivars, for a total of 600 cuttings. Cuttings were
taken Oct. 14 and stored in cold storage at MSU (5 °C) until stuck on Nov. 22.
Harvest took place May 21. Chlorophyll fluorescence readings were taken, ten
per cultivar, on Oct 14, Oct. 30, and Nov. 18. Four readings were taken per
cultivar, per block on Dec. 3, Dec. 18, Jan. 6, Jan. 29, Mar. 3, April 6, and May 6.

The 1997-1998 trial at Zelenka Nursery was designed similarly to the MSU
trial, however, only four of the Six blocks were harvested. Cuttings were taken
Oct. 14, stored in cold storage at Zelenka in open buckets at 2.5 °C, and stuck
Nov. 20. Harvest took place May 27. Chlorophyll fluorescence readings were
taken, ten per cultivar, on Oct. 14, Oct. 30, and Nov. 20. Four readings were
taken per cultivar, per block, on Dec. 4, Jan. 8, Jan. 30, Feb. 27, April 3, and May
7.

The 1997-1998 chlorophyll fluorescence measurements were taken with a
Morgan CF-1000 (P.K Morgan Instruments, Inc., Andover, MA). For each
measurement (F.,/Fm). the underside of a single, randomly selected needle was
measured at a light level of 700 mol Imzls. Samples were dark adapted for 15
minutes before measurement in the manufacturer's plastic/foam clips.
Chlorophyll fluorescence measurements were usually taken the same day as the
needles were collected, although occasionally logistics required needles to be

stored overnight. This was accomplished in a germination tray covered with moist

38

paper towels and stored at 5 °C. Needles were acclimated to room temperature
at least one hour prior to fluorescence measurements. No distortion of values
was observed as a result of overnight storage.

The 1998-1999 trial at Zelenka was performed similarly to the previous
trials with a few exceptions. Cuttings were taken Oct. 29 and a fluorescence
reading taken for each individual cutting. Cuttings were rolled in plastic, to
preserve their order, and placed in MSU cold storage (5°C) until Dec. 1, when
they were stuck at Zelenka. The experimental design was a randomized
complete block with four blocks and nine cultivars, using 540 cuttings total. Dark
Green Pyramidalis was not tested due to a shortage of material. Chlorophyll
fluorescence readings were taken on each individual cutting on Oct. 30, Nov. 30,
and Mar. 10, and a sample of 6 readings per cultivar, per block, was taken Jan. 7
and Feb. 3. Needles for all tests were randomly selected. Cuttings were
harvested Mar. 10, which was three months earlier than the previous year in
efforts to measure greater differences in rooting percentage between cuttings.

The 1998-1999 chlorophyll fluorescence measurements were taken with a
Plant Efficiency Analyzer (PEA) (Hansatech Instruments Ltd., Norfolk, England,
UK). Samples were dark adapted for 15 minutes in the manufacturer‘s
plasticlfoam clips, and a fluorometer light level of 1200 umollm’ls (40% of
maximum capacity) was determined sufficient to saturate PSII, according to the
manufacturer's instructions.

Statistical Analysis. Data was subjected to statistical analysis consisting of

ANOVA and LSD tests performed on each trial of the harvest and chlorophyll

39

fluorescence data. Correlation calculations were done between individual cutting
chlorophyll fluorescence and harvest data, overall and within cultivars, for the
1998 -1999 season (proc anova, proc corr, SASIPC software, SAS Institute Inc.,
Cary, NC).

RESULTS AND DISCUSSION
Initial Chlorophyll Fluorescence

Differences were found in FJF... values among cultivars for the 1997-1998
data (T able 1), however no individual mean was different from all others. Values
ranged from .730 (relative units) for Wardii to .873 for Dark Green Pyramidalis.
Subsequent readings for the 1997-1998 year never exceeded these initial values.

Similarly, differences were found among cultivars for the 1998-1999
season, but cultivar ranges overlapped extensively (T able 2). Values ranged from
.778 for Brownii to .833 for Dark Green Spreader. Numbers are higher than for
the 1997-1998 season, however, betvveen-year comparisons are dangerous
because different fluorometers were used for each season.

These readings suggest relatively healthy cutting material. Bjorkman and
Demmig (1987) found the FJF... ratio of unstressed, healthy conifers to be about
.853 +l- .004. It is possible that larger cultivar differences may have been found if
a non-ratio parameter had been used to measure chlorophyll fluorescence, such

as F. or F...

ChlorOphle Fluorescence Over the Course of Propagation

Changes in FJF... over the course of propagation were measured biweekly
and then monme in the first year trials at Zelenka (Fig. 1) and MSU (Fig. 2), and
monthly in the second year trial at Zelenka (Fig. 3). Generally, F.,/F... starts out
high, declines over the cold storage duration and sticking, and than increases as
rooting occurs and growth resumes.

The 1997-1998 Zelenka data (Figure 1) represents the most ideal
environmental conditions of the three trials. There is a clear decline in FJF... over
the 37 days of storage as the cuttings enter dormancy. This decline continues for
more than 13 days after sticking, pointing to the creation of new stresses in the
planting process. Rooting occurred approximately 75 days after severance from
the stock plant, at which point an increase in F.,/F... is observed. This increase
continues as a general trend overall, until harvest when there is a slight drop in
FJF... This drop could be caused by any number of factors associated with the
warm, sunny May weather (photoinhibition, heat stress) or transplanting
preparation (water stress).

The 1997-1998 MSU data shows a different picture (Fig. 2). There is a
decrease in FJF... over cold storage (39 days), however, it is not as consistent as
in me Zelenka trial, perhaps due to the warmer storage conditions at MSU (5 °C
vs. 2.5 °C). Readings increased quickly after sticking and peaked by day 50, long
before rooting occurred. Readings then show a slight, gradual decline until

harvest. This pattern may be a reflection of the wanner greenhouse conditions at

41

MSU, where days were commonly above 21 °C. As a result, cuttings resumed
needle growth before rooting, which occurred over a month later than at Zelenka.
Although there were fewer data collection dates in the 1998 -1999 Zelenka
trial, it shows a new picture as wall (Fig. 3). A clear decrease in F.,/F... can be
seen during the 32 storage days. At sticking, we see the most pronounced FJF...
differences in cultivars of any time during the trial (T able 3). In contrast to the
other bials, which started out with their highest chlorophyll fluorescence levels,
these F.,/F... values are higher 38 days after sticking than at any other point
measured in the trial. A slight decline in values continues until harvest, when
cultivar differences are, once again, indistinct (T able 4). The rapid increase in
photosynthetic efficiency after sticking may be a result of the unseasonably warm
and sunny weather during the 1998-1999 spring, disabling Zelenka from keeping

greenhouse temperatures as cool as desired.

Rooting Data

Root dry weight, number, and length data provide information on the
quality of the rooted cutting, which could influence future growth when planted in
the field as liners. Root dry weight, root number, and root length were correlated
so only data for root dry weight are presented.

The 1997-1998 MSU harvest data presented here includes rooting
percentages (T able 5), and root dry weights (T able 6). Differences in these
measurements were seen among cultivars, however, individual cultivar means

were generally indistinct Rooting pemntages ranged from a very low LC.

42

Bobbink (31.7%) to Densifonnis (96.7%), with much variation within cultivars.
Root dry weight values ranged from .042 g (L.C. Bobbink) to .225 g (Densifonnis
Gem) and root numbers from 2.5 (L.C. Bobbink) to 15 (Dansifonnis).

The 1997-1998 Zelenka harvest resulted in extremely high rooting
percentages. There were no detectable differences in rooting percentages
among cultivars, which ranged from 97.5 to 100% rooting (data not presented).
Cultivar differences were found with dry weight values, and root length ratings,
which were both slightly higher than at MSU, and root numbers, which were twice
as high as at MSU (data not presented). These results may be a response to the
more favorable environmental conditions at Zelenka, where the lack of
environmental stress did not allow for separation among cultivars in regards to
tolerance as it did at MSU. Increases may reflect the much earlier rooting and
depth of the propagation beds at Zelenka Nursery.

