Andrew Holsinger Thesis

Southern Illinois University Carbondale
OpenSIUC
Theses Theses and Dissertations
1-1-2008
INFLUENCE OF BENZYLADENINE ON
SHOOT FORCING AND TISSUE CULTURE
OF JUGLANS NIGRA L. AND QUERCUS
RUBRA L.
Andrew Craig Holsinger
Southern Illinois University Carbondale, andrewholsinger@hotmail.com
Recommended Citation
Holsinger, Andrew Craig, "INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND TISSUE CULTURE OF
JUGLANS NIGRA L. AND QUERCUS RUBRA L." (2008). Theses. Paper 501.
http://opensiuc.lib.siu.edu/theses/501
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INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND TISSUE
CULTURE OF JUGLANS NIGRA L. AND QUERCUS RUBRA L.
by
Andrew Craig Holsinger, Jr.
B.S., Southern Illinois University, 2006
A Thesis
Submitted in Partial Fulfillment of the Requirements for the
Master of Science Degree
Department of Plant, Soil and Agricultural Systems

in the Graduate School
December 2008
THESIS APPROVAL
INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND TISSUE

CULTURE OF JUGLANS NIGRA L. AND QUERCUS RUBRA L.
By
Andrew Craig Holsinger Jr.
A Thesis Submitted in Partial
Fulfillment of the Requirements
for the Degree of
Master of Science
in the field of Plant and Soil Science
Approved by:
Dr. John E. Preece, Chair
Dr. Paul Henry
Dr. James Zaczek
Graduate School
December 2008
AN ABSTRACT OF THE THESIS OF
Andrew Craig Holsinger, Jr., for the Master of Science degree in PLANT AND
SOIL SCIENCE, presented on *October 31, 2008, at Southern Illinois University
Carbondale.
TITLE: INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND

TISSUE CULTURE OF JUGLANS NIGRA L. AND QUERCUS RUBRA L.
MAJOR PROFESSOR: Dr. John E. Preece
Shoot production and in vitro performance of Juglans nigra L and Quercus
rubra L. was studied where 0, 3, 10, 30, or 100mM benzyladenine (BA) in a 20%
white exterior latex paint diluted with deionized water were applied separately to
40 cm branch segments to determine the most effective concentration of
benzyladenine on bud break and shoot growth. Softwood shoot production was
maximized in the harvest months of March and April for J. nigra. Softwood shoot
production was maximized in the harvest months of April and August for Q.
rubra. Both shoot number and shoot length of softwood shoots decreased
linearly with increasing BA concentrations applied to the branch segments of
both species. Shoot production also decreased for both species during the
dormant season September-December. The softwood shoots were surface
disinfested and established on either 0 or 5µM Long Preece medium. When all
BA treated softwood shoots were compared to the controls, the BA in the
medium caused a significant increase in the number of shoots produced by
explants obtained from the branch segments painted with BA. Painting with BA
also increased shoot production in vitro, only if BA was also in the medium.
Nodal explants cultured on 5µM LP medium taken from softwood shoots forced
from branch segments painted with 3mM BA produced more shoots than any
i
other BA concentration applied to branch segments except nodal explants on
5µM LP medium taken from softwood shoots forced from branch segments
painted with 30mM BA.
ii
DEDICATION
I would like to sincerely thank my wife, Shana Holsinger, for her
unconditional love and encouragement.
iii
ACKNOWLEDGMENTS
I would like to express my gratitude to my major professor, Dr. John E.
Preece, for his patience, encouragement, and his necessary guidance for the
successful completion of my thesis. I also wish to thank my committee members
Dr. Paul Henry and Dr. James Zaczek for their guidance, support, and for serving
on my committee. They have both greatly helped to increase my knowledge of
trees. A wish to give special thank you to my fellow graduate student and friend
Anthony Fulford.
iv
TABLE OF CONTENTS
CHAPTER PAGE
ABSTRACT ...................................................................................................... i
DEDICATION ...................................................................................................iii
ACKNOWLEDGMENTS .................................................................................. iv
LIST OF TABLES ........................................................................................... viii
LIST OF FIGURES .......................................................................................... ix
CHAPTERS
CHAPTER 1 – INTRODUCTION ........................................................... 1

Goal ............................................................................................. 3
Objectives .................................................................................... 3
Null Hypotheses ........................................................................... 3
Shoot Forcing..................................................................... 3
In vitro. ............................................................................... 6
CHAPTER 2 – REVIEW OF LITERATURE ........................................... 9

Species Overview ........................................................................ 9
Juglans nigra L................................................................... 9
Juglans nigra L. Propagation ........................................... 10
Juglans nigra L. Micropropagation ................................... 10
Quercus rubra L. ............................................................. 11
Quercus rubra L. Propagation .......................................... 12
Quercus rubra L. Micropropagation ................................. 12
Types of Woody Stem Cuttings.................................................. 13
Vegetative Propagation .............................................................. 14
Epicormic Shoots ....................................................................... 15
Phase Change ........................................................................... 17
Shoot Forcing............................................................................. 19
Shoot Tip Forcing in Solution ........................................... 19
Shoot Forcing of Large Branch Segments ....................... 20
Advantages of Shoot Forcing ..................................................... 22
Shoot Forcing Limitations .......................................................... 23
Plant Growth Regulators ............................................................ 23
Cytokinins .................................................................................. 24
CHAPTER 3 – MATERIALS AND METHODS ..................................... 27

Stock Plants ............................................................................... 27
Application of Benzyladenine (BA) ............................................. 28
v
Shoot Forcing Environments ...................................................... 29
Tissue Culture ............................................................................ 30
Experimental Design, Data Collection, and Statistical Analysis ........... 31

Shoot Forcing Experiment................................................ 32
In vitro Experiment ........................................................... 32
Data Collection................................................................. 32
Statistical Analysis ........................................................... 32
CHAPTER 4 – RESULTS AND DISCUSSION .................................... 34

Shoot Forcing............................................................................. 34
Comparison between Painted and Non-painted Controls 34
Effect of Month of Harvest on Shoot Forcing ................... 35
BA Concentration ............................................................. 40
In vitro Results ........................................................................... 43
Culture Initiation ............................................................... 43
Influence of BA................................................................. 43
Influence of Explant Source ............................................. 46
CHAPTER 5 – Summary, Conclusion, Recommendation .................... 49
LITERATURE CITED ..................................................................................... 52
APPENDICIES
Appendix Table 1: Stock Solution Preparation Long and Preece Media

(LP Media, Long et al., 1995; Preece et al., 1995) ............................... 67
Appendix Table 2: Analysis of variance for the effects of 20% white

exterior latex paint applied to 40 cm long branch segments of Juglans
nigra L. on the mean number and length of shoots (≥ 3 cm long). ....... 68
Appendix Table 3: Analysis of variance for the effects of 20% white

exterior latex paint applied to 40 cm long branch segments of Quercus
rubra L. on the mean number and length of shoots (≥ 3 cm long). ...... 69
Appendix Table 4: Analysis of variance for the effects of Month of Harvest

and BA concentrations (only including painted) applied in paint to 40 cm
long branch segments of Juglans nigra L. on the mean shoot number.
Shoots were forced in a greenhouse in flats of vermiculite with drip
irrigation ............................................................................................... 70
vi
long branch segments of Juglans nigra L. on the mean length of shoots (≥
3 cm long). Shoots were forced in a greenhouse in flats of vermiculite with
drip irrigation. ....................................................................................... 71

long branch segments of Quercus rubra L. on the mean shoot number.
Shoots were forced in a greenhouse in flats of vermiculite with drip
irrigation. .............................................................................................. 72

long branch segments of Quercus rubra L. on the mean length of shoots
(≥ 3 cm long). Shoots were forced in a greenhouse in flats of vermiculite
with drip irrigation. ................................................................................ 73
Appendix Table 8: Analysis of variance for the effects of Month of

Harvest, Explant Source (Tip or Nodal), Media (0 or 5µM BA), and Shoot
forcing BA treatments (including painted and non-painted controls) applied
in paint to 40 cm long branch segments of Quercus rubra L. on the mean
shoot number of explants cultured in vitro from softwood shoots. ....... 74
Appendix Table 9: Analysis of variance for the effects of Month of

Harvest, Explant Source (Tip or Nodal), Media (0 or 5µM BA), and Shoot
forcing BA treatments (including painted and non-painted controls) applied
length of shoots of explants cultured in vitro from softwood shoots.. ... 75
Appendix Table 10: Analysis of variance for the pooled effects of BA

treatments (including combined painted and non-painted controls) applied
shoot number of explants cultured in vitro from softwood shoots.. ...... 76
Appendix Table 11: Analysis of variance for the pooled effects of BA

treatments (including combined painted and non-painted controls) applied
length of shoots (≥0.5 cm long) of explants cultured in vitro from softwood
shoots. ................................................................................................. 77
VITA ............................................................................................................. 78
vii
LIST OF TABLES
TABLE PAGE
Table 1: Main effect of month of harvest on shoot number and shoot length
(≥ 3cm long) of softwood shoots forced from branch segments of Juglans nigra L.
over a twelve month period............................................................................. 37
(≥ 3cm long) of softwood shoots forced from branch segments of Quercus rubra
L. over a twelve month period......................................................................... 38
Table 3: Main effect of BA concentration applied in latex paint to 40 cm branch

segments on mean shoot number and shoot length (≥ 3cm long) from softwood
shoots of Juglans nigra L. forced over a twelve month period. ....................... 41

shoots of Quercus rubra L. forced over a twelve month period. ..................... 42
Table 5: Effect of Month of Harvest x BA interaction with BA in the in vitro culture

medium on mean in vitro shoot number and shoot length (≥0.5 cm long) of
explants from the forced softwood shoots of Quercus rubra L. branch segments
treated with application of BA applied in 20% white exterior latex paint. ........ 45
Table 6: Effect of BA x Explant x Media interaction on mean in vitro shoot

number and shoot length (≥0.5 cm long) of explants forced from the forced
softwood shoots of Quercus rubra L.. ............................................................. 47
viii
LIST OF FIGURES
FIGURE PAGE
Figure 1: Softwood shoot forced from a J. nigra branch segment harvested in

