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Theses, Dissertations, and Student Research in
Agronomy and Horticulture Department
Agronomy and Horticulture
Summer 8-2014
EFFECTS OF CULTURE MEDIA AND PLANT

GROWTH REGULATORS ON
MICROPROPAGATION OF WILLOW (Salix
matsudana ‘Golden Spiral’) AND HAZELNUT
(Corylus colurna ‘Te Terra Red)
Dongxue Shi
University of Nebraska – Lincoln
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Shi, Dongxue, "EFFECTS OF CULTURE MEDIA AND PLANT GROWTH REGULATORS ON MICROPROPAGATION OF
WILLOW (Salix matsudana ‘Golden Spiral’) AND HAZELNUT (Corylus colurna ‘Te Terra Red)" (2014). Theses, Dissertations, and
Student Research in Agronomy and Horticulture. 79.
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EFFECTS OF CULTURE MEDIA AND PLANT GROWTH
REGULATORS ON MICROPROPAGATION OF WILLOW (Salix
matsudana ‘Golden Spiral’) AND HAZELNUT (Corylus colurna
‘Te Terra Red)
By
Dongxue Shi
A THESIS
Presented to the Faculty of
The Graduate College at the University of Nebraska
In partial Fulfillment of Requirements
For the Degree of Master of Science
Major: Horticulture
Under the supervision of Professor Paul E. Read
Lincoln, Nebraska
August, 2014
EFFECTS OF CULTURE MEDIA AND PLANT GROWTH
REGULATORS ON MICROPROPAGATION OF WILLOW (Salix
matsudana) AND HAZELNUT (Corylus colurna)
Dongxue Shi, M. S.
University of Nebraska, 2014
Advisor: Paul E. Read
The branches and leaves of Salix matsudana ‘Golden Spiral’ , willow, grow in a
twisted manner, which makes it an important bonsai plant. Its mature stems have
potential for the woody cut floral industry. Corylus colurna ‘ Te Terra Red’, hazelnut,
distinguished by red or purple leaves in the spring, has potential ornamental value
in the horticulture industry for landscape use. Micropropagation of these two plants
could provide more and healthier plantlets for rapid commercial scale-up by the
nursery industry. Nas and Read Medium (NRM) is a newer medium developed
specifically for hazelnut species by Nas and Read based on the composition of the
seed. In this study, experiments were conducted to test whether NRM (with added
plant growth regulators) is superior to other media such as Murashige and Skoog
Medium (MS), Woody Plant Medium (WPM) and Driver-Kuniyuki Walnut medium
(DKW) for shoot and root production of Salix and Corylus. The results showed that
Salix explants grown in NRM with BA at 2.0mg/L and IBA at 0.05 mg/L produced a
greater number of new shoots and longer stems. Explants grown in NRM with IBA at
0.05 mg/L produced more roots, which is necessary before transplanting to the soil.
In addition, these plantlets exhibited deeper green color and acclimatized
successfully into potential transplants. The hazelnut explants had a very high
contamination rate and grew slowly on the various media. By improving
disinfestation methods and by adding antibiotics in forcing solution and media, the
contamination rate and browning rate can be decreased greatly.

i
THIS THESIS IS DEDICATED TO MY PARENTS, GUANGZHI SHI AND LIJING CHENG
I LOVE YOU
ii
Acknowledgements
My sincere gratitude goes to my adviser, Dr. Paul Read, who provided me an
opportunity to study abroad, and his guidance, kindness and patience are more than
I can ever asked for.
My great appreciation is also extended to my committee members Dr. Ellen
Paparozzi and Dr. Gary Yuen for their friendship and valuable criticism.
I would also like to thank to the viticulture group members: Thanks Ben Loseke
for his help in training me, and thanks Christina Bavougian and Stephen Gamet for
extending my study experience.
I express my deep appreciation to my parents Guangzhi Shi and Lijing Cheng for
their great material and spiritual support. Our love to each other are more than
words can say.