The 1998-1999 Zelenka cuttings were harvested 132 days after cutting,
instead of 225 as in the previous year, in an attempt to find larger differences in
the harvest data. Rooting percentages ranged from 45.0% (Densifonnis) to
96.6% (Brownii) (Table 7). Dry weights (T able 8), root numbers, and root lengths
(data not presented) were lower than previously, likely due to the earlier harvest
data. Even though Dark Green Spreader (48.5%), Runyan (46.7%), and
Densiforrnis (45.0%) rooted poorly, the results from the previous year suggest
that with sufficient time, they would have rooted in high percentages.

Unexpectedly, the relationships between cultivars differed greatly in all

three trials. The lowest rooting cultivars at MSU during 1997 - 1998 were some of

43

the highest rooting cultivars at Zelenka during the 1998 - 1999 trial.
Environmental or environmental/cultivar interactions seem to play greater roles
determining chlorophyll fluorescence and harvest characteristics than cultivar
genetics. One can only speculate on the many environmental factors which may
have differed between the trials, years, and locations. Differing greenhouse
conditions were surely a factor between the two locations and the two years were
accompanied by seasonal weather differences. Cultivar traits affecting timing of
rooting may have come into play in the early harvest imposed the second year
and results may not be representative of what final May harvest results would

have been.

Correlations

For the 1998-1999 data, each individual cutting is associated with a
specific collection, sticking, and harvest F.,/F... reading, and specific harvest data.
No strong correlations were found, experiment-wide (T able 9) or within-cultivar
'(T able 10) between any of the FJF... measurements and any of the harvest data.
However, some very weak correlations existed at the P<= .05 level. In the
experiment as a whole, correlations existed between initial chlorophyll
fluorescence and root number (-.142) and root rating (-.208), chlorophyll
fluorescence at time of sticking and dry weight (.103), and harvest chlorophyll
fluorescence and rooting percentage (.127). At the cultivar level, L.C. Bobbink
showed a correlation of initial chlorophyll fluorescence and root rating (-.269).

Chlorophyll fluorescence at sticking was found correlated with root number in

Hicksii (.326). Harvest chlorophyll fluorescence was found correlated with rooting
percentage (.395) and root number (-.796) in Dark Green Spreader, rooting
percentage (.430) in Hicksii, and rooting percentage (.274) in Tauntoni. These

correlations, althth statistically significant, are too small to be useful.

Conclusions

Although differences in FJF... exist among cultivars, they are not
sufficiently distinct to enable chlorophyll fluorescence to be used as a cultivar
identification tool. Since all cuttings showed initially relatively healthy readings,
perhaps chlorophyll fluorescence is not a good tool for identifying unhealthy stock
(or perhaps there was no unhealthy stock).

Trends in FJF... values traced over time seem to be highly affected by
local environmental conditions. This makes comparisons between years and
locations difficult because of the multitude of different factors to consider. It also
complicates attempts to use chlorophyll fluorescence as a stock plant quality
measure, since each field will have its own unique set of local conditions.

There Is a lack of correlation between chlorophyll fluorescence, measured
as FJF..., and rooting percentage, root number, root dry weight, and root length in
the ten Taxus cultivars examined. This may indicate that photosynthetic stress is
not a significant factor in determining Taxus rooting characteristics or that FJF...
is not an accurate measurement of stress in Taxus. Regardless, we would not
consider chlorophyll fluorescence measurements a practical method for

determining stock plant rooting ability in Taxus.

45

Inadequate control of unwanted variation is a potential source of error.
Chlorophyll fluorescence measurements have been shown to be affected by
temperature differences, photoinhibition, and other seasonal environmental
effects, as well as top and bottom leaf surfaces, sun and shade leaves, needle
age, storage time before measurement, and dehydration. Chlorophyll
fluorescence values may be far too sensitive to these immediate conditions for
the general type of health rating that we’re looking for.

Only one chlorophyll fluorescence parameter was used, FJF... This may
nOt have been the best parameter for our purposes. Perhaps F. or F... individually
would have served our purposes better or a measurement which included
quenching effects such as an Rfd value (F... — F./ F... ).

A useful approach for further studies might be one which determines the
amount of variation present in a branch, individual plant, Or field of Taxus. This
would enable estimations of necessary sample size and help determine the
feasibility of potential chlorophyll fluorescence uses, in both logistical and
cost/benefit terms.

Unknown are the daily and seasonal fluctuations of FJ F... in Taxus.
lnforrnation on these is vital to the appropriate evaluation of stand-alone
measurements taken in different years or under different climatic conditions. ls
F.,! F... affected by stress in Taxus, and at what stress intensities? What stresses
affect F.,! F... the most in Taxus? Cold stress? Dorrnancy? Photoinhibition? How

does this stress detection compare with visual observation?

Although common in stress studies, F.,! F... may not be the best chlorophyll
fluorescence parameter for examining Taxus. Other parameters may be more

effective and should be at least minimally investigated.

47

LITERATURE CITED

Bjorkman, O. and B. Demmig. 1987. Photon yield of 02 evolution and chlorophyll
fluorescence characteristics at 77K among vascular plants of diverse origins.
Planta 170:489-504.

Davidson, H. and A Olney. 1964. Clonal and sexual differences in the
propagation of Taxus, p. 156-161. In: lntemational Plant Propagator‘s Society
Combined Proceedings, vol. 14.

Eccher, T. 1988. Response of cuttings of 16 Taxus cultivars to rooting
treatments. Acta Horticulturae 227:251-253.

F isker, S., R. Rose, and D.L. Haase. 1995. Chlorophyll fluorescence as a
measure of cold hardiness and freezing stress in 1+1 Douglas-Fir seedlings.
Forest Sci. 41 (3):564-575.

Hallgren, J.E., T. Lundmark, and M. Strand. 1990. Photosynthesis of Scots pine
in the field after night frosts during summer. Plant Physiol. Biochem. 28(4):437-
445. .

Hawkins, C.D.B. and GR. Lister. 1985. In vitro chlorophyll fluorescence as a
possible indicator of the dormancy state in Douglas-fir seedlings. Can. J. For.
Res. 15:607-612.

Heinstein, PF. and OJ. Chang. 1994. Taxol. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 45:663-674. '

Lichtenthaler, H.K and U. Rinderle. 1988. Chlorophyll fluorescence signatures as
vitality indicator in forest decline research, p. 143-149. In: H.K Lichtenthaler
(ed.). Applications of Chlorophyll Fluorescence. Kluwer Academic Publ.,
Dordrecht, The Netherlands.

Lindgren, K and J.E. Hallgren. 1993. Cold acclimation of Pinus contorta and
Pinus sylvestris assessed by chlorophyll fluorescence. Tree Physiol. 13297-106.

Saarinen, T. 1993. Chlorophyll fluorescence, and nitrogen and pigment content
of Scots pine (Pinus sylvestris) needles in polluted urban habitats. Ann. Bot.
Fennici 30:1 -7.

Saarinen, T. and J. Liski. 1993. The effect of industrial air pollution on chlorophyll
fluorescence and pigment contents of Scots pine (Pinus sylvestris) needles.
Euro. J. For. Pathol. 232353-361.

Van Huylenbroech, J. and P. Degergh. 1992. Acclimation of micropropagated

Gerbera jamesonii, Use of chlorophyll fluorescence. Forum for Applied
Biotechnology. Brugge (Belgium). 24-25 Sept. Mededelingen-van-de-Faculteit-
Landbouwwetenschappen-Rijksuniversiteit-Gent (Belgium). 57(4a):1595-1579.

Welander, N.T., P. Gemmel, O. Hallgren, and B. Ottosson. 1994. The
consequences of freezing temperatures followed by high irradiance on in vivo
chlorophyll fluorescence and growth in Picea abies. Physiol. Plant. 91:121-127.

Westin, J., LG. Sundblad, and J.E. Hallgren. 1995. Seasonal variation in

photochemical activity and hardiness in clones of Norway spruce (Picea abies).
Tree Physiol. 15:685-689.

49

TABLE 1. 1997 -1998 Initial chlorophyll fluorescence (F.,/F...)

often cultivars Of Taxus xmedia from stock plant material

 

at Zelenka Nursery

Cultivar FJF...