Apr. (a), multiple softwood shoots forced from branch segments of Q. rubra
harvested in Apr. (b). ...................................................................................... 39
Figure 2: In vitro explants from shoot forcing control cultured on 5µM LP medium
(a), In vitro explants from branch segments painted with 3mM BA cultured on
5µM LP medium (b). ....................................................................................... 48
ix
1
CHAPTER 1
INTRODUCTION
Many tree and shrub species important to the nursery industry are
propagated clonally by rooting leafy softwood or semihardwood cuttings (Henry
and Preece, 1997a, b; Preece et al., 2002). Actively growing softwood shoots
are often better sources for rooting cuttings and establishing explants in vitro
than dormant hardwood cuttings for many difficult-to-root woody species
(Chalupa, 1987; Henry and Preece, 1997a, b; Preece et al., 2002). The difficulty
in obtaining softwood cuttings from temperate zone tree species has been the
lack of availability during the winter months (Henry and Preece, 1997a, b). In
temperate climates softwood shoots are produced during the late spring to early
summer.
Improving vegetative propagation of mature cultivars of Juglans nigra L.
(Coggeshall and Beineke, 1997; Van Sambeek et al., 1997) and Quercus rubra
L. could have several advantages over the current propagation methods used for
these species. The traditional methods of propagating J. nigra are with budding
and grafting on seedling rootstocks which have unknown genetic potential. Q.
rubra is traditionally propagated by seed because of graft incompatibility as the
grafts mature (Dirr and Heuser, 1987) and poor rooting percentages of softwood
cuttings. Superior characteristics could be selected from a single source plant or
ortet with an elite phenotype to reproduce a clonal population or ramets with the
desired traits if vegetative propagation methods were improved.
With J. nigra and Q. rubra being primarily propagated by seed because of

2
difficulties that occur using other propagation methods, most cultivars of these
species that are selected as clones are genetically heterozygous or due not
“come true” from seed. If sexual propagation were chosen a high percentage of
unsatisfactory genotypes among the progenies would occur. Thus, the uniformity
and unique characteristics desired for clonal selection would be immediately lost
if the cultivars chosen were sexually propagated by seed (Hartmann et al., 2002).
The time period to root and overwinter softwood cuttings successfully can
be extended by growing stock plants under higher air temperatures than field-
grown plants (MacDonald, 1986). Keeping stock plants in the greenhouse can
increase production costs and decrease the amount of useable space in a
nursery. Techniques to improve cost and propagation efficiency have been
desired to extend the time period to propagate softwood cuttings and extend the
availability of explants for micropropagation.
Shoot forcing is a method of vegetative propagation in which softwood
shoots are forced to grow from latent or epicormic buds. These epicormic buds
are forced from the lower portions of the tree or woody shrub as to exploit
juvenility characteristics found within the cone of juvenility. Softwood shoots are
desired by propagators because for some woody plants softwood cuttings are
more reliable to root than semi-hardwood or hardwood cuttings.
The focus of this thesis is to improve the vegetative propagation methods
of shoot forcing and tissue culture of adult J. nigra and adult Q. rubra.
3
Goal: To detect the year round influence of benzyladenine (BA) applied in
20% white exterior latex paint to branch segments on new softwood shoot
production and in vitro performance of explants of Juglans nigra L. and Quercus
rubra L.
Objectives:
1. To determine the most effective concentration of benzyladenine (BA)
on bud break and shoot growth applied to branch segments.
2. To determine if explants obtained were “primed” or more responsive
for tissue culture by application of exogenous cytokinin (BA).
3. To study the monthly effects for one year of (BA) on the production
and in vitro performance of forced epicormic shoots for stages I and II
micropropagation.
Null Hypotheses:
The results from the experiments presented in this work are formulated into the
following hypotheses:
Shoot Forcing
Ho1: There is no difference between the painted and non-painted control in
number of shoots produced by J. nigra using shoot forcing methods.
Ha1: There are differences between the painted and non-painted control in
number of shoots produced by J. nigra using shoot forcing methods.

4
length of shoots produced by J. nigra using shoot forcing methods.
length of shoots produced by J. nigra using shoot forcing methods.
number of shoots produced by Q. rubra using shoot forcing methods.
number of shoots produced by Q. rubra using shoot forcing methods.
length of shoots produced by Q. rubra using shoot forcing methods.
length of shoots produced by Q. rubra using shoot forcing methods.
Ho5: There is no difference among months of harvest for shoot number when
J. nigra branch segments were harvested for shoot forcing in a
greenhouse in flats of vermiculite with drip irrigation.
Ha5: There are differences among months of harvest for shoot number when
Ho6: There is no difference among months of harvest for shoot length when

5
Ha6: There is are differences among months of harvest for shoot length when
Ho7: There is no difference among months of harvest for shoot number when
Q. rubra branch segments were harvested for shoot forcing in a
Ha7: There are differences among months of harvest for shoot number when
Ho8: There is no difference among months of harvest for shoot length when
Ha8: There are differences among months of harvest for shoot length when
Ho9: There is no difference among BA treatments for shoot number when
Ha9: There are differences among BA treatments for shoot number when

6
Ho10: There is no difference among BA treatments for shoot length when J.
nigra branch segments were harvested for shoot forcing in a greenhouse
in flats of vermiculite with drip irrigation.
Ha10: There is no difference among BA treatments for shoot length when J.
nigra branch segments were harvested for shoot forcing in a greenhouse
in flats of vermiculite with drip irrigation.
Ho11: There is no difference among BA treatments for shoot number when
Ha11: There are differences among BA treatments for shoot number when
Ho12: There is no difference among BA treatments for shoot length when Q.
rubra branch segments were harvested for shoot forcing in a
Ha12: There are differences among BA treatments for shoot length when Q.
rubra branch segments were harvested for shoot forcing in a
In vitro
Ho13: There is no difference among months of harvest for shoot number of
explants cultured in vitro from softwood shoots of Q. rubra.

7
Ha13: There are differences among months of harvest for shoot number of
Ho14: There is no difference among months of harvest for shoot length of
Ha14: There are differences among months of harvest for shoot length of
Ho15: There is no difference among shoot forcing BA treatments for shoot
number of explants cultured in vitro from softwood shoots of Q. rubra.
Ha15: There are differences among shoot forcing BA treatments for shoot
number of explants cultured in vitro from softwood shoots of Q. rubra.
Ho16: There is no difference among shoot forcing BA treatments for shoot length
of explants cultured in vitro from softwood shoots of Q. rubra.
Ha16: There are differences among shoot forcing BA treatments for shoot length
of explants cultured in vitro from softwood shoots of Q. rubra.
Ho17: There is no difference between shoot tip and nodal explants of Q. rubra.
Ha17: There are differences between shoot tip and nodal explants of Q. rubra.
Ho18: There is no difference between media with 0µM BA and media with 5µM
BA on shoot number of explants cultured in vitro from softwood shoots of
Q. rubra.
Ha18: There are differences between media with 0µM BA and media with 5µM
BA on shoot number of explants cultured in vitro from softwood shoots of
Q. rubra.
8
Ho19: There is no difference between media with 0µM BA and media with 5µM
BA on shoot length of explants cultured in vitro from softwood shoots of Q.
rubra.
Ha19: There are differences between media with 0µM BA and media with 5µM
BA on shoot length of explants cultured in vitro from softwood shoots of Q.
rubra.
Ho20: There is no difference between pooled shoot forcing BA treatments and
pooled controls (painted and non-painted) on shoot number of explants
cultured in vitro from softwood shoots of Q. rubra.
Ha20: There are differences between pooled shoot forcing BA treatments and
pooled controls (painted and non-painted) on shoot number of explants
Ho21: There is no difference between pooled shoot forcing BA treatments and
pooled controls (painted and non-painted) on shoot length of explants
Ha21: There are differences between pooled shoot forcing BA treatments and
pooled controls (painted and non-painted) on shoot length of explants

9
CHAPTER 2
REVIEW OF LITERATURE
Species Overview
Juglans nigra L.
The genus Juglans L. (family Juglandaceae), although numerous and
widely distributed during the Tertiary geological period, now only comprises
about 20 species of deciduous trees (Harlow et al., 1979). Walnuts are
monoecious trees found in North, Central, and South America, the West Indies,
southern Europe, and southern and eastern Asia (Harlow et al., 1979). Six
species of Juglans are native to the United States (Harlow et al., 1979). Of these
species two are important sources of lumber J. nigra L. (eastern black walnut)
and J. cinerea L. (butternut) (Harlow et al., 1979). Walnuts are among the most
economically important nut trees in the world, and two species, J. nigra and J.
regia L. (Persian walnut), are widely cultivated for this reason (Jaynes, 1969;
Woodruff, 1979).
Eastern black walnut is a highly valued hardwood species found
throughout the eastern half of the United States (Harlow et al., 1979). This
species is not dominant in most forests, but rather is generally found as scattered
single trees or as small isolated groups within hardwood stands (Fischer, 1982)
on deep well drained, pH neutral soils (Dickerson, 2002). Black walnut has a
large geographical range but a narrow span of suitable sites where it can grow at
an adequate rate. Black walnut is relatively slow growing and when mature may
reach a maximum height of 30 - 37 m on good sites (Williams, 1990). The dark-

10
colored, fine-grained wood is highly valued for cabinet work, furniture, gunstocks,
interior trim, and veneer. The nuts of J. nigra are valued by humans for
confectionary uses as well as by wildlife as a source of food.
Flowering of black walnut occurs in mid April and early June with male and
female flowers maturing at different times (McDaniel, 1956). Leafing out occurs
at approximately the same time as flowering and both are early enough for
possible damage by late spring frosts (Funk, 1979). The average length of the
juvenility period in black walnut is 12 years (Brinkman, 1974).
Juglans nigra L. Propagation
Propagation of black walnut is usually by seed (Dirr, 1998). Dormancy of
the seed must be broken by cold stratification before germination can occur.
Vegetative propagation has been largely unsuccessful through rooting of cuttings
(Coggeshall and Beineke, 1997). Cultivars of Juglans spp. are vegetatively
propagated by grafting onto seedling rootstocks.
Juglans nigra L. Micropropagation
Micropropagation of J. nigra using axillary shoots from adult tissues has
been reported unsuccessful by many authors (Cummins and Ashby, 1968;
Huetteman et al., 1986; Lenartowicz and Millikan, 1977, Somers et al., 1982) and
unfortunately those with success have only found it with juvenile material. A
standardized axillary shoot culture protocol for black walnut has yet to be
11
developed for J. nigra, although protocols have recently been developed for J.
regia.
Quercus rubra L.
The genus Quercus L. (family Fagaceae), known as oak, includes some
500 species with 58 of these in the United States (Little, 1979). The genus
consists of three major groups; red or black oaks (Quercus section Lobatae),
white oaks (Quercus section Quercus), and the third section, known as the
intermediate group (Quercus section Protobalanus) (Nixon, 1997). The red oaks
and white oaks include evergreen and deciduous species, whereas the
intermediate oaks are all evergreen (Johnson et al., 2002).
Northern red oak (Quercus rubra L.) is a forest tree species found
throughout the eastern half of the United States (Harlow et al., 1979). This
species is found on a range of sites from xeric (very dry) to mesic (moderately
moist). The best site conditions to grow northern red oak are deep, well-drained
loams to silty clay loams (Sander, 1990). Northern red oak often reaches mature
heights of 20 - 30 m. Northern red oak is a valuable species with timber used for
flooring, furniture making, and veneer. Northern red oak is also an urban forestry
species and is valuable to wildlife for its nuts.
Northern red oaks are monoecious having both staminate (male) and
pistillate (female) flowers on the same tree. Flowering of northern red oak begins
at approximately at 15 - 25 years old (Johnson et al., 2002). The staminate and
pistillate flowers are produced at the same time from April - June. The pistillate
12
flowers are formed from tissues located in leaf axils and usually are more
abundant in the upper crown (Cecich and Larsen, 1997). The staminate flowers
are usually found in the top and middle portions of the crown (Johnson et al.,
2002). Fertilization occurs in mid-June for northern red oak in the 2nd yr. (Cecich
and Haenchen, 1995). Northern red oak requires two growing seasons between
pollination and seed maturation.
Quercus rubra L. Propagation
Northern red oak is propagated by seed. A good seed crop is produced
every 3 to 5 years (Dirr and Heuser, 1987). The seeds of oaks are particularly
difficult to store and manage and cause seedling variation in phenotypic traits.
Quercus rubra L. Micropropagation
There have been difficulties in finding an efficient micropropagation
method of northern red oak. A recent protocol for micropropagation of Quercus
spp. was published in which Q. rubra was included (Ostrolucka, et al., 2007).
However, the disinfestation process described in this protocol for mature material
included washing for 1 h, treating for 5-10 min in 70% ethanol and 10-15 min in
0.1% solution of mercuric chloride with 3 drops of Tween, followed by rinsing in
sterile distilled water (3 x 15 min) under aseptic conditions. The stem cuttings
used were only collected from Feb. to mid Mar., and were obtained from the
shoot tips, which are more adult. Only nodal explants were used in this protocol.
The culture medium used was Woody Plant Medium (WPM; Lloyd and McCown,
13
1980) supplemented with plant growth regulators 2.22-4.44 µM benzyladenine
(BA) and 0.54-2.69 µM naphthaleneacetic acid (NAA). Other components used
in the media were 20 g⋅L-1 sucrose and 6 g⋅L-1 Difco-Bacto Agar with pH adjusted
to 5.5-5.7 before autoclaving at 121 °C and 108 kPa for 20 min.
Vieitez et al., (1993) also used an initiation medium consisting of WPM
with 4.44 µM benzyladenine, although the sucrose was 30 g⋅L-1 in their protocol,
the same type and amount of agar was used with a similar pH adjustment to 5.5-
5.6. The surface disinfestation procedure called for successive immersion for 30
s in 70% alcohol (presumably ethanol) and 10 min in 12% commercial sodium
hypochlorite (40 g⋅L-1 of active chlorine), followed by three sterile rinses in sterile
distilled water (time not listed). The mature material was collected in Dec. and
cut into 15-20 cm lengths and stored at 4 °C until Apr. or May., when the cuttings
were forced to flush in a climate chamber with a temperature of 24 °C, a 16-h
photoperiod and a relative humidity of 80-85%. Similar cold storage was
reported by Sanchez et al., (1996).
Types of Woody Stem Cuttings
The stems of woody plants are often divided morphologically into three
stages of growth: softwood, semi-hardwood and hardwood (Hartmann et al.,
2002). Softwood cuttings are taken from current season‟s growth before
extensive lignification has occurred. Semi-hardwood cuttings can be subdivided
into two groups, soft and firm, based on the degree of lignification. With soft
semi-hardwood cuttings, the shoot is still growing but lignification occurs

14
acropetally, whereas with firm semi-hardwood cuttings, the entire stem
undergoes varying degrees of lignification. Hardwood cuttings are usually taken
in the fall or winter when the previous season‟s growth is complete. Hardwood
stems are firm and woody, usually exposed to at least one frost, and leaves of
deciduous plants have abscised or can be pulled off easily without tearing the
bark (MacDonald, 1986).
Vegetative Propagation
Vegetative propagation is primarily used for woody perennial plants that are
heterozygous (Hartmann et al., 1988). Since most woody plants do not “come
true” from seed it is often desired to propagate them clonally. Determining
whether vegetative propagation of a woody plant is desired is ultimately based on
phenotypic traits when maturity is reached. Softwood cuttings from field-grown
trees are available from stump sprouts, epicormic shoots, and the new growth
from the crown of the tree. Cuttings excised from these sources are more
susceptible to contamination in vitro than from softwood shoots obtained from
stock plants in a greenhouse or forced stem segments (Preece and Reed, 2003).
Trees with superior phenotypic traits may be selected for forcing shoots to
enable their mass propagation (Harmer, 1988; Henry and Preece, 1997a, b).
Vegetative propagation of woody plants has proven easier by using propagules
from sources of juvenile origin rather than adult. Ontogenetic juvenile-mature
gradients occur in trees acropetally from base to apex of the main stem (Fishel et
al., 2003). Juvenile material can be obtained from the lower branches or select
15
stems of intact trees and woody shrubs by forcing epicormic buds to break and
produce softwood to semihardwood shoots (Cameron and Sani, 1994; Harmer,
1988; Henry and Preece, 1997a, b; Preece et al., 2002). Sections of lower
branches from trees can be forced in a greenhouse or other controlled
environments such as a laboratory (Aftab et al., 2005; Mansouri and Preece,
2006). Epicormic shoots can be forced by cutting the trunk of a tree into sections
although this may eliminate future opportunities to propagate from the original
stock plant. The ability to force new softwood growth from stem segments of
mature trees in a greenhouse or laboratory allows for less in vitro contamination
than from field grown woody plants (Aftab et al., 2005).
Epicormic Shoots
Epicormic buds may be adventitious in origin but are usually formed from
dormant axillary buds; occurring in most angiosperm tree species and a few
gymnosperms (Gordon et al., 2006). Axillary buds are formed from pockets of
meristematic cells in the axils of leaves. These meristematic cells remain
spatially associated with the leaf axil even when appearing detached from the
apical meristem because of vacuolation of intervening cells; these are called
detached meristems (Evert, 2006). In some plants, axillary meristems undergo
immediate development to form an axillary shoot. In other plants, axillary
meristems might initiate a few leaves and then become developmentally arrested
or dormant because the terminal bud inhibits the growth of axillary buds. This
type of control over axillary buds is known as apical dominance.

16
Dormant axillary buds may become embedded in the stem of woody
plants as the periderm (bark) tissue forms around them (Gordon et al., 2006).
These epicormic axillary buds are often referred to as dormant, latent, or
suppressed although they are active during much of the growing season laying
down new leaf and scale primordia (Brown, 1971).
Epicormic buds elongate keeping pace with the radial expanding growth of
the vascular cambium through which they make their vascular connection or bud
trace (Brown, 1971; Gordon et al., 2006). This bud trace connects the bud all the
way to the pith in epicormic axillary buds (Pallardy, 2008). These dormant
axillary buds resume development at a later time depending on their
developmental program or in response to environmental cues (Shimizu-Sato and
Mori, 2001). Some of the main causes for the initiation of epicormic bud growth
are exposure to increased light levels, breakage, pruning, or fire (Helms, 1998).
Adventitious buds (and shoots) arise in irregular patterns from any part
other than leaf axils or apical meristems (Evert, 2006; Hartmann et al., 2002).
Adventitious buds may develop on root, internode, hypocotyl, leaf, callus or
lignotuber tissues (Evert, 2006). Unlike dormant buds, adventitious buds do not
have a bud trace all the way to the pith (Pallardy, 2008). On rare occasion,
adventitious buds resulting from localized injury may become embedded in the
periderm (Brown, 1971).
Epicormic, axillary, or adventitious buds can remain latent just beneath the
bark for many years. Fontaine et al. (1999), examining Quercus petraea Matt.
17
Liebl., showed that small and large individual epicormic buds can survive for 40
years.
Visible swelling before bud break occurs and can be observed with both
types of epicormic buds (Books and Tubbs, 1970; Church and Godman, 1966;
and Preece et al., 2002). When epicormic bud break occurs the new shoot must
push its way through the bark. The bark becomes an increasing mechanical
barrier with its increasing age and may decrease the ability of epicormic buds to
sprout.
Phase Change
There are three phases during post-embryonic development in plants: a
juvenile vegetative phase, an adult vegetative phase, and a reproductive phase
(Poethig, 1990). These three phases represent ontological changes and can be
distinguished by various vegetative characteristics, including leaf shape, leaf
thickness, leaf retention throughout the winter in temperate climates, leaf
epidermal characteristics, phyllotaxis, thorniness, shoot orientation, vigor of shoot
growth, anthocyanin pigmentation, disease and insect resistance; and
competence to form adventitious buds, roots and somatic embryos (Hackett and
Murray, 1993).
Flowering cannot be induced under normal conditions in the juvenile
phase. When flowering does occur under normal conditions, the juvenile phase
is ended and the adult reproductive phase begins (Poethig, 1990, Hacket, 1985).
18
The juvenile phase can last up to 30-40 years for certain forest trees but length of
juvenility is ultimately dependent upon species (Hackett, 1985).
Differences in the maturation of tissues along the vertical axis of a tree
can be represented by the “cone of juvenility”. The cone of juvenility refers to the
innermost portion of a mature seed-propagated tree (Schaffalitzky de Muckadell,
1954; Preece, 2008). Juvenile growth maintains its position near the bottom of a
tree. As trees mature, a transition zone can be observed along the middle of the
vertical axis where both juvenile and adult traits can be found. The reproductive
structures and other adult characteristic traits are found toward the outer crown
of trees. Thus the conical shape exhibited is from the oldest part of the tree
when the tree was still in the juvenile phase of growth. The lower portions of a
tree, the lower trunk and branches, exhibit juvenile characteristics; whereas the
outer portions of a tree, the upper trunk and crown, exhibit adult characteristics.
The juvenile portion of the tree is readily identifiable in some species, such as
Quercus (oak), Fagus (beech), and some Acer (maple), in which leaf retention
(marcescence) in the winter is apparent in the juvenile portions of the tree
(Preece, 2003).
Vegetative propagation is generally easier when plants are in the juvenile
phase than when they have reached the adult phase (Preece, 2008). Cuttings
taken from adult shoots of plants can be rooted, but the frequency of success is
often low, especially in woody plants (Preece, 2008, 2003). It was observed that
cuttings taken from shoots formed in the basal region had a higher capacity to
19
form adventitious roots than those taken from shoots in the upper part of the
plant (Hackett, 1985).
Shoot Forcing
Shoot forcing can be defined as a method of vegetative propagation that
results in the initiation and growth of new softwood shoots from on a wide variety
of woody ornamental plants. There are differences in shoot forcing techniques
depending on differences in size and morphololgy of stems, branches, or twigs.
Successful vegetative propagation has been achieved by forcing new growth on
stems, branches, or twigs.
Shoot Tip Forcing in Solution. The technique of forcing softwood shoots from
dormant woody shoot tips using forcing solutions that are similar to those used
as floral preservatives has been shown to extend the season of availability for
rooting softwood cuttings and obtaining explants for use in micropropagation
(Preece and Read, 2003). Stems to be forced can be collected in the fall and
held in cold storage at 4 – 5 °C thus saving valuable greenhouse space and
reducing the need to keep stock plants in the greenhouse during the winter
months (Yang and Read, 1992).
The stem tips harvested for forcing are usually 20-25 cm long and of
deciduous origin (Read and Yang, 1991). Before the cut stems are placed into
forcing solutions, the basal 1/3 of the stems are soaked for 15 min in a solution
containing 15% bleach (0.78% NaClO) in water and 20 drops of Tween-20