Dark Green Pyramidalis 0.873 a
Tauntoni 0.851 a b
Densifonnis 0.826 b c
Hicksii 0.819 b c
Runyan 0.817 b c
Dark Green Spreader 0.815 b c
Densifonnis Gem 0.791 c d
Brownii 0.772 d
L.C. Bobbink 0.760 d e
Wardii 0.730 e

 

Mean separation among cultivars by LSD, P 0.05.

TABLE 2. 1998-1999 Initial chlorophyll fluorescence (FJF...)

of nine cultivars of Taxus xmedia from stock plant material

 

at Zelenka Nursery

Cultivar FJF...

Dark Green Spreader 0.833 a
Hicksii 0.832 a
Densifonnis Gem 0.828 a b
LC. Bobbink 0.825 a b c
Runyan 0.822 a b c d
Densiformis 0.821 b c d
Wardii 0.814 c d
Tauntoni 0.811 d
Brownii 0.778 e

 

Mean separation among cultivars by LSD, P 0.05.

51

TABLE 3. 1998 -1999 Chlorophyll fluorescence (FJF...) of stem cuttings

at sticking (after 32 days of cold storage at 5 °C).

Nine cultivars of Taxus xmedia at Zelenka Nursery.

 

Cultivar FJF...

Hicksii 0.732 a

Dark Green Spreader 0.719 a b

LC. Bobbink 0.700 b c
Wardii 0.693 c
Runyan 0.659 d
Densifonnis Gem 0.653 ' d
Tauntoni 0.61 1 e
Brownii 0.563 ' r
Densiformis 0.553 f

 

Mean separation among cultivars by LSD, P 0.05.

52

TABLE 4. 1998 -1999 Chlorophyll fluorescence (F.,/F...) of stem cuttings

at harvest (after 32 days of cold storage at 5 °C
and 100 days in the propagation bed).

Nine cultivars Of Taxus xmedia at Zelenka Nursery.

 

Cultivar FJF...

Hicksii 0.848 a
Dark Green Spreader 0.843 a b
Runyan 0.840 a b
LC. Bobbink 0.835 a b
Brownii 0.835 a b
Densifonnis Gem 0.834 a b
Densifonnis 0.832 b
Wardii 0.828 b
Tauntoni 0.807 c

 

Mean separation among cultivars by LSD, P 0.05.

53

TABLE 5. 1997 -1998 Rooting percentage often cultivars

of Taxus xmedia at Michigan State University.

 

Cultivar Rootingj

Densifonnis 96.7 a

Wardii 88.3 a b
Densifonnis Gem 85.0 a b c

Dark Green Pyramidalis 76.3 b c d
Runyan 70.0 c d e
Hicksii 65.0 d a
Dark Green Spreader 61.7 d e f
Tauntoni 56.7 . e f
Brownii 46.7 f g
LC. Bobbink 31.7 g

 

Mean separation among cultivars by LSD, P 0.05.

54

TABLE 6. 1997 -1998 Mean root dry weights often cultivars

Of Taxus xmedia at Michigan State University.

 

 

Cultivar Dry Weight (g)
Densifonnis Gem 0.225 a

Wardii 0.223 a
Densiformis 0.202 a b
Runyan 0.184 a b
Brownii 0.166 b c d
Tauntoni 0.133 c d
Hicksii 0.131 c d a
Dark Green Spreader 0.117 d a
Dark Green Pyramidalis 0.087 e
LC. Bobbink 0.042 f

 

Mean separation among cultivars by LSD, P 0.05.

55

TABLE 7. 1998 -1999 Rooting percentage of nine cultivars

of Taxus xmedia at Zelenka Nursery.

 

Cultivar Rooting%

Brownii 96.6 a

LC. Bobbink 95.0 a

Hicksii 86.7 a b
Densifonnis Gem 83.3 a b c
Tauntoni 75.0 b c
Wardii 71.7. c
Dark Green Spreader 48.5 d
Runyan 46.7 d
Densifonnis 45.0 d

 

Mean separation among cultivars by LSD, P 0.05.

TABLE 8. 1998 -1999 Mean root dry weights of nine cultivars

of Taxus xmedia at Zelenka Nursery.

 

 

Cultivar Dry Weim

LC. Bobbink 0.087 a
Brownii 0.084 a

Hicksii 0.075 a
Wardii 0.058 b
Densifonnis Gem 0.057 b
Runyan 0.053 b
Dark Green Spreader 0.045 b c
Densifonnis 0.033 c d
Tauntoni 0.023 d

 

Mean separation among cultivars by LSD, P 0.05.

57

TABLE 9. 1998 -1999 Taxus xmedia chlorophyll fluorescence (FJF...)
correlated with harvest data at Zelenka Nursery at the
whole-experiment level. Initial (F.,/F...) was taken at cutting
collection, sticking (F.,/F.,.) after 32 days cold storage at 5 °C,
and harvest (FJF...) after 100 days in the propagation bed.

Pearson correlation coefficients
Root

Rooting Root dry Root length

Chlorophyll fluorescence percentage weight number rating_

Initial (FJF...) -0.029 -0.079 -0.142‘ -0.208**
Sticking (FJF...) 0.029 0.103" 0.096 0013
Harvest (FJF...) 0.127” 0.040 0.007 0.038

 

*, ” Significant at P <= 0.05 or 0.01, respectively

58

TABLE 10. 1998 -1999 Taxus xmedia chlorophyll fluorescence (FJF...)
correlated with harvest data at Zelenka Nursery at the
cultivar level. Initial (FJF...) was taken at cutting
collection, sticking (F.,/F...) after 32 days cold storage at 5 °C,

and harvest (FJF...) after 100 days in the propagation bed.

 

Chlorophyll Root

fluorescence Rooting Root dry Root length

Cultivar (F.,/F...) percentage weight number rating
Brownii initial 0.135 -0.053 0.140 -0.066
sticking -0.127 0.039 -0.039 0.174

harvest -0.063 -0.089 -0.142 -0.238

Dark Green Spreader . initial 0.136 0.034 -0.086 -0.012
sticking 0.112 -0.150 -0.132 -0.185

harvest 0.395“ 0.017 -0.796* 0.172

Densifonnis initial 0.052 -0.020 0.779 0.005
sticking 0.000 -0.015 0.529 0.138

harvest 0.240 -0.130 0.393 -0.134

Densifonnis Gem initial 0.124 0.091 0.240 0.194
sticking -0.068 0.048 ' 0.224 0.105

harvest -0.029 0.009 -0.043 0.112

59

Hicksii

L.C. Bobbink

Runyan

Tauntoni

Wardii

*, *" Significant at P <= 0.05 or 0.01, respectively

initial
sticking
harvest
initial
sticking
harvest
initial
sticking
harvest
initial
sticking
harvest
initial
sticking

harvest

0.091

0.067

0.430“

-0.151

-0.140

0.159

-0.014

0.186

0.189

0.060

-0.080

0.274‘

0.125

-0.161

0.228

-0.066
0.130
0.109
0.144
0.085
0.179
0.091
0.176
0.246
0.055
0.134
-0.108
0.156
0.059

0.185

0.059

0.326‘

0.137

-0.025

-0.139

-0.019

0.154

-0.372

0.035

-0.737

-0.773

0.898

-0.454

-0.082

0.190

-0.068

-0.036

0.090

-0.269*

0.026

-0.075

0.096

0.193

0.005

0.058

-0.006

0.093

0.187

-0.064

0.215

LIST OF FIGURES

Figure Page

1 Chlorophyll fluorescence in Taxus over the course of propagation at
Zelenka Nursery Oct. 14, 1997 - May 7, 1998. Cuttings were
collected from stock plants in the field and placed in cold storage at
2.5 °C for 37 days and then stuck in rooting beds (100% perlite) at 18 °C
with bottom heat at 21 °C. Chlorophyll Fluorescence readings (FJFm) were
taken periodically until harvest. The first three points per cultivar are each
a mean of 10 readings, while all other points represent means of 24
readings. Vertical bars represent + SE.