20
(wetting agent) liter-1 to provide cleaner cuttings and enhance bud break (Read
and Yang, 1989). Following this disinfestation, the basal 0.3 to 1.0 cm of the
stem is excised; this is repeated every 2-3 days (Yang and Read, 1997). The
basal 1/3 of the stems is placed in a forcing solution containing 200 mg⋅L-1 8-
hydroxyquinoline citrate (8-HQC) and 2% sucrose (Yang and Read, 1993 &
1997).
Plant growth regulators (PGRs) can be added to the forcing solution.
This was found to be an effective way to stimulate bud break, enhance in vitro
shoot proliferation, and promote rooting (Read et al., 1984, 1986; Yang and
Read, 1990).
Shoot Forcing of Large Branch Segments
The history of forcing epicormic shoots to develop on large stem segments
is quite diverse, spanning many years. Europeans used large stem segments
obtained from pruned limbs, called truncheons, to produce olive trees (Brown,
1916). Truncheons of olive can be split in half, or quartered depending on
diameter of the stem segment. Diameter can vary from 1 – 7.5 cm. These
truncheons are then buried horizontally 7.5 – 10 cm below the surface of the
propagation bed.
Lower branches from intact trees, boles or large stems from shrubs can be
removed and cut into sections for shoot forcing (Henry and Preece, 1997a, b).
The lower branches or stems are cut into segments to be placed horizontally in a
medium suitable for the forcing conditions selected. The medium can be
21
contained in horticultural flats, greenhouse benches or in frames built with a
mesh material such as shade cloth or screen. The lower branch or stem
segments are recommended to be at least 30 cm long and 1 cm in diameter
(Preece and Read, 2003). Varying differences in shoot production were found
for species of Acer based upon diameter and length (Henry and Preece, 1997b).
Forcing conditions may include intermittent mist, fog, drip irrigation, or hand-
watering in a greenhouse or laboratory.
Growth chambers have also been used in a laboratory setting to force
softwood shoots (Vieitez et al., 1994). Perlite is the most suitable medium for
forcing under intermittent mist because of its excellent drainage (Aftab et al.,
2005). Coarse vermiculite is the most suitable medium for forcing using drip
irrigation because of its excellent water retention. Interestingly, Ikemori (1987)
using Eucalyptus grandis W. Hill ex Maid. Forced shoots 3 cm long after 15 to 20
days using no medium under intermittent mist with the ends of the branch
segments sealed with paraffin wax. Aftab et al. (2005) also reported successful
forcing without medium. They found that when forcing under fog conditions more
shoots were harvested from empty flats than flats with perlite, vermiculite, or a 1
perlite : 1 vermiculite mix (by volume).
The technique of forcing softwood shoots from large branch and or stem
segments has allowed for an extended production time from late winter to early
autumn compared to late spring through summer on field-grown plants (Preece
et al., 2002). Harmer (1988) forced large stem segments of Quercus robur L.
and found that the largest numbers of shoots were produced on segments taken
22
in late winter and spring. Late winter to early spring rooting of softwood shoots
allows for an extended growing season compared to traditional methods of
obtaining softwood shoots (Preece et al., 2002). The larger segments can also
be held in cold storage for extended time until propagation (Henry and Preece,
1997a).
Advantages of Shoot Forcing
Advantages of using shoot forcing as a propagation method whether using
shoot tips or large segments are: decreasing the heavy spring workload in
nurseries, increasing the amount of work in the winter to employ nursery workers
year round, and allowing for some species, e.g. Acer palmatum Thunb., to
become established in the first year of propagation (Henry and Preece, 1997).
Excision of cuttings can occur when greenhouse or laboratory forced shoots are
of softwood morphology. This is advantageous over obtaining them from the
field where growing conditions are less controlled.
Ikemori‟s (1987) conclusions for using this technique are still valid today.
“The advantages of the technique are:
1. It makes it unnecessary to fell the selected tree.
2. The explants are subject to less contamination because they
develop in a glasshouse.
3. The morphology of the leaves produced from the proventitious
buds are of the juvenile type that can enhance multiplication and
rooting in vitro.”
23
A proventitious epicormic bud originates from an existing bud and is
connected to the pith of the main stem (Fontaine et al., 1999).
Shoot Forcing Limitations
Branch or stem segments that are cut for forcing early in the dormant
season (October-December) tend to produce few if any visible buds or epicormic
shoots of viable use for micropropagation (Van Sambeek et al., 2002). Carya
illinoinensis (Wangenh.) C. Koch (Pecan) was unable to break bud when forced
under lab conditions during August to November, 2004 (Aftab and Preece, 2007).
The onset of dormancy in field-grown woody plants limits the ability to force an
adequate number of softwood shoots during the dormant season. Shoot forcing
becomes an adequate method for forcing softwood shoots when the dormancy
requirement is met for a species.
Plant Growth Regulators
Plant growth regulators (PGRs) are a group of organic substances that
may occur naturally or be synthesized to influence physiological processes at low
concentrations (Davies, 2004). The plant growth substances that occur naturally
in plants are referred to as plant hormones. These plant hormones may or may
not be translocated from their source of origin. The importance of plant growth
regulators in plant tissue culture is well documented. The two classes of plant
growth regulators primarily used in plant tissue culture are auxins and cytokinins.
The classes of gibberellins, ethylene, and abscisic acid have had only a
24
secondary role in plant tissue culture compared to auxins and cytokinins. Auxins
and cytokinins have been found to be necessary for plant growth and
development. It must be understood that explants contain endogenous levels of
both auxin and cytokinins. The balance of auxins and cytokinins is therefore
influenced by those included in the medium and within the explant.
Cytokinins
Cytokinins were first discovered in the 1950s as a class of plant-specific
hormones named for their ability to promote cytokinesis - cell division (Miller et
al., 1955). The first cytokinin discovered was kinetin. Kinetin was first isolated
and purified from autoclaved herring sperm DNA (Miller et al., 1956); it is not
naturally produced and has not been found in living plants (Sakakibara, 2004).
Naturally occurring cytokinins are all N6-substituted adenine derivatives. These
adenines have substituted at the N6 terminal either an isoprene-derivied side
chain (isoprenoid cytokinins), or an aromatic derivative side chain (aromatic
cytokinins) (Sakakibara, 2004). The first naturally occurring cytokinin to be
discovered was zeatin, which was isolated from immature endosperm from seeds
of Zea mays L. in the 1960s (Letham, 1963; Miller, 1961). Other natural
occurring cytokinins include isopentenyladenine, dihydrozeatin, trans-zeatin, (4-
hydroxy-3-methyl-trans-2-butenylaminopurine), and (6-(4-hydroxy-3-methyl-
trans-2- butenly)aminopurine) (Staden et al., 2008).
Benzyladenine (BA), an aromatic cytokinin and its derivatives have been
isolated and identified in some plant issues (Ernst et al., 1983, Nandi et al., 1989
25
a,b). This cytokinin was once thought to be purely synthetic. It is still generally
viewed as a synthetic compound. Benzyladenine is the most frequently and
successful cytokinin used in micropropagation. One of the reasons for
benzyladenine being frequently used is because it is easy to produce and thus
has a lower cost than many of the other available cytokinins.
Cytokinins are believed to be synthesized in highest concentrations in
young or meristematic tissues, e.g., root apices, cambial tissue, developing
endosperm in seeds, young fruits, and developing shoot buds (Srivastava, 2002).
The function of cytokinins may be stimulatory or inhibitory in a number of different
development processes including root growth and branching, release of apical
dominance in the shoot (lateral buds), chloroplast development, and delay of leaf
senescence (Mok, 1994). The major sites of natural cytokinin biosynthesis
appear to be in the root apices (Staden et al., 2008). It is from the roots that
natural cytokinins are transported through the xylem and are distributed
throughout the plant.
Another group of cytokinins are the phenylureas. They are of synthetic
origin and are not produced by plants (Mok and Mok, 2001) although they were
first reported to be isolated from liquid coconut endosperm (Sakakibara, 2004).
A phenylurea with high cytokinin activity is the thiadiazole-substitued phenylurea:
thidiazuron (TDZ) (Mok et al., 1982). It was originally developed and registered
as a cotton defoliant under the trade name of „Dropp‟ (Arndt et al., 1976).
Thidiazuron has been shown to be more effective in some plants than adenine
26
based compounds for stimulating growth of callus or shoot proliferation
(Huetteman and Preece, 1993; Sakakibara, 2004).