62

2 Chlorophyll fluorescence in Taxus over the course Of propagation at
Michigan State University Oct. 14, 1997 — May 6, 1998. Cuttings were
collected from stock plants in the field and placed in cold storage at 5 °C
for 39 days and then stuck in rooting beds (100% perlite) at 18-21 °C.
Chlorophyll Fluorescence readings (FJF...) were taken periodically until
harvest. The first three points per cultivar are each a mean of 10 readings,
while all other points represent means of 24 readings. Vertical bars
represent + SE.

63

3 Chlorophyll fluorescence in Taxus over the course of propagation at
Zelenka Nursery Oct. 29, 1998 - March 10, 1999. Cuttings were
collected from stock plants in the field and placed in cold storage at
2.5 °C for 33 days and then stuck in rooting beds (100% perlite) at 18 °C
with bottom heat (21 °C). Chlorophyll Fluorescence readings (Ft/Fm) were
taken periodically until harvest. The first three points per cultivar are each
a mean of 60 readings, while all other points represent means of 24
readings. Vertical bars represent + SE.

64

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Chapter Two
Chlorophyll Fluorescence and Cold Storage of Taxus Cuttings

(Formatted according to publication guidelines of the American Society of
Horticultural Science)

65

Chlorophyll Fluorescence and Cold Storage of Taxus Cuttings

S. E. Bruce‘ and 0. B. Rowez
Department of Horticulture, Michigan State University, East Lansing, MI 48824

Received for publication . Accepted for publication . This paper is a
portion of a MS. thesis submitted by S. E. Bruce.

Acknowledgement. This experiment was a Michigan State University study
funded by Zelenka Nursery in Grand Haven, MI, International Plant Propagator's
Society, Michigan Nursery and Landscape Association, and Michigan Agricultural
Experiment Station.

1Graduate research assistant
2Assistant professor

Propagation and Tissue Culture
Chlorophyll Fluorescence and Cold Storage of Taxus Cuttings
Additional index words. Yew, vegetative propagation, stem cuttings

Abstract. Propagators routinely use a cold storage treatment in the propagation
of Taxus via stern cuttings. Since storage conditions are a major factor in cutting
quality and subsequent rooting, the objective of this study was to examine the
effect of different storage temperatures, desiccations, and durations in four
cultivars of Taxus xmedia (Taxus baccata L. x T. cuspidata Sieb. 8. Zucc.),
quantifying stress in relation to subsequent rooting using chlorophyll fluorescence
measurements (F.,/F...) Storage temperatures used included -30, -2.5, 0, 2.5, 5,
10, and 20 °C. Desicwtion levels were created with closed, perforated, and open
plastic bags, and storage durations consisted of 34, 70, 107 days. Cultivars
Brownii, Dark Green Spreader, Hicksii, and Wardii were used. Chlorophyll
fluorescence readings were taken initially, at sticking, and at harvest and rooting
percentage, root dry weight, root number, and root length were measured.
Temperatures of -2.5 to 2.5 were found to be ideal, and desiccation was not
found to influence rooting at these temperatures. Longer storage durations (70
and 107 days) had negative effects on all rooting data. Some differences were
found to exist between cultivars in response to the various treatments.
Chlorophyll fluorescence measurements could detect substandard storage

conditions only at temperature and desiccation extremes.

67

Propagation of Taxus is achieved primarily through vegetative stern
cuttings. Traditionally, 15 - 20 cm cuttings are taken between November and
February, after natural exposure to cold temperatures (Sabo, 1976; Scheer,
1976; Verkade, 1976). Cuttings are stripped at their base, treated with an auxin-
based rooting compound, and placed in a sand, perlite, or sand/perlite media
under mist (Hartman at al., 1990). Nurseries involved in large-scale production
today have adjusted this basic process to make it more efficient and cost
effective. Smaller cuttings tend to be used, often 10 - 15 cm, and stripping is rare
(Richey, 1986). Ideal hormone treatments are relatively low (~0.25 mM) IBA
(Nandi at al., 1996) and some research indicates that a 0.08% thiamine addition
may be beneficial (Chee, 1995). Although impractical for most nurseries, studies
indicate that apical cuttings produce the highest rooting percentages (Eccher,
1988). Perhaps the mast significant change has been the use of a cold storage
treatment to ensure dormancy instead of mid-winter harvest of cuttings.

In order to tap into a large seasonal work force, harvest cuttings in
hospitable weather conditions, and provide for a longer rooting period in the
propagation beds, nurseries collect cuttings in early fall (September or October),
often before the first frost, and store them in a cooler at 2 - 5 °C (Richey, 1986).
Humidity is kept at 85 - 90% (Richey, 1986). Sticking is done a month or so later.
This process allows propagators to control cold exposure cuttings receive,

ensuring sufficient cold duration and consistency among cuttings.

Chlorophyll Fluorescence

Chlorophyll fluorescence measurements were utilized to measure stress
because of their potential as a quantitative and objective method for evaluating
storage conditions.

Chlorophyll fluorescence is created when light energy, absorbed by
chlorophyll a, exceeds the photochemical processing capacity of photosystem II
(PSII). One way this ‘extra’ energy is dissipated is by being re-emitted as light,
which we call chlorophyll fluorescence. The fluorescence measured at
physiological temperatures is largely a product of chlorophyll a molecules
involved in PSII, although other light capturing pigments do fluoresce, and PSI
fluorescence can be measured. Since chlorophyll fluorescence levels are tied to
the amount of light energy not used for photosynthetic processes, they are
inversely related to the amount Of energy that is used for photosynthesis, and
serve as indicators of plant photosynthetic potential.

Chlorophyll fluorescence measurement is increasingly used as an
estimate of photosynthetic health. The emitted light signal follows a general
intensity pattern known as the Kautsky Effect. Pre-darkened samples, with a
minimum fluorescence level (F.), Show a ‘fast rise’ in fluorescence to a maximum
value (F...) upon exposure to a light source. As photochemical processing Of the
light energy increases, fluorescence values are gradually reduced to a steady
state (F.), somewhere between F. and F.... A common parameter used in stress
studies is F.,/Fm, F., being the variable fluorescence, calculated by subtracting F.

from F... Numerous studies have shown FF... and other chlorophyll

69

fluorescence parameters, to be effective measurements of the photoefficiency of
PSII. Bjorkman and Demmig (1987) linearly correlated FJF... with the quantum
yield of PSII, as determined by oxygen evolution, in a variety of stressed plants.

Studies utilizing chlorophyll fluorescence to quantify plant stresses include
heat and cold stress, especially tolerance studies, photoinhibition, mineral
nutrition, pollution, and water stress. Studies on conifers have found effects of
vehicle and oil refinery pollution on Scotch pine (Pinus sylvestris L.) (Saarinen,
1993; Saarinen and Liski, 1993), photoinhibition effects in Scotch pine (Hallgren
at al., 1990), and forest decline and photoinhibition effects in Norway spruce
(Picea abies (L) Karst.)(Lichtenthaler and Rinderle, 1988; Welander et al., 1994).
Seasonal changes in FJF... values have been used to assess dormancy in
Douglas fir (Pseudotsuga menziesii (Mirbel) Franco) (Hawkins and Lister, 1985),
Scotch and lodgepole pines (Pinus contorta Dougl. ex Loud.) (Lindgren and
Hallgren, 1993), and Norway spruce (Westin at al., 1995). Fisker at al. (1995)
found chlorophyll fluorescence to be an accurate estimate of freeze damage in
needles and seedling survival in Douglas fir, and could detect non-visible
damage. However, fluorescence measurements were unable to predict cold
hardiness prior to the temperature treatments. Likewise, Welander et al. (1994)
were unable to use chlorophyll fluorescence to predict growth response after a
night frost followed by high irradiation. Camm and Lavender (1993) found cold-
storage light levels to affect FvlFm in white spruce seedlings (Picea glauca
[Moench] Voss) while jack pine seedlings (Pinus banksiana Lamb.) remained
unaffected.

70

The only fluorescence study involving propagation examined stress
levels during the acclimatization of micropropagated Transvaal daisy (Gerbera
jamesonii Bol. ex Adlam) (Van Huylenbroech and Debergh, 1992). To our
knowledge, no studies of Taxus or stem cutting storage have involved chlorophyll
fluorescence. Since storage conditions are a major factor in cutting quality and
subsequent rooting, the objective of this study was to examine the effect of
different storage temperatures, desiccations, and durations in four cultivars of
Taxus xmedia (Taxus baccata L. x T. cuspidata Sieb. 8. Zucc.), quantifying stress

in relation to subsequent rooting using chlorophyll fluorescence measurements.

MATERIALS AND METHODS

Studies were conducted over the 1997-98 and 1998-99 propagation
seasons. Although similar in many respects, test condition and treatment
differences were such that the studies must be considered separate experiments.

The 1997-98 study examined four factors: cultivar, storage duration,
storage desiccation, and storage temperature. Brownii, Dark Green Spreader,
Hicksii, and Wardii cultivars of-Taxus xmedia were used. Seven hundred and
twenty, 10 - 15 cm cuttings were collected from each cultivar on Oct. 14, 1997,
from fields at Zelenka Nursery, Grand Haven, MI. Chlorophyll fluorescence
readings (FJFm), were taken, 10 per cultivar, and cuttings were randomly divided
into the desiccation and temperature treatments. The three desiccation
treatments consisted of sealed plastic bags (low desiccation), sealed plastic bags

with holes punched in them (medium desiccation), and open plastic bags (high

71

desiccation). These were then further divided into 5 coolers at Michigan State
University, East Lansing, MI, set at 0, 2.5, 5, 10, and 20 °C. After 34 days in
storage, half the cuttings from each treatment were removed to a Michigan State
University greenhouse where they were cut to 11.4 cm (apical and basal portions
removed), treated with Woods Rooting Hormone (IBA 1.03%; NAA 0.66%) at
2800 ppm (1 :5 ratio), and placed in 100% perlite media in 7.6 cm deep growth
trays. The experimental layout consisted of a split-plot design with three splits
(duration, cultivar, and desiccation) and four blocks. Cuttings were stuck in rows
1.3 cm apart and the rows spaced 3.8 cm apart. Sixty-five days after the original
cutting collection (Dec. 18), the remainder of the cuttings were removed from
storage and planted similarly to the first set (rooting data from the 65 day
duration not included in this analysis). All cuttings were watered as needed until
spring. Greenhouse temperatures tended to range higher than desired, often
rising above 21 °C on sunny days. At harvest, June 11-18, cuttings were gently
uprooted, and root lengths rated (0=less than 2.54 cm [1 inch], 1=less than 5.08
cm [2 inch], 2=less than 7.62 cm [3 inch], 3=greater than 7.62 cm) and root
numbers recorded. Roots were dried at 28 °C for 3 days and weighed (Explorer
Balance, Ohaus Corp., Florham Park, NJ). Chlorophyll fluorescence
measurements were taken of storage material at cutting collection, 10 per
cultivar, and than 5 per treatment at 24, 35, 50, and 65 day storage duration (Oct.
14, Nov. 7, Nov. 18, Dec. 3, and Dec. 18, respectively).

The 1997-98 chlorophyll fluorescence measurements were taken with a

Morgan CF-1000 (P.K Morgan Instruments, Inc., Andover, MA). For each

72

measurement (FJF...), the underside of a single, randomly selected needle was
measured at a light level of 700 umollmzls. Samples were dark adapted for 15
minutes before measurement in the manufacturer's plasticlfoam clips.
Chlorophyll fluorescence measurements were usually taken the same day as the
needles were collected, although occasionally logistics required needles to be
stored overnight. This was accomplished in a germination tray covered with moist
paper towels and stored at 5 °C. Needles were acclimated to room temperature
at least one hour prior to fluorescence measurements. No distortion of values
was observed as a result of overnight storage.

The 1998-99 study examined three factors, cultivar, storage duration, and
storage temperature. Cultivars were the same as in the 1997-98 study. Two
hundred eighty-eight cuttings per cultivar were collected, Oct. 29, 1998, from
Zelenka Nursery. Cuttings were randomly divided into 3 duration treatments (34,
70, and 107 days in storage), 4 temperatures (-30, -2.5, 0, and 2.5 °C), and 4
blocks. A sample of 3 chlorophyll fluorescence (Fv/Fm) readings per treatment,
per block was taken, and cuttings bagged in plastic and placed in their respective
cold storage temperatures at MSU. After the determined amount of cold storage,
cuttings were placed at Zelenka Nursery, Grand Haven, MI, in 15 cm deep
propagation beds of 100% perlite. Propagation procedures were the same as in
the 1997-98 study. The experimental layout consisted of a split-plot design with
two splits (storage duration and cultivar) and four blocks. Sticking occurred on
Dec. 3 (34 days cold storage), Jan. 7 (70 days cold storage), and Feb. 3 (107

days cold storage). Greenhouse temperatures were held at 18 °C with bottom

73

heat (21 °C) provided in the benches. Cuttings were watered as needed. The
staggered planting dates led to staggered harvest dates of Mar. 8, April 15, and
May 13, respectively, resulting in 96 - 99 days in the propagation bed for each
storage duration level. The propagation season was shortened from the previous
year in efforts to measure greater treatment differences. Harvest was conducted
similarly to the 1997-98 season. In addition to the initial measurement,
chlorophyll fluorescence readings were taken of the material in storage at 34, 70,
and 107 days storage duration (Dec. 2, Jan. 5, and Feb. 3), and their respective
harvest dates (5 samples per block per treatment).

The 1998-99 chlorophyll fluorescence measurements were taken with a
Plant Efficiency Analyzer (PEA) (Hansatech Instruments Ltd., Norfolk, England,
UK). Samples were dark adapted for 15 minutes in the manufacturer’s
plastic/foam clips, and a fluorometer light level of 1200 timollmzls (40% of
maximum capacity) was determined to be ideal, according to the manufacturer’s
instructions.

Statistical Analysis. Harvest and chlorophyll fluorescence data were
subjected to statistical analysis consisting of an analysis of variance (ANOVA)
and LSD tests (least significant difference) performed overall and at the individual
cultivar level. Correlation calculations were done between the harvest data and
fluorescence data (all dates) for each season (Proc anova, proc corr, SASIPC

software, SAS Institute Inc., Cary, NC).

74

RESULTS AND DISCUSSION

The storage treatments, cultivar, duration, desiccation (1997-1998 season
only), and temperature, combine with one another to create unique yet
inseparable effects. Due to the interactions between all treatments, analysis is
difficult This report will examine each treatment individually with respect to its

interaction with other treatments.

Storage Desiccation

Low, medium, and high storage desiccation treatments were performed in
1997-1998 (Figs. 1, 2). Results for root number and root length rating tended to
correlate with root dry weight, so only root dry weight data are presented. Few
desiccation effects were found in Brownii due to its low rooting percentages. Dark
Green Spreader showed a decrease in harvest data as a result of desiccation at
0, 5, and 10 °C, with rooting percentage only being affected at 5 and 10 °C.
Hicksii was little affected by desiccation, showing a decrease in root dry weight
and length at 0 °C (data not presented) and rooting percent at 10 and 20 °C. Low
desiccation tended to decrease the negative effects of higher temperatures, so
that it was only at low desiccation that some rooting occurred at 20 °C. The sole
effect of desiccation in Wardii was a decrease in rooting percentage at 10 and 20
°C. Wardii appeared more resistant to high temperatures at low desiccation than
other cultivars, showing rooting percentages in the 10 and 20 °C treatments.

Other cultivars succumbed to fungal rot in these treatments.

75

Effects of medium and high desiccation treatments were generally
indistinct, suggesting that the actual desiccation levels may have been similar.
Medium and high desiccations combined with 10 and 20 °C temperatures were
universally lethal. Low desiccation allows minimal rooting at higher temperatures,
especially in rot-resistant cultivars such as Wardii. At lower temperatures (0 to 5

°C) desiccation treatments had little effect.