27
CHAPTER 3
MATERIALS AND METHODS
Stock Plants
Lower branches were harvested from the lowest 3 m of northern red oak
and black walnut trees for one year during the first week of each month (March
2007-February 2008) from two different plantations. The northern red oak
plantation is an abandoned plantation (USDA Forest Service) near Carbondale,
IL established on SIUC property and the black walnut plantation is located at the
Tree Improvement Center at the Southern Illinois University Carbondale Forestry
Department Forest Education & Research Station (ForestERS), Carbondale, IL .
The northern red oak trees were 18 yr old seed-origin trees ranging from 10 - 12
m tall. The black walnut trees were 37 yr old seed-origin trees ranging from 20 -
23 m tall. All secondary branches were removed and the resulting branches
were transported to the Tree Improvement Center. The branches were then cut
into 40 cm long segments with a minimum distal diameter of 2cm.
Once cut, branch segments of each species were sorted and placed into
five groups according to their diameter to facilitate blocking in the greenhouse
and to use blocking to reduce the effect of diameter during statistical analysis.
The black walnut and northern red oak branch segments were randomized
together within each of the five blocks. Blocking was also used to reduce the
effect of greenhouse variation across the benches due to variation of
temperature.
28
Application of Benzyladenine (BA)
The plant growth regulator 6-benzyladenine (BA) was used in the shoot
forcing experiments. The BA was dissolved in 1N KOH and diluted with
deionized water unless otherwise noted. Treatments were randomly drawn and
assigned to branch segments. The treatment concentrations of 0, 3, 10, 30 or
100 mM BA were applied to the branch segments in a 20% white exterior latex
paint1. The entire surface of each of the branch segments with the exception of a
non-painted control were painted and allowed to air dry. Application of the BA to
the branch segments of the northern red oak was less difficult than application to
the black walnut as the black walnut bark was more furrowed than the northern
red oak bark. Although, equal amounts of each concentration of BA by volume
were distributed to the surface of each branch segment with only one application
of paint per branch segment with the 20% white exterior latex paint as the carrier
for the BA. The experiments all had two controls, painted with 0 BA and a non-
painted control.
50 mL of white exterior latex paint was used in each of the treatments
containing paint, and then the appropriate BA concentration was added following
dilution with deionized water until the solution reached a final volume of 250 mL
resulting in a 20% paint solution. The treatments were mixed in 1000 mL
canning jars2 with wooden paint stirrers each labeled for the appropriate
concentration of BA used. A different nylon brush was used to apply each
1
The Sherwin-Williams Company, Exterior latex super white, Cleveland, OH.
2
Ball Corp., Muncie IN.
29
treatment separately to the branch segments.
Shoot Forcing Environments
The experiments were conducted in the eastern greenhouse located at the
Tree Improvement Center. Three benches were used for shoot forcing in a
newly renovated greenhouse range which allowed for the next run of the
experiment to occur simultaneously with previous runs allowing adequate time to
determine if shoot production had been exhausted. Temperature in this range
was controlled with a newly installed pad and fan system along the west wall
controlled with a Procom greenhouse controller3. With fans along the east wall
pulling air from the west, it was expected there would be a west-east temperature
gradient created. On 10 May 2007 temperature and light levels were reduced by
the application of Koolray® Ultra White liquid shade compound4 to the glass of
the greenhouse. Approximately 30% shading was achieved. The shade
compound was removed on 15 Oct. 2007 to raise light levels. The greenhouse
experiments were conducted from Mar. 2007-Feb. 2008 under a natural
photoperiod with night interruption from 10:00 PM to 2:00 AM provided by light from
400w metal halide bulbs.
The shoot forcing environment consisted of black polyethylene 10205 flats
with drainage holes (52.2x25.9x6.0 cm, LengthxWidthxHeight) filled with
3
Manufactured by Micro Grow Greenhouse Systems, INC, Temecula, CA
4
The Continental Products Company, Euclid, OH
5
T.O. Plastics, Inc., Minneapolis, Minn.
30
horticultural-grade coarse vermiculite approximately 4cm deep. The branch
segments of both species were placed horizontally in the flats and pushed half
way down into the vermiculite and then drenched with water. The vermiculite
was irrigated using drip irrigation (to avoid microbial contamination) for 30 min, 3
times a day, using three drip tubes (1.0 mm diameter) per flat. The drip irrigation
system consisted of a network of tubes (1.5 cm inner diameter), made of black
polyvinyl chloride to carry water at a low flow rate under low pressure to the
branch segments. Drip tubes were attached to the black pvc pipes and had lead
weights at the end of the tubes to keep them in place. Data were collected on
the number and length of softwood shoots that grew from the large branch
segments.
Tissue Culture
The number of softwood shoots harvested weekly during each month
varied. Softwood shoots were harvested when at least 8-10 shoots ≥ 3 cm long
grew from any of the branch segments. All leaves were removed from shoots
with the exception of one to aid in handling during excision of the softwood shoot
from the branch segment. Once shoots were excised the remaining leaf was
removed and the cutting was then labeled according to the treatment applied to
the branch segment. After each cutting was collected and labeled they were
wrapped with wet paper towels placed into a (26.8 cm x 27.3 cm) plastic storage
bag to prevent dehydration until transport to the tissue culture lab.
Softwood cuttings of both species were separately disinfested using the

31
same disinfestation procedure. The cuttings were disinfested for 20 min with
0.6% NaClO plus 0.1% Tween 20; then rinsed three times (5 min each) with
sterile deionized water in a laminar flow hood6. The disinfested cuttings were cut
into 1.5 cm long nodal and shoot tip explants. These explants were cultured on
Long-Preece (LP) medium (Long et al., 1995) (Appendix Table 1) with 0 or 5 µM
BA, supplemented with 30 g⋅L-1 sucrose and the pH was adjusted to 5.8 prior to
the addition of 6.5 g⋅L-1 plant tissue culture grade agar7 and autoclaved for 20
min at 121°C and 1 kg⋅cm-2 pressure. Explants were placed vertically on 15 mL
of medium in glass culture tubes8 (25x150 mm) under aseptic conditions. Caps
were sealed to the tube with white Viscose-Celons9 (30.5x25 mm, WxH),
randomized and incubated under Cool White® fluorescent lamps10 that provided
a 16-h photoperiod at 22 ±1°C with photosynthetic photon flux of 35 to 47 µmol s-

1
m-2 provided by 40-w cool white fluorescent lamps. The tubes were labeled
according to treatment applied to the large branch segments and explant type.
The run and harvest date were labeled on the culture tube racks.
Experimental Design, Data Collection, and Statistical Analysis
6
Edge Guard, The Baker Company, Sanford, ME.
7
Phytotechnology Laboratories, Shawnee Mission, KS.
8
Bellco Glass, Inc., Vineland NJ.
9
Kaufman Container, Cleavland, OH.
10
Westinghouse Elect. Corp., Bloomington, NJ.
32
Shoot Forcing Experiment . The shoot forcing experiment was arranged in a
randomized complete block design with five blocks for each run (harvest month)
of the experiment and six treatments and both species within each block. Branch
segments were blocked according to diameter to reduce variation in the
analyses. Data of number of shoots and the length of each shoot were collected
weekly from softwood shoots ≥ 3 cm long produced from the branch segments
and cultured in vitro.
In vitro Experiment. In vitro experiments were arranged in a completely
randomized design and replication depended on the number and length of
softwood shoots that were produced during forcing. Data were taken on number
of shoots ≥ 0.5 cm long growing from explants and their length.
Data Collection.
Shoot Forcing Data were collected weekly on each run in the greenhouse until
there were a total of ≤ 8 softwood shoots produced during a one week period.
Tissue Culture Data were taken biweekly on the number of shoots ≥ 0.5 cm long
growing from the explants and shoot length after transfer of non-contaminated
explants to fresh medium every two weeks. Final analyses are based on data of
non-contaminated explants after 12 weeks of in vitro.
Statistical Analysis. Data were transformed using √(y+0.5) when ≥ 50% of the
33
values were zero (Steel and Torrie, 1980). Data were analyzed using using proc
glm and mixed procedures (SAS Institute, Inc., 2002-2003). Trend analyses
were done to determine whether there was a linear or quadratic trend on shoot
number and length for month of harvest of J. nigra and Q. rubra. Trend analyses
was done to determine whether there was a linear or quadratic trend on shoot
number and length for BA concentrations applied to branch segments of J. nigra
and Q. rubra. The species were analyzed separately because of differences in
shoot production for each species. Data will be analyzed as a split plot with
harvest months as the main plots and BA treatments as the split plot.
34
CHAPTER 4
RESULTS AND DISCUSSION
Shoot Forcing
Comparison between Painted and Non-painted Controls
The shoot forcing experiment had two controls, painted and non-painted
branch segments for each monthly run of the experiment. Two controls were
used to determine if there was any effect of white exterior latex paint on softwood
shoot production. There were significant differences between the two controls on
softwood shoot production in the 12 runs of the experiment for Juglans nigra L.
shoot number (P=0.0066) and shoot length (P=0.0018) (Appendix Table 2).
Although these results indicate that 20% white exterior latex paint had a
statistically significant effect on shoot production for J. nigra, the horticultural
significance was small as only a 0.1 increase in shoot number compared to non-
painted control and only a 0.7cm increase in shoot length. There were no
significant differences between the two controls for Quercus rubra L. shoot
number (P= 0.7805) or shoot length (P= 0.9783) (Appendix Table 2). Thus,
painted controls are used throughout the analyses reported in results and
discussion.
The effect of the 20% white exterior latex paint on Juglans nigra L. slightly
increasing both shoot number and length may be because of a conservation of
moisture by the coat of paint applied or because of a reduction of temperature
with white paint reflecting more light than the non-painted control. In Quercus
rubra L., however, there were no differences between either painted or non-
painted controls. Mansouri (2006) found similar results in her study of Acer
35
saccharinum L. with no difference between either painted or non-painted
controls.
The differing results of shoot number and length between species indicate
differences in the ability of each species to respond to the shoot forcing
environment in a greenhouse using drip irrigation. Van Sambeek et al. (2002)
and Van Sambeek and Preece (1999) both reported differences among black
walnut and red oak species in number of shoots produced by various shoot
forcing environments. In comparison to the results of Fishel et al. (2003)
northern red oak produced a greater amount of shoots using mist than reported
in this thesis using drip irrigation.
Effect of Month of Harvest on Shoot Forcing
The main effect of month of harvest was found to be significant for both
shoot number (P= 0.0001) and shoot length (P= 0.0001) of softwood shoots
produced by J. nigra branch segments (Appendix Tables 4 and 5). The main
effect of month of harvest was found to be significant for both shoot number
(P= 0.0002) and shoot length (P= 0.0001) of softwood shoots produced by Q.
rubra branch segments (Appendix Tables 6 and 7).
J. nigra shoot production followed a quadratic trend and was similar from
the harvest months of Mar 2007-Aug 2007; thereafter few, if any shoots were
produced (Table 1). Specifically, there were no statistical differences between
the harvest months of March and April which had the highest shoot production
when compared to any of the other months for shoot number and length (Table
36
1 and Fig 1). Shoot production was lower and not significantly different among
any other months for J. nigra according to the 5% t test (Table 1).
Q. rubra shoot production decreased linearly in both shoot number and
length from Mar 2007-Feb 2008 (Table 2). There were no statistical differences
between the harvest months of April and August which had the highest shoot
number when compared to any of the other months for Q. rubra according to the
5% t test (Table 2). There were no significant differences between the harvest
months of April and September which had the longest shoot lengths when
compared to any of the other months for Q. rubra according to the 5% t test
(Table 2).
Branch segments of J. nigra when cut early in the dormant season (Sept.-
Dec.) exhibited the same effects of endodormancy as reported by Van Sambeek
et al., (1997) in that they failed to produce softwood shoots. Branch segments of
Q. rubra also exhibited endodormancy during the dormant season (Sept.-Dec.)
with a decline in shoot number although some softwood shoots did continue to
be produced.
J. nigra branch segments from the harvest months of Sept.-Feb. produced
few if any visible buds or epicormic shoots of sufficient length for culture in vitro.
It was reported by Jacobs et al. (2008) that the chilling requirement for J. nigra
seedlings held at 3 °C cold storage was 119 days. These results correspond
with those of Van Sambeek et al., (1997), who conducted a yearlong study using
30 cm branch segments from 30 year old grafted J. nigra of three different nut
cultivars forced under laboratory conditions. They found that after 90 days of
37
(≥ 3cm long) of softwood shoots forced from branch segments of Juglans nigra L.
over a twelve month period.
Harvest Meanz
x
Month Shoot number Shoot lengthy,x (cm)
Mar. 0.4 1.3

Apr. 0.6 1.9
May. 0.2 0.7
Jun. 0.1 0.5
Jul. 0.0 0.0
Aug. 0.2 0.7
Sept. 0.0 0.2
Oct. 0.0 0.0
Nov. 0.0 0.0
Dec. 0.0 0.0
Jan. 0.0 0.0
Feb. 0.0 0.2
Significancew *** ***