Storage Duration

Storage durations of 34, 70, and 107 days were tested in the 1998-1999
study. Decreases in rooting percentage (Fig. 3), root dry weight (Fig. 4), root
number (Fig. 5), and root length (Fig. 6) were found with each increase of
storage duration. Decreases in rooting permntages were found in all cultivars at
-2.5, 0, and 2.5 °C. The -30 °C treatment was lethal to all cuttings stored at that
temperature. Brownii rooting percentages dropped in the 70 day extension of
storage, but 70 and 107 day duration effects remained indistinguishable. Wardii,
however, showed no significant difference in rooting percentages between the 34
and 70 day durations, only to decline with the 107 day duration. Dark Green
Spreader and Hicksii showed a decrease in rooting percentage with each
extension of duration. Root dry weight and root length decreased with duration in
all cultivars, except Dark Green Spreader, where treatment effects were not
significant. Root number showed an overall decrease with extended storage

duration.

76

Preliminary studies showed storage duration effects to be of little
consequence under unsuitable conditions such as higher desiccation and 10 and
20 °C temperatures. At low desiccation and cooler temperatures (-2.5 to 2.5 °C),
increasing duration, from 34 to 70 days and 70 to 107 days, had negative effects

on rooting percentage, root dry weight, root number, and root length.

Storage Temperature
Storage treatment temperatures in the 1997-1998 study consisted of 0,

2.5, 5, 10, and 20 °C (Figs. 1, 2). Brownii rooting percentages were so low that
few effects of temperature could be seen. Dark Green Spreader rooting
percentages showed little difference between 0 and 10 °C at low desiccation,
however decreases in dry weight (Fig. 2), root number, and root length were
observed (data not presented). At medium desiccation, 0 to 5 °C temperatures
produced indistinguishable rooting percentages. At high desiccation, 0 and 2.5
°C produced the highest rooting data. Dry weight, root number, and root length
showed no clear trend in most treatments. In Hicksii, decreases in dry weight,
root number, and root length are seen with increasing temperature. Wardii dry
weight, root number, and root length were little affected by temperature
treatments. Rooting percentages decreased with increasing temperatures at
medium and high desiccations. Overall, the highest root numbers and dry
weights were produced at 0 °C. The clearest effect of temperature is seen in the

high desiccation treatments.

Storage temperature was negatively correlated with rooting percent in
Brownii (0535), Dark Green Spreader (-0.842), and Hicksii (-0.766) (T able 1). In
Hicksii, it was also conelated with root length (-0.584), root number (0709), and
root (by weight (0722).

The 1998-1999 storage temperature treatments consisted of -30, -2.5, 0
and 2.5 °C. No rooting was seen in the -30 °C treatments. Brownii rooting
percentages peaked at increasing temperatures with increasing storage duration:
in 34 day duration, -2.5 and 0 °C were ideal; in 70 day duration -2.5, 0, and 2.5
°C were ideal; in 107 day duration 2.5 °C was ideal. Rooting percentage was the
only harvest parameter with significant temperature effects. In Dark Green
Spreader, Hicksii, and Wardii, rooting data at -2.5, 0, and 2.5 °C was not
significantly different.

Storage temperatures were negatively correlated with root number at the
34 day storage duration in Dark Green Spreader (-0.727), and root dry weight in
the 70 day storage duration of Dark Green Spreader (-0.898) and 107 day
storage duration of Hicksii (0632).

Little rooting occurred with the 10 and 20 °C treatments, except at low
desiccation and short duration, and no rooting occurred with the -30 °C
treatments. Slight freezing (-2.5 °C) showed no detrimental effects in most cases,
however, the Brownii data suggests that slightly warmer temperatures may be
best for longer term storage (107 day). Ideal storage temperatures fall between
-2.5 and 2.5 °C, with 5 °C treatments often showing high rooting in low

desiccation as well.

78

Chlorophyll Fluorescence during Storage

Data for chlorophyll fluorescence in 1997-1998 is pooled for the four
cultivars (Fig. 7). At low desiccations, a decrease in chlorophyll fluorescence
values is seen only in the 20 °C treatment. This decrease was visible by day 35.
At medium and high desiccations, however, a decrease in chlorophyll
fluorescence in the 20 °C treatment can be detected by day 24, and by day 35 or
day 50, 10 °C shows a decrease in values as well. In general, 0, 2.5, and 5 °C
showed almost no difference in chlorophyll fluorescence readings during storage.
Wardii, as a cultivar, showed the most resilient chlorophyll fluoreswnce levels.

In the 1997-1998 data, chlorophyll fluorescence readings correlated with
temperature from day 24 in all cultivars (T able 1). Correlations also existed with
rooting percentages in Dark Green Spreader (day 24 = 0.539, day 35 = 0.709),
Hicksii (day 24 = 0.660, day 35 = 0.723), and at day 35 in Wardii (0.556). These
correlations grew stronger with increased duration in storage. In Hicksii, the day
35 chlorophyll fluorescence values were also correlated with root length (0.619)
and dry weight (0.562).

The 1998-1999 storage study chlorophyll fluorescence data combines
temperatures (-2.5, 0, and 2.5 °C) where no significant differences in chlorophyll
fluorescence values were found (Fig. 8). Data for -30 °C treatments was
eliminated due to 0 percent rooting (chlorophyll fluorescence levels declined

sharply in storage, reaching the fluorometer detectable minimum by 107 days).

79

No significant decline in chlorophyll fluorescence levels over storage was found
in any cultivar at -2.5, 0, or 2.5 °C.

In the 1998-1999 data, Brownii, Dark Green Spreader, Hicksii, and Wardii
chlorophyll fluorescence values are sporadically correlated with temperature and
the harvest parameters. Strangely, correlations made at day 34 or day 70 often
are not found at day 107 or harvest. No correlation continues in time from the
point of its appearance until harvest. Correlations found with the rooting data
exist at harvest in Brownii (34 day storage root length rating {-0.618}, 107 day
storage rooting percent [0.805], root length rating [0.759], and root dry weight
[0918]), Dark Green Spreader (70 day storage rooting percent [0.701] and root
length rating [0972]), and Hicksii (70 day storage rooting percent [0.643] and
107 day storage root number [0844]). Wardii plant material was appareme not
randomly distributed, since initial chlorophyll fluorescence (before cuttings placed

into storage) is correlated with storage temperature.

Potential error in these studies may lie in the wide temperature ranges
used and the frequency of chlorophyll fluorescence measurements. An excess of
interacting treatments complicated analysis and reduced the extent of possible
conclusions. The majority of treatments showed non-significant trends in means,

which suggests larger sample sizes be used in the future.

CONCLUSIONS

Grower storage conditions usually consist of a month of 2.5 to 5 °C
temperatures with low desiccation. These conditions are ideal. Slightly cooler
temperatures may be desirable (-2.5 to 2.5) although they may not be cost
effective. A shorter, colder storage treatment is more desirable than a longer,
warmer one, although slight increases in temperature or duration may have
negligible effects, especially in certain cultivars. Cultivars Hicksii and Wardii are
more resilient in undesirable storage conditions than cultivars Brownii and Dark
Green Spreader. if warmer temperatures are necessary (5 to 10 °C) normal
rooting percentages may be preserved with high humidity. Cuttings are
unaffected by desiccation at 2.5 °C and below. Longer storage durations are to
be avoided, as rooting percentages are often almost halved with each extra
month of storage. Rooting percentages and root quality show large decreases .
with time in storage exceeding 34 days in ideal conditions, indicating that the
negative effects of extended duration are not purely effects of plant material
desiccation.

This study shows chlorophyll fluorescence measurements can detect high
or low storage temperatures (20 and -30 °C) and desiccation effects in Taxus
cutting material by 3 weeks into storage. Since temperature and desiccation
closely affect rooting, chlorophyll fluorescence can serve as an indicator of
rooting in extreme temperature and desiccation conditions. However, at low
desiccation, chlorophyll fluoreswnce failed to consistently distinguish between -

2.5, 0, 2.5, 5, and 10 °C treatments, 8 range within which there are clear rooting

81

differences. Chlorophyll fluorescence readings also failed to detect longer
storage durations which lead to decreased rooting potential. Factors causing
decreases in rooting of Taxus as a result of these stresses do not affect plant
photosynthetic efficiency in a comparative manner. Chlorophyll fluorescence
values, as measured by F.,/F... ratios, are not useful indicators of storage

conditions in Taxus.

82

LITERATURE CITED

Bjorkman, O. and B. Demmig. 1987. Photon yield of 02 evolution and chlorophyll
fluorescence characteristics at 77K among vascular plants of diverse origins.
Planta 170:489-504.

Camm, EL. and DP. Lavender. 1993. Photosynthetic apparatus in cold-stored
conifer seedlings is affected by nursery and storage photoperiod. Forest Sci.
39(3):546-560.

Chee, RP. 1995. Stimulation of adventitious rooting of Taxus species by
thiamine. Plant Cell Reports 14:753-757.

Eccher, T. 1988. Response of cuttings of 16 Taxus cultivars to rooting
treatments. Acta Horticulturae 227:251-253.

Fisker, S., R. Rose, and D.L. Haase. 1995. Chlorophyll fluorescence as a
measure of cold hardiness and freezing stress in 1+1 Douglas-Fir seedlings.
Forest Sci. 41 (3):564-575.

Hallgren, J.E., T. Lundmark, and M. Strand. 1990. Photosynthesis of Scots pine
in the field after night frosts during summer. Plant Physiol. Biochem. 28(4):437-
445.

Hartman, H.T., D.E. Kestar, and F.T. Davies, Jr. 1990. Plant Propagation:
principles and practices, p. 600-601. Prentice Hall: Englewood Cliffs, NJ.

Hawkins, C.D.B. and GR. Lister. 1985. In vitro chlorophyll fluorescence as a
possible indicator of the dormancy state in Douglas-fir seedlings. Can. J. For.
Res. 151607-612.

Lichtenthaler, H.K and U. Rinderle. 1988. Chlorophyll fluorescence signatures as
vitality indimt0r in forest decline research, p. 143-149. In: H.K Lichtenthaler
(ed.). Applications of Chlorophyll Fluorescence. Kluwer Academic Publ.,
Dordrecht, The NetherIands.

Lindgren, K and J.E. Hallgren. 1993. Cold acclimation of Pinus contorta and
Pinus sylvestris assessed by chlorophyll fluorescence. Tree Physiol. 13297-106.

Nandi, S.K, L.M.S. Palni, and HG. Rikhari. 1996. Chemical induction of

adventitious root formation in Taxus baccata cuttings. Plant Growth Regulation
19:1 1 7-122.

Richey, M. 1986. Sticking Taxus as unstripped cuttings, an update, p. 597-599.
In: lntemational Plant Propagator's Society Combined Proceedings, vol. 36.

83

Saarinen, T. 1993. Chlorophyll fluorescence, and nitrogen and pigment content
of Scots pine (Pinus sylvestris) needles in polluted urban habitats. Ann. Bot.
Fennici 30:1-7.

Saarinen, T. and J. Liski. 1993. The effect of industrial air pollution on chlorophyll
fluorescence and pigment contents of Scots pine (Pinus sylvestris) needles.
Euro. J. For. Pathol. 23:353-361.

Sabo, J.E. 1976. Propagation of Taxus in northern Ohio, p. 174-176. In:
lntemational Plant Propagator’s Society Combined Proceedings, vol. 26.

Scheer, C. 1976. Taxus propagation by cuttings, p. 173-174. In: lntemational
Plant Propagator’s Society Combined Proceedings, vol. 26.

Van Huylenbroech, J. and P. Degergh. 1992. Acclimation of micropropagated
Gerbera jamesonii, Use of chlorophyll fluorescence Fonim for Applied
Biotechnology. Brugge (Belgium). 24-25 Sept. Mededelingen-van-de—Faculteit-
Landbouwwetenschappen-Rijksuniversiteit-Gent (Belgium). 57(4a):1595-1579.

Verkade, G. 1976. Propagation of Taxus by cuttings, p. 177. In: lntemational
Plant Propagator's Society Combined Proceedings, vol. 26.

Von Komya, JP. 1976. Propagation of Taxus cuttings, p.178-179. In:
lntemational Plant Propagator’s Society Combined Proceedings, vol. 26.

Welander, N.T., P. Gemmel, O. Hallgren, and B. Ottosson. 1994. The
consequences of freezing temperatures followed by high irradiance on in vivo
chlorophyll fluorescence and growth in Picea abies. Physiol. Plant. 91:121-127.

Westin, J., LG. Sundblad, and J.E. Hallgren. 1995. Seasonal variation in
photochemical activity and hardiness in clones of Norway spruce (Picea abies).
Tree Physiol. 15:685-689.

TABLE 1. 1997 -1998 Taxus xmedia chlorophyll fluorescence (F.,/F...)

and storage temperature treatments (0, 2.5, 5, 10, and 20 °C)

correlated with harvest data (Pearson correlation coeffecients).

Storage duration = 34 days.

 

 

Brownii
Root
Temperature Rooting Length Root Root Dry
°C Percent Rating Number WeigIL
F.,/F... Day 24 -0.765** 0.286 -0.178 -0.048 -0.421
FJF... Day 35 -0.811"* 0.361 -0.584 0.148 0685
Temperature -0.535* -0.015 -0.127 -0.240
Dark Green Spreader
Root
Temperature Rooting Length Root Root Dry
°C Percent Rating Number Weigh;
FJF... Day 24 -0.714** 0.539“ -0.307 -0.499 -0.402
FJF... Day 35 -0.787” 0.709“ -0.398 -0.536 0556
Temperature -0.842*' -0.412 -0.204 -0.547

*, *“ Significant at P <= 0.05 or 0.01, respectively

85

Hicksii

Temperature Rooting

Root

Length Root Root Dry

 

 

°C Percent Rating Number Weight
FJF... Day 24 0736“ 0.660” 0.306 0.200 0.347
FJF... Day 35 -0.786** 0.723“ 0619* 0.532 0562*
Temperature -0.766*“ -0.584* 0709*" 0722""
Wardii
Root
Temperature Rooting Length Root Root Dry
°C Percent RatinL Number Weigll
FJF... Day 24 0666“ 0.502 0.248 -0.182 0.464
F.,/F... Day35 -0.770** 0.556" 0.215 -0.326 0.204
Temperature -0.494 0.168 0.443 0.137

*, *" Significant at P <= 0.05 or 0.01, respectively

LIST OF FIGURES
Page

Effect of storage temperature and desiccation on rooting of four cultivars
of Taxus (1997 - 1998). Storage duration = 34 days. Stern cuttings were
collected from stock plants in the field, placed in cold storage treatments,
and then placed in perlite propagation beds. Rooting percentages were
measured 96 - 99 days after sticking. Points represent means of 24
readings. Vertical bars represent + SE.

89

Effect of storage temperature and desiccation on root dry weight of four
cultivars of Taxus (1997 - 1998). Storage duration = 34 days. Stem
cuttings were collected from stock plants in the field, placed in cold
storage treatments, and then placed in perlite propagation beds. Roots
were harvested 96 - 99 days after sticking, dried at 28 0C for 3 days and
weighted. Points represent means of 24 readings. Vertical bars represent
+ SE.

90

Effect of storage duration on rooting of four cultivars of Taxus (1998 -
1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were collected
from stock plants in the field, placed in cold storage for treatment duration,
and then placed in perlite propagation beds at 18 °C with bottom heat
(21 °C). Rooting percentages were measured 96 - 99 days after sticking.
Points represent means of 72 readings. Vertical bars represent + SE.

91

Effect of storage duration on root dry weight of four cultivars of Taxus
(1998 — 1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Roots were harvested 96 - 99 days after sticking, dried at 28
°C for 3 days and weighted. Points represent means of 72 readings.
Vertical bars represent + SE.

92

Effect of storage duration on root number of four cultivars of Taxus (1998
- 1999). Storage temperature = -2.5 — 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Root numbers were measured 96 - 99 days after sticking.
Points represent means of 72 readings. Vertical bars represent + SE.