P value 0.0001 0.0001
5% t testv 0.22 0.65
1% t test 0.30 0.86
Contrastu
Month
Linear *** ***
Quadratic *** ***
z
Each mean is based on 25 replications with non-painted control removed.
y
Shoots ≥ 3cm long.
x
Data in these columns were transformed for analysis using √(y + 0.5) Non-
transformed means are presented.
w
Significant at the P≤ 0.001 level according to F test with 11 and 192 df.
v
t test for paired comparisons.
u
Significant contrast at the level P≤ 0.001 according to F test with 1 and 192 df.
NS, *** Nonsignificant or significant at P ≤ 0.001 respectively.
38
(≥ 3cm long) of softwood shoots forced from branch segments of Quercus rubra
L. over a twelve month period.
Harvest Meanz
x
Month Shoot number Shoot lengthy,x (cm)
Mar. 1.4 3.6

Apr. 3.2 8.3
May. 1.2 3.8
Jun. 1.2 3.7
Jul. 0.2 0.8
Aug. 2.2 4.8
Sept. 1.6 7.1
Oct. 0.8 2.2
Nov. 1.1 2.9
Dec. 0.2 1.7
Jan. 1.6 5.2
Feb. 0.5 3.1

P value 0.0002 0.0001
5% t testv 0.88 2.39
1% t test 1.16 3.16
Contrastu
Month
Linear *** **
Quadratic NS NS
z
Each mean is based on 25 replications with non-painted control removed.
y
x
transformed means are presented.
w
Significant at the P≤ 0.001 level according to F test with 11 and 192 df.
v
u
Significant contrast at the P≤ 0.001 level for shoot number and at the P≤ 0.01
level for shoot length according to F test with 1 and 192 df.
NS, **, *** Nonsignificant or significant at P ≤ 0.05, 0.001 respectively.
39
(a)
(b)
Fig. 1: Softwood shoot forced from a J. nigra branch segment harvested in Apr.
(a), multiple softwood shoots forced from branch segments of Q. rubra harvested
in Apr. (b).
40
exposure to low temperatures in the field, J. nigra branch segments became
ecodormant (quiescent) and began breaking bud slowly under laboratory
conditions. Buds remain endodormant until their chilling requirement is satisfied,
which varies from species to species and also by cultivar.
BA Concentration
The main effect of BA concentrations was found to be significant for both
shoot number (P= 0.0104) and shoot length (P= 0.0002) of softwood shoots
produced by J. nigra branch segments (Appendix Tables 4 and 5). The main
effect of BA concentrations was found to be significant for both shoot number (P
= 0.0079) and shoot length (P= 0.0194) of softwood shoots produced by Q. rubra
branch segments (Appendix Tables 6 and 7).
Both softwood shoot number and shoot length of J. nigra and Q. rubra
decreased linearly with increasing BA concentration applied to branch segments
(Tables 3 and 4). The level of 100µM BA was the most inhibitory to shoot
production when applied to the branch segments of J. nigra in 20% white exterior
latex paint according to the 5% t test (Table 3). The levels of 30 and 100µM BA
were the most inhibitory to shoot production when applied to the branch
segments of Q. rubra in 20% white exterior latex paint according to the 5% t test
(Table 4). The results of Mansouri (2006) were similar when BA was applied in
20% white exterior paint to stem segments of Acer saccharinum L.; application of
BA did not improve the number of shoots forced from the harvest months of Jul.
or Aug. 2005. The combination of BA and GA3 however resulted in greater shoot
41

shoots of Juglans nigra L. forced over a twelve month period.
Meanz
BA Shoot numberx Shoot lengthy,x (cm)
(mM)
non-painted 0.1 0.4

0 0.2 1.1
3 0.2 0.5
10 0.1 0.2
30 0.2 0.3
100 0.0 0.1
Significancew * ***
P value 0.0104 0.0002
5% t testv 0.15 0.42
1% t test 0.55
Contrastsu
BA Conc.
Linear ** **
Quadratic NS *
z
Each mean is based on 60 replications.
y
x
transformed means presented.
w
Significant at the P≤ 0.05 level for shoot number and at the P≤ 0.001 level for
shoot length according to the F test with 4 and 192 df.
v
u
NS,*, **, ***Nonsignificant or significant at P ≤ 0.05, 0.01, 0.001 respectively.
42

shoots of Quercus rubra L. forced over a twelve month period.
Meanz
x
BA Shoot number Shoot lengthy,x (cm)
(mM)
non-painted 2.0 4.5

0 1.5 4.6
3 1.4 5.1
10 1.7 3.9
30 1.2 3.5
100 0.7 2.7
Significancew ** *
P value 0.0079 0.0194
5% t testv 0.57 1.55
1% t test 0.75
Contrastsu
BA Conc.
Linear *** **
Quadratic NS NS
z
Each mean is based on 60 replications.
y
x
w
Significant at the P≤ 0.01 level for shoot number and at the P≤ 0.05 level for
shoot length according to the F test with 4 and 192 df.
v
u
NS, *, **, ***Nonsignificant or significant at P ≤ 0.05, 0.01, 0.001 respectively.
43
production in the Mansouri study. Therefore the addition of GA3 with BA also
may be necessary to improve shoot production of J. nigra and Q. rubra.
It should be noted that greenhouse conditions are variable not only within
the greenhouse but from month to month as external conditions of light and
temperature change throughout the year. Light has been found to have an effect
on epicormic bud break when forest stands are thinned. The effect of light on
epicormic bud break on branch segments has not been thoroughly investigated.
The white pigment in the paint may have occluded the irradiance of the branch
segments. The temperature of a greenhouse also fluctuates throughout the year;
data should be collected on temperature and analyzed in future shoot forcing
studies.
In vitro Results
Culture Initiation: Culture initiation was successful for Q. rubra and explants
grew and survived for 12 weeks. Culture initiation of J. nigra however was
unsuccessful as no explants survived as a consequence of high rates of
endogenous contamination. Therefore data are only presented for Q. rubra.
Influence of BA: Explants taken from softwood shoots forced from branch
segments were cultured in LP medium with 0 or 5µM BA to test the effect of BA
applied in paint on branch segments on explant performance in vitro. In the
harvest months of Mar., Apr., May., Aug., Sept., Oct., and Nov. inclusion of BA in
the basal medium had no effect on number or length of shoots produced from Q.
44
rubra explants (Table 5). However, inclusion of BA in the medium for explants
from the harvest months of Dec., Jan., and Feb. resulted in more shoots
produced than if no BA were included and also resulted in longer shoots being
produced on explants that began to be forced in Dec. and Jan. (Table 5).
An analysis of variance was done where all BA treatments applied to the
branch segments and both controls were separately pooled together (Appendix
Tables 10 and 11). In the pooled analysis the BA x media interaction was found
to be significant (P ≤ 0.05) according to the F test with 1 and 895 df for shoot
number (Appendix Tables 10 and 11). When there was no BA in the medium
explants from the control branch segments produced 0.6 shoots; whereas when
BA was included in the medium explants of the control branch segments
produced 0.8 shoots. When there was no BA in the medium explants from the
branch segments painted with BA produced 0.5 shoots; whereas when there was
BA in the medium the branch segments painted with BA produced 1.1 shoots.
The t test value for paired comparisons at the 5% level is 0.1, thus BA in the
medium caused a significant increase in the number of shoots produced by
explants obtained from the branch segments painted with the varying
concentrations of BA. Also, painting with BA increased shoot production in vitro,
especially if BA was also in the medium. Although, the morphological and
physiological differences between the control and the shoot forcing BA
treatments appeared to negatively affect the quality of the shoots with increasing
BA concentrations. Similar results of have been found by Romano et al. (1992)

45
Table 5: Effect of Month of Harvest x BA interaction with BA in the in vitro culture

medium on mean in vitro shoot number and shoot length (≥0.5 cm long) of
explants from the forced softwood shoots of Quercus rubra L. branch segments
treated with application of BA applied in 20% white exterior latex paint.
Harvest Mean
Month Media Nz Shoot numberx Shoot lengthy,x (cm)
BA(µM)
Mar. 0 48 0.5 0.5

5 41 0.4 0.3
Apr. 0 117 0.6 0.4

5 110 0.8 0.4
May. 0 25 0.6 0.4

5 27 0.7 0.4
Aug. 0 38 0.9 0.6

5 23 1.2 0.6
Sept. 0 119 0.6 0.5

5 41 0.6 0.3
Oct. 0 21 1.2 0.5

5 20 0.9 0.3
Nov. 0 46 0.7 0.6

5 50 0.5 0.3
Dec. 0 13 0.5 0.4

5 11 2.6 1.2
Jan. 0 74 0.1 0.0

5 75 1.9 1.1
Feb. 0 33 0.0 0.1

5 34 0.8 0.4
P value 0.0001 0.0001
5% t testv 0.70 0.47
1% t test 0.91 0.62
z
Data based on non-contaminated explants after 12 weeks of in vitro.
y
Shoots ≥0.5 cm long.
x
Data in these columns were transformed for analysis using √(y+0.5) Non-
w
Significant at the P≤ 0.001 level according to the F test with 9 and 784 df.
v
***
Significant at P≤ 0.001.
46
with Quercus suber L. where at higher concentrations of BA morphological
abnormalities occurred.
Influence of Explant Source: Softwood cuttings were cut into either tip or single-
node explants to determine the effect of explants source on shoot proliferation.
The number of explants forming axillary shoots differed significantly (P ≤ 0.001)
between tip and nodal explants sources (Appendix Tables 8 and 9). Over the
entire in vitro experiment tip explants produced a mean of 0.4 shoots, whereas
nodal explants produced a mean of 0.8 shoots. The analysis of variance
detected an interaction BA x explants x media (P = 0.0581) which can be
explained by nodal explants cultured on 5µM LP medium taken from softwood
shoots forced from branch segments painted with 3mM BA producing more
shoots than any other BA concentration except nodal explants on 5µM LP
medium taken from softwood shoots forced from branch segments painted with
30mM BA (Table 6 and Fig 2).

47
Table 6: Effect of BA x Explant x Media interaction on mean in vitro shoot

number and shoot length (≥0.5 cm long) of explants forced from the forced
softwood shoots of Quercus rubra L.
Mean
Media BA Explant BA Conc. Nz Shoot number,x Shoot
(µM) (mM) lengthy,x (cm)
0 Tip Control 27 0.6 0.1

Paint 17 0.2 0.2
3 14 0.1 0.1
10 22 0.3 0.1
30 14 0.3 0.1
100 11 0.2 0.1
Nodal Control 98 0.5 0.4
Paint 87 0.7 0.5
3 72 0.3 0.4
10 70 0.8 0.8
30 65 0.8 0.5
100 37 0.4 0.3
5 Tip Control 30 0.4 0.2
Paint 13 0.6 0.8
3 11 0.3 0.2
10 16 0.7 0.3
30 14 0.3 0.4
100 4 0.8 0.6
Nodal Control 69 0.8 0.4
Paint 75 0.9 0.5
3 55 1.9 0.8
10 61 1.0 0.6
30 52 1.3 0.6
100 32 0.6 0.3
Significancew P value NS
P value 0.0581 0.1502
5% t testv 0.94
z
Data based on non-contaminated explants after 12 weeks of in vitro.
y
Shoots ≥ 0.5 cm long.
x
Data in these columns were transformed for analysis using √(y+0.5)1/2 Non-
w
Significant at P≤ 0.0581 level according to the F test with 9 and 784 df.
v
NS, Nonsignificant
48
(a)
(b)
Fig 2: In vitro explants from shoot forcing control cultured on 5µM LP medium (a),
In vitro explants from branch segments painted with 3mM BA cultured on 5µM LP
medium (b).
49
CHAPTER 5
SUMMARY
Shoot Forcing
1. The 20% white exterior latex paint had a minimal effect on shoot production
for J. nigra and there were no significant differences between the painted
and non-painted controls on softwood shoot production for Q. rubra from
large branch segments during the 12 runs of the experiment.
2. Shoot production of Quercus rubra L. declined when branch segments were
harvested during the dormant season (Sept.-Dec.).
3. Juglans nigra L. failed to produce shoots from branch segments harvested
during the dormant season (Sept.-Dec.).
4. Shoot number and length of J. nigra reached a maximum on branch
segments harvested in Mar. and Apr.
5. Both shoot number and shoot length decreased linearly with increasing BA
concentrations applied to the branch segments of J. nigra and Q. rubra.
In Vitro
1. Culture initiation was successful for Q. rubra.
2. Culture initiation was unsuccessful for J. nigra as a consequence of high
rates of endogenous contamination.
3. For Q. rubra explants produced from branch segments harvested in Mar.,
Apr., May., Aug., Sept., Oct., and Nov., inclusion of BA in the basal medium
had no effect on number or length of shoots produced.
4. Inclusion of BA in the medium during harvest months Dec., Jan., and Feb.
50
resulted in more shoots produced than if no BA were included in the medium
and also influenced longer shoots produced in Dec. and Jan.
5. Where all BA treatments applied to the branch segments and both controls
were separately pooled together, BA in the medium caused a significant
increase in the number of shoots produced by explants obtained from the
branch segments painted with BA.
6. Painting with BA increased shoot production in vitro, only if BA was also in
the medium.
7. Nodal explants generally produced greater axillary shoots than tip explants.
8. Nodal explants cultured on 5µM LP medium taken from softwood shoots
forced from branch segments painted with 3mM BA produced more shoots
than any other BA concentration applied to branch segments except nodal
explants on 5µM LP medium taken from softwood shoots forced from branch
segments painted with 30mM BA.

51
CONCLUSION
Shoot forcing
The results of this research show that Q. rubra can be established and
multiplied successfully in vitro by using explants obtained from softwood shoots
forced from branch segments in a greenhouse using drip irrigation.
Benzyladenine was unsuccessful in overcoming the endodormancy
imposed on either J. nigra or Q. rubra in the shoot forcing study and caused a
decrease in shoot production for both species as concentration increased.
In vitro
Inclusion of BA in the medium for explants from the harvest months of
Dec., Jan., and Feb. resulted in more shoots produced than if no BA were
included and also resulted in longer shoots being produced on explants that
began to be forced in Dec. and Jan.

52
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APPENDICES
67
Appendix Table 1: Stock Solution Preparation Long and Preece Media (LP
Media, Long et al., 1995; Preece et al., 1995).
Component Stock Amount Final

Solution (ml/l) Conc.
(g/l) (mg/L)
Nitrates
NH4NO3 45.40 908.0
Ca(NO3)2 • 4H2O 63.10 20 1262.0
Zn(NO3)2 • 6H2O 0.425 8.5
Sulfates
K2SO4 63.725 1274.5
MgSO4 • 7H2O 27.75 555.0
MnSO4 • H2O 1.395 20 27.9
ZnSO4 • 7H2O 0.215 4.3
CuSO4 • 5H2O 0.0125 0.25
Phosphates
KH2PO4 10.875 217.5
H3BO3 0.275 20 5.5
NaMoO4 • 2H2O 0.016 0.32
Calcium
CaCl2 • 2H2O 6.125 20 122.5
Iron
Na2 EDTA 2.0675 41.35
20
FeSO4 • 7H2O 1.54 30.8
Organics
Thiamine • HCl 0.075 1.5
Nicotinic acid 0.0375 0.75
Glycine 0.10 20 2.0
Pyridoxine • HCl 0.0125 0.25
Myo-inositol 5.00 100.0
68
Appendix Table 2: Analysis of variance for the effects of 20% white exterior latex
paint applied to 40 cm long branch segments of Juglans nigra L. on the mean
number and length of shoots (≥ 3 cm long).
Source of variation DF MS F Sig P value

Variable: Transformedz shoot number
Month 11 0.119 2.62 * 0.0106
Block(Month) 48 0.046 - - -
Paint 1 0.119 8.06 ** 0.0066
Month x Paint 11 0.052 3.52 ** 0.0012
Error 48 0.015 - - -
DF MS F Sig P value
Source of variation
Variable: shoot number
Month 11 0.457 2.49 * 0.0145
Block(Month) 48 0.183 - - -
Paint 1 0.408 7.00 * 0.0110
Month x Paint 11 0.208 3.57 *** 0.0010
Error 48 0.058 - - -

z
Variable: Transformed shoot length (cm)
Month 11 0.875 2.36 * 0.0204
Block(Month) 48 0.371 - - -
Paint 1 1.339 10.90 ** 0.0018
Month x Paint 11 0.422 3.44 ** 0.0014
Error 48 0.123 - - -
DF MS F Sig P value
Source of variation
Variable: shoot length (cm)
Month 11 7.651 2.03 * 0.0462
Block(Month) 48 3.775 - - -
Paint 1 13.002 9.75 * 0.0030
Month x Paint 11 4.148 3.11 * 0.0031
Error 48 1.333 - - -
z
Data were transformed for analysis using √(y + 0.5).
NS, *, **, ***
Nonsignificant or significant at P ≤ 0.05, 0.01, 0.001 respectively.
69
Appendix Table 3: Analysis of variance for the effects of 20% white exterior latex
paint applied to 40 cm long branch segments of Quercus rubra L. on the mean
number and length of shoots (≥ 3 cm long).

Month 11 1.375 3.02 ** 0.0039
Block(Month) 48 0.455 - - -
Paint 1 0.648 2.08 NS 0.1562
Month x Paint 11 0.202 0.65 NS 0.7805
Error 48 0.312 - - -

Month 11 14.270 2.83 ** 0.0063
Block(Month) 48 5.050 - - -
Paint 1 8.533 2.50 NS 0.1206
Month x Paint 11 2.224 0.65 NS 0.7760
Error 48 3.417 - - -

Variable: Transformedz shoot length (cm)
Month 11 2.289 1.54 NS 0.1501
Block(Month) 48 1.491 - - -
Paint 1 0.001 0.00 NS 0.9783
Month x Paint 11 1.305 1.21 NS 0.3052
Error 48 1.077 - - -

Month 11 35.461 1.32 NS 0.2434
Block(Month) 48 26.887 - - -
Paint 1 0.320 0.02 NS 0.8986
Month x Paint 11 25.843 1.32 NS 0.2406
Error 48 19.514 - - -
z
NS, *, **, ***
70
Appendix Table 4: Analysis of variance for the effects of Month of Harvest and
BA concentrations (only including painted) applied in paint to 40 cm long branch
segments of Juglans nigra L. on the mean shoot number. Shoots were forced in
a greenhouse in flats of vermiculite with drip irrigation.

Month 11 0.214 4.80 *** 0.0001
Block(Month) 48 0.044 - - -
BAtreat 4 0.107 3.39 * 0.0104
Month x BAtreat 44 0.022 0.71 NS 0.9087
Error 192 0.031 - - -
Contrasts
BA Concentration
Linear 1 0.229 7.28 ** 0.0076
Quadratic 1 0.015 0.47 NS 0.4922
Month
Linear 1 1.410 44.83 *** 0.0001
Quadratic 1 0.376 11.96 *** 0.0007

Month 11 0.949 4.52 *** 0.0001
Block(Month) 48 0.210 - - -
BAtreat 4 0.447 2.76 * 0.0292
Error 192 0.162 - - -
Contrasts
BA Concentration
Linear 1 0.942 5.81 * 0.0169
Quadratic 1 0.012 0.07 NS 0.7880
Month
Linear 1 6.127 37.80 *** 0.0001
Quadratic 1.585 9.78 ** 0.0020
z
NS, *, **, ***
71
segments of Juglans nigra L. on the mean length of shoots (≥ 3 cm long).
Shoots were forced in a greenhouse in flats of vermiculite with drip irrigation.

Month 11 1.132 5.38 *** 0.0001
Block(Month) 48 0.210 - - -
BAtreat 4 0.860 5.80 *** 0.0002
Error 192 0.148 - - -
Contrasts
BA Concentration
Linear 1 1.417 9.56 ** 0.0023
Quadratic 1 0.612 4.13 * 0.0435
Month
Linear 1 7.340 49.52 *** 0.0001
Quadratic 1 2.267 15.29 *** 0.0001

Month 11 9.305 5.12 *** 0.0001
Block(Month) 48 1.817 - - -
BAtreat 4 8.265 6.12 *** 0.0001
Error 192 1.35 - - -
Contrasts
BA Concentration
Linear 1 12.461 9.23 * 0.0027
Quadratic 1 6.840 5.07 * 0.0255
Month
Linear 1 59.834 44.32 *** 0.0001
Quadratic 1 18.704 13.86 *** 0.0003
z
NS, *, **, ***
72
segments of Quercus rubra L. on the mean shoot number. Shoots were forced in
a greenhouse in flats of vermiculite with drip irrigation.

Month 11 1.995 4.39 *** 0.0002
Block(Month) 48 0.454 - - -
BAtreat 4 0.912 3.56 ** 0.0079
Error 192 0.256 - - -
Contrasts
BA Concentration
Linear 1 3.213 12.55 *** 0.0005
Quadratic 1 0.015 0.06 NS 0.8118
Month
Linear 1 3.940 15.39 *** 0.0001
Quadratic 1 0.160 0.62 NS 0.4305

Month 11 17.540 3.62 *** 0.0009
Block(Month) 48 4.847 - - -
BAtreat 4 8.830 3.53 ** 0.0083
Error 192 2.500 - - -
Contrasts
BA Concentration
Linear 1 29.275 11.71 *** 0.0008
Quadratic 1 0.009 0.00 NS 0.9523
Month
Linear 1 42.110 16.84 *** 0.0001
Quadratic 1 1.441 0.58 NS 0.4486
z
NS, *, **, ***
73
segments of Quercus rubra L. on the mean length of shoots (≥ 3 cm long).
Shoots were forced in a greenhouse in flats of vermiculite with drip irrigation.