93

87

Figure Page

6 Effect of storage duration on root length rating of four cultivars of Taxus
(1998 — 1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Root length ratings were measured 96 - 99 days after
sticking. (0=less than 2.54 cm [1 inch], 1=less than 5.08 cm [2 inch],
2=less than 7.62 cm [3 inch], 3=greater than 7.62 cm) Points represent
means of 72 readings. Vertical bars represent + SE.

94

7 Effect of storage temperature and desiccation on chlorophyll fluorescence
of cuttings of Taxus (1997 - 1998). Chlorophyll fluorescence (FJFM) was
measured on the lower surface of individual dark-adapted (15 minutes)
needles with fluoromater (Morgan CF-1000) light level set at 700
timol/mzls. The first point of each line represents a mean of 40 readings,
all other points represent means of 80 readings. Vertical bars represent
+ SE.

95

8 Effect of storage duration on chlorophyll fluorescence of cuttings of Taxus
(1998 —1999). Storage temperature = -2.5 — 2.5 °C. Chlorophyll
fluorescence (FJF...) was measured on the lower surface of individual
dark-adapted (15 minutes) needles with fluorometer (PEA, Hansatech
Instruments Ltd.) light level set at 1200 umollm’ls. Points represent
means of 20 readings. Vertical bars represent + SE.

96

Fig. 1 Effect of storage temperature and desiccation on rooting of four cultivars
of Taxus (1997 - 1998). Storage duration = 34 days. Stern cuttings were
collected from stock plants in the field, placed in cold storage treatments,
and then placed in perlite propagation beds. Rooting percentages were
measured 96 - 99 days after sticking. Points represent means of 24
readings. Vertical bars represent +I- SE.

Brownii + Low desiccation
5° —0— Medium desiccation *1
—v-— High desiccation

 

 

   

50‘
40-1
30-
20-
10-

0..

 

 

 

 

 

 

 

 

0.0 2.5 5.0 10.0 20.0
Dark Green Spreader

 

140
120 '1
100 '1
80 a
60 J
401
20 -
o -I

 

 

 

 

0.0 2.5 5.0 10.0 20.0
Hicksii

Rooting Percentage

 

140
120‘
100d
80-
50.
4o-
20-

0 %

 

 

 

 

0.0 2.5 5.0 10.0 20.0
Wardii

 

 

 

 

 

 

0.0 2.5 5.0 10.0 20.0
Storage Temperature (°C) ‘

89

Fig. 2 Effect of storage temperature and desiccation on root dry weight of four

Dry Weight (g)

cultivars of Taxus (1997 — 1998). Storage duration = 34 days. Stem
cuttings were collected from stock plants in the field, placed in cold
storage treatments, and then placed in perlite propagation beds. Roots
were harvested 96 - 99 days after sticking, dried at 28 °C for 3 days and
weighted. Points represent mean of 24 readings. Vertical bars represent

+ SE.
0.4

 

+ Low desiccation

 

0.3

0.31
0.2 -
0.2 -
0.1 -
0.1 -
0.0 ~

 

Brownii —0— Medium desiccation

+ High desiccation

 

 

 

 

 

I l U

0.0 2.5 10.0

 

0.7
0.6 a
0.5 -
0.4 -
0.3 -
0.2 -
0.1

 

Dark een Spreader

 

 

I I I T

0.0 2.5 5.0 10.0

 

0.7
0.6 -
0.5 n
0.4 -
0.3 -
0.2 -‘
0.1 4
0.0 d

 

Hicksii

 

 

 

0.5

I I l I I

0.0 2.5 5.0 10.0

 

0.4 -
0.4 a
0.4 a
0.3 a
0.3 n
0.2 a
0.2 -

Wardii

 

0.1

 

 

I T

5.0 10.0 20.0
Storage Temperature (°C)

 

 

 

 

Fig 3. Effect of storage duration on rooting of four cultivars of Taxus (1998 -
1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were collected
from stock plants in the field, placed in cold storage for treatment duration,
and then placed in perlite propagation beds at 18 °C with bottom heat
(21 °C). Rooting percentages were measured 96 - 99 days after sticking.
Points represent means of 72 readings. Vertical bars represent + SE.

 

120 4

100-

l

 

l

 

Percent Rooted

 

3

20-

 

 

 

 

Days in Storage

 

+ Brownii
—0— Dark Green Spreader
+ Hicksii
-v— Wardii

 

 

 

91

Fig. 4 Effect of storage duration on root dry weight of four cultivars of Taxus

Root Dry Weight (g)

(1998 - 1999). Storage temperature = -2.5 - 2.5 °C. Stern cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Roots were harvested 96 - 99 days after sticking, dried at 28
°C for 3 days and weighted. Points represent means of 72 readings.
Vertical bars represent + SE.

 

0.20 -

0.15 -

 

0.10 ‘

 

 

0.05 -

 

 

 

 

a.“ 1 I I

 

Days in Storage

 

+ Brownii
-O— Dark Green Spreader
+ Hicksii
-v— Wardii

 

 

 

92

Fig. 5

Root Number

Effect of storage duration on root number of four cultivars of Taxus (1998
- 1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Root numbers were measured 96 - 99 days after sticking.
Points represent means of 72 readings. Vertical bars represent 4» SE.

 

20‘

104

 

 

 

 

Days in Storage

 

+ Brownii
—O— Dark Green Spreader
+ Hicksii
—v— Wardii

 

 

 

93

Fig. 6 Effect of storage duration on root length rating of four cultivars of Taxus
(1998 - 1999). Storage temperature = -2.5 - 2.5 °C. Stem cuttings were
collected from stock plants in the field, placed in cold storage for treatment
duration, and then placed in perlite propagation beds at 18 °C with bottom
heat (21 °C). Root length ratings were measured 96 - 99 days after
sticking. (0=less than 2.54 cm [1 inch], 1=less than 5.08 cm [2 inch],
2=less than 7.62 cm [3 inch], 3=greater than 7.62 cm) Points represent
means of 72 readings. Vertical bars represent + SE.

 

Root Length Rating

 

 

 

 

 

 

 

I l

34 70 107
Days in Storage

 

+ Brownii
—0— Dark Green Spreader
—v— Hicksii
—v— Wardii

 

 

 

 

Fig. 7 Effect of storage temperature and desiccation on chlorophyll fluorescence

Chlorophyll Fluorescence, FvlFm (relative units)

of cuttings of Taxus (1997 - 1998). Chlorophyll fluorescence (Ft/Fm) was
measured on the lower surface of individual dark-adapted (15 minutes)
needles with fluorometer (Morgan CF-1000) light level set at 700
umollm’ls. The first point of each line represents a mean of 40 readings,
all other points represent means of 80 readings. Vertical bars represent
+ SE.

Low Desiccation
1.000

 

0.800 1

 
  

 

0.600-l +000
0.400- +25
+5

03°01 —v— 10
0.000- +20

 

 

 

 

 

 

I I r

0 24 35
Medium Desiccation
1.000

g-

65

 

0.600 -

 

0.600 -
0.400 -
0.200 a
0.000 -‘ I

I I I

24 35

 

 

 

 

g.
8

0
High Desiccation
1.000

0.600 r ' _.
0.600 ‘

 

 

 

0.400 '-
0200 -
0.000 -1 I

I r T I I

24 35 50 65
Days in Storage

 

 

 

 

O

Fig. 8 Effect of storage duration on chlorophyll fluorescence of cuttings of Taxus
(1998 -1999). Storage temperature = -2.5 — 2.5 °C. Chlorophyll
fluorescence (FJF...) was measured on the lower surface of individual
dark-adapted (15 minutes) needles with fluorometer (PEA, Hansatech
instruments Ltd.) light level set at 1200 umollmzls. Points represent
means of 20 readings. Vertical bars represent + SE.

Chlorophyll Fluorescence, FvlFin (relative units)

 

(1900
0.850 -
0.800 1
0.750 4
(L700'-
0.650 -

0.600q

 

 

 

 

 

 

 

 

(L550

I I I

0 34 70 107

Days in Storage

 

—O— Brownii
—0— Dark Green Spreader
+ Hicksii
—v— Wardii