Month 11 7.149 4.74 *** 0.0001
Block(Month) 48 1.507 - - -
BAtreat 4 3.108 3.01 * 0.0194
Error 192 1.033 - - -
Contrasts
BA Concentration
Linear 1 10.226 9.90 ** 0.0019
Quadratic 1 2.396 1.35 NS 0.2463
Month
Linear 1 7.348 7.12 ** 0.0083
Quadratic 1 1.270 1.23 NS 0.2688

Month 11 117.307 4.07 *** 0.0003
Block(Month) 48 28.838 - - -
BAtreat 4 52.228 2.83 * 0.0258
Error 192 18.426 - - -
Contrasts
BA Concentration
Linear 1 161.922 8.79 ** 0.0034
Quadratic 1 23.409 1.27 NS 0.2611
Month
Linear 1 89.389 4.85 * 0.0288
Quadratic 1 20.491 1.11 NS 0.2930
z
NS, *, **, ***
74
Appendix Table 8: Analysis of variance for the effects of Month of Harvest,

Explant Source (Tip or Nodal), Media (0 or 5µM BA), and Shoot forcing BA
treatments (including painted and non-painted controls) applied in paint to 40 cm
long branch segments of Quercus rubra L. on the mean shoot number of
explants cultured in vitro from softwood shoots.

Month 9 0.476 2.45 ** 0.0095
BA 5 0.143 0.73 NS 0.5976
Month x BA 43 0.157 0.81 NS 0.8104
Explant 1 2.538 13.04 *** 0.0003
Month x Explant 7 0.127 0.65 NS 0.7125
BA x Explant 5 0.153 0.78 NS 0.5614
Month x BA x Explant 29 0.181 0.93 NS 0.5622
Media 1 0.310 1.59 NS 0.2071
Month x Media 9 1.158 5.95 *** 0.0001
BA x Media 5 0.198 1.02 NS 0.4049
Month x BA x Media 39 0.128 0.66 NS 0.9484
Explant x Media 1 0.507 2.61 NS 0.1069
Month x Explant x Media 6 0.210 1.08 NS 0.3724
BA x Explant x Media 5 0.418 2.15 NS 0.0581
Month x BA x Explant x Media 16 0.235 1.21 NS 0.2552
Error 784 0.195

Month 9 3.710 2.40 * 0.0110
BA 5 0.813 0.53 NS 0.7571
Month x BA 43 1.129 0.73 NS 0.9012
Explant 1 14.265 9.22 ** 0.0025
BA x Explant 5 1.180 0.76 NS 0.5764
Media 1 3.216 2.08 NS 0.1497
Month x Media 9 8.346 5.40 *** 0.0001
BA x Media 5 1.355 0.88 NS 0.4967
Error 784 1.546 - - -
z
NS, *, **, ***
75
Appendix Table 9: Analysis of variance for the effects of Month of Harvest,

Explant Source (Tip or Nodal), Media (0 or 5µM BA), and Shoot forcing BA
treatments (including painted and non-painted controls) applied in paint to 40 cm
long branch segments of Quercus rubra L. on the mean length of shoots of

Month 9 0.172 1.81 NS 0.0628
BA 5 0.130 1.37 NS 0.2349
Month x BA 43 0.090 0.95 NS 0.5676
Explant 1 1.315 13.84 *** 0.0002
BA x Explant 5 0.051 0.54 NS 0.7486
Media 1 0.091 0.96 NS 0.3281
Month x Media 9 0.583 6.13 *** 0.0001
BA x Media 5 0.075 0.79 NS 0.5538
Month x BA x Explant x Media 16 0.169 1.77 * 0.0304
Error 784 0.095

Month 9 0.924 1.77 NS 0.0701
BA 5 0.728 1.40 NS 0.2238
Month x BA 43 0.482 0.92 NS 0.6123
Explant 1 5.584 10.70 ** 0.0011
BA x Explant 5 0.353 0.68 NS 0.6419
Media 1 0.664 1.27 NS 0.2597
Month x Media 9 28.085 5.98 *** 0.0001
BA x Media 5 0.364 0.70 NS 0.6256
Month x BA x Explant x Media 16 1.043 2.00 * 0.0113
Error 784 0.522 - - -
z
NS, *, **, ***
76
Appendix Table 10: Analysis of variance for the pooled effects of BA treatments
(including combined painted and non-painted controls) applied in paint to 40 cm
long branch segments of Quercus rubra L. on the mean shoot number of

Month 9 0.494 2.53 ** 0.0072
BA 1 0.092 0.47 NS 0.4938
Month x BA 9 0.148 0.76 NS 0.6574
Explant 1 3.687 18.88 *** 0.0001
BA x Explant 1 0.054 0.28 NS 0.5989
Media 1 1.257 6.44 * 0.0113
Month x Media 9 1.323 6.77 *** 0.0001
BA x Media 1 0.764 3.91 * 0.0482
Error 895 0.195

Month 9 4.081 2.66 ** 0.0048
BA 1 0.698 0.45 NS 0.5003
Month x BA 9 0.950 0.62 NS 0.7816
Explant 1 21.874 14.25 *** 0.0002
BA x Explant 1 0.122 0.08 NS 0.7777
Media 1 9.315 6.07 * 0.0139
Month x Media 9 9.729 6.34 *** 0.0001
BA x Media 1 5.846 3.81 NS 0.0513
Error 895 1.535 - - -
z
NS, *, **, ***
77
Appendix Table 11: Analysis of variance for the pooled effects of BA treatments
(including combined painted and non-painted controls) applied in paint to 40 cm
long branch segments of Quercus rubra L. on the mean length of shoots (≥0.5
cm long) of explants cultured in vitro from softwood shoots.

Month 9 0.168 1.72 NS 0.0796
BA 1 0.003 0.03 NS 0.8589
Month x BA 9 0.116 1.19 NS 0.3003
Explant 1 1.849 18.94 *** 0.0001
BA x Explant 1 0.009 0.09 NS 0.7677
Media 1 0.248 2.54 NS 0.1110
Month x Media 9 0.622 6.37 *** 0.0001
BA x Media 1 0.179 1.84 NS 0.1756
Error 895 0.098

Month 9 0.887 1.65 NS 0.0977
BA 1 0.020 0.04 NS 0.8478
Month x BA 9 0.594 1.10 NS 0.3570
Explant 1 8.328 15.48 *** 0.0001
BA x Explant 1 0.010 0.02 NS 0.8910
Media 1 1.144 2.13 NS 0.1452
Month x Media 9 3.293 6.12 *** 0.0001
BA x Media 1 0.719 1.34 NS 0.2480
Error 895 0.538 - - -
z
NS, *, **, ***
78
VITA
Graduate School
Southern Illinois University
Andrew Craig Holsinger, Jr. Date of Birth: November 24, 1978
1006 East Poplar Street, West Frankfort, Illinois 62896

Bachelor of Science, Plant and Soil Science, May 2006
Thesis Title:
Influence of Benzyladenine on Shoot Forcing and Tissue Culture of
Juglans Nigra L. and Quercus Rubra L.
Major Professor: John E. Preece

Andrew Holsinger Thesis

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Southern Illinois University Carbondale

Andrew Craig Holsinger, Jr.

B.S., Southern Illinois University, 2006

Department of Plant, Soil and Agricultural Systems

INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND TISSUE

Andrew Craig Holsinger Jr.

A Thesis Submitted in Partial

Fulfillment of the Requirements

for the Degree of

in the field of Plant and Soil Science

Dr. John E. Preece, Chair

Dr. Paul Henry

Dr. James Zaczek

TITLE: INFLUENCE OF BENZYLADENINE ON SHOOT FORCING AND

MAJOR PROFESSOR: Dr. John E. Preece

Shoot production and in vitro performance of Juglans nigra L and Quercus

40 cm branch segments to determine the most effective concentration of

linearly with increasing BA concentrations applied to the branch segments of

dormant season September-December. The softwood shoots were surface

BA treated softwood shoots were compared to the controls, the BA in the

medium caused a significant increase in the number of shoots produced by

painted with 30mM BA.

I would like to sincerely thank my wife, Shana Holsinger, for her

unconditional love and encouragement.

I would like to express my gratitude to my major professor, Dr. John E.

successful completion of my thesis. I also wish to thank my committee members

on my committee. They have both greatly helped to increase my knowledge of

LIST OF TABLES ........................................................................................... viii

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

CHAPTER 1 – INTRODUCTION ........................................................... 1

CHAPTER 2 – REVIEW OF LITERATURE ........................................... 9

CHAPTER 3 – MATERIALS AND METHODS ..................................... 27

Experimental Design, Data Collection, and Statistical Analysis ........... 31

CHAPTER 4 – RESULTS AND DISCUSSION .................................... 34

CHAPTER 5 – Summary, Conclusion, Recommendation .................... 49

LITERATURE CITED ..................................................................................... 52

Appendix Table 1: Stock Solution Preparation Long and Preece Media

Appendix Table 2: Analysis of variance for the effects of 20% white

Appendix Table 3: Analysis of variance for the effects of 20% white

Appendix Table 4: Analysis of variance for the effects of Month of Harvest

Appendix Table 6: Analysis of variance for the effects of Month of Harvest

Appendix Table 7: Analysis of variance for the effects of Month of Harvest

Appendix Table 8: Analysis of variance for the effects of Month of

Appendix Table 9: Analysis of variance for the effects of Month of

Appendix Table 10: Analysis of variance for the pooled effects of BA

Appendix Table 11: Analysis of variance for the pooled effects of BA

Table 3: Main effect of BA concentration applied in latex paint to 40 cm branch

Table 4: Main effect of BA concentration applied in latex paint to 40 cm branch

Table 5: Effect of Month of Harvest x BA interaction with BA in the in vitro culture

Table 6: Effect of BA x Explant x Media interaction on mean in vitro shoot

Figure 1: Softwood shoot forced from a J. nigra branch segment harvested in

propagated clonally by rooting leafy softwood or semihardwood cuttings (Henry

than dormant hardwood cuttings for many difficult-to-root woody species

Improving vegetative propagation of mature cultivars of Juglans nigra L.

and grafting on seedling rootstocks which have unknown genetic potential. Q.

rubra is traditionally propagated by seed because of graft incompatibility as the

cuttings. Superior characteristics could be selected from a single source plant or

desired traits if vegetative propagation methods were improved.

With J. nigra and Q. rubra being primarily propagated by seed because of

increase production costs and decrease the amount of useable space in a

nursery. Techniques to improve cost and propagation efficiency have been

availability of explants for micropropagation.

Shoot forcing is a method of vegetative propagation in which softwood

more reliable to root than semi-hardwood or hardwood cuttings.