iii
Table of Contents
Introduction .............................................................................................................................................. 1
Literature Review ................................................................................................................................... 3
Literature cited ..................................................................................................................................... 14
Chapter 1: Salix matsudana.............................................................................................................. 19
Introduction ........................................................................................................................................... 19
Literature Review ................................................................................................................................ 19
Objectives................................................................................................................................................ 23
Materials and Methods ...................................................................................................................... 23
Results and Discussion ...................................................................................................................... 27
Conclusion .............................................................................................................................................. 30
Literature Cited for Chapter 1: ....................................................................................................... 46
Chapter 2: Corylus colurna ............................................................................................................... 48
Introduction ........................................................................................................................................... 48
Literature review ................................................................................................................................. 49
Objectives................................................................................................................................................ 53
Materials and Methods ...................................................................................................................... 54
Results and Discussion ...................................................................................................................... 58
Conclusion .............................................................................................................................................. 62
Literature Cited for Chapter 2 ........................................................................................................ 78
Appendix ................................................................................................................................................. 81
List of Figures
Figure 1-1: Shoot number of Salix matsudana 'Golden Spiral' explants cultured in
vitro on 4 media containing 2.5mg BA/L + 0.05 mg IBA/L ....................................... 31
vitro on 4 media containing 2.5 mg BA/L + 0.01 mg IBA/L ..................................... 32
vitro on 4 media containing 2.0 mg BA/L + 0.01 mg IBA/L ...................................... 33
vitro on 4 media containing 0.05 mg IBA/L..................................................................... 34
Figure 1-5: Growth comparison of Salix matsudana ‘Golden Spiral’ explants cultured
in vitro ............................................................................................................................................ 36
Figure 1-6: Stem length of Salix matsudana 'Golden Spiral' explants cultured in vitro
on 4 media containing various PGR .................................................................................... 37
Figure 1-7a: Pantone Matching System Color Chart for comparing healthy-colored
explants of Salix matsudana ‘Golden Spiral’ cultured in vitro .................................. 38
Figure 1-7b: Number of healthy-colored explants of Salix matsudana 'Golden Spiral'
cultured in vitro on 4 media containing various PGR .................................................. 39
Figure 1-8: Shoot number of Salix matsudana 'Golden Spiral' explants cultured on
DKW and NRM containing 2.5 mg BA/L+ 0.01 mg IBA/L for shoot
iv
multiplication and transferred to DKW and NRM containing 0.05 mg IBA/L for
root initiation ............................................................................................................................... 40
Figure 1-9: 1 month after Salix matsudana ‘Golden Spiral’ explants transplanted
from Nas&Read Medium containing 0.05 mg IBA/L to soil....................................... 42
Figure 2-1: Effects of cooler storing time of branches on the contamination and
browning incidence for Corylus colurna ‘Te Terra Red’ .............................................. 63
Figure 2-2: Effects of tap water rinsing time of branches on the contamination and
Figure 2-3: Effects of 10% bleach soaking time of branches on the contamination
and browning incidence for Corylus colurna ‘Te Terra Red’ ..................................... 65
Figure 2-4: Effects of 70% ethanol spray time of branches on the contamination and
Figure 2-5: Effects of explant materials (softwood cutting/buds) on the
contamination and browning incidence for Corylus colurna ‘Te Terra Red’ ....... 67
Figure 2-6: Contamination and browning of Corylus colurna ‘Te Terra Red’ buds
cultured in vitro .......................................................................................................................... 68
Figure 2-7: Corylus colurna 'Te Terra Red' explant contamination and browning
incidence of two mother plants ............................................................................................ 69
Figure 2-8: Browning rate of Corylus colurna 'Te Terra Red' explants cultured on 4
media containing various PGR ............................................................................................. 70
Figure 2-9: Corylus colurna ‘Te Terra Red’ explants (axillary buds) cultured in vitro
that were able to grow shoots successfully ..................................................................... 71
Figure 2-10: Corylus colurna 'Te Terra Red' explants cultured on 4 media containing
2.5mg BA/L+0.05mg IBA/L.................................................................................................... 72
Figure 2-11: Corylus colurna ‘Te Terra Red’ explants that were able to initiate roots
but failed to grow in soil. ......................................................................................................... 73
Figure 2-12: Effects of forcing solution supplemented with streptomycin on the
Figure 2-13: Effects of media supplemented with streptomycin on the
List of Tables
Table 1-1: Mean shoot number±SE of Salix matsudana ‘Golden Spiral’ in 4 media
containing PGRs in Experiment 1,2,3, and 4(Figure 1-1, 1-2, 1-3, and 1-4) ........ 43
Table 1-2: P value of comparisons of treatment (media) differences Least Squares
Means (LSM) at =0.05 in experiment 1, 2, 3, and 4 .................................................... 43
Table 1-3: Mean shoot length ± SE of Salix matsudana ‘Golden Spiral’ in 4 media
containing PGRs in Experiment 1, 2, 3, and 4 (Figure 1-6) ........................................ 43
Table 1-4: Simple effect comparisons of medium*PGR Least Squares Means of stem
length in Experiment 1, 2, 3, and 4 (Figure 1-6) ............................................................ 44
v
Table 1-5 Mean shoot or root number ± SE of Salix matsudana ‘Golden Spiral’ on
DKW and NRM for shoot multiplication and root initiation in Experiment 5
(Figure 1-6a, 1-6b)..................................................................................................................... 45
Table 1-6: Growth rate of Salix matsudana ‘Golden Spiral’ after transplanted into soil
........................................................................................................................................................... 45
Table 2-1: P value comparisons of cooler storing time effect on contamination and
browning incidence for Corylus colurna ‘Te Terra Red’ (=0.05) ........................... 76
Table 2-2: P value comparisons of tap water rinsing time effect on the
contamination and browning incidence for Corylus colurna ‘Te Terra Red’
(=0.05) ......................................................................................................................................... 76
Table 2-3: P value comparisons of 10% bleach soaking time effect on the
(=0.05) ......................................................................................................................................... 76
Table 2-4: P value comparisons of 70% ethanol spray time effect on the
(=0.05) ......................................................................................................................................... 76
Table 2-5: P value comparisons mother plants effect on contamination and
browning incidence of Corylus colurna ‘Te Terra Red’ (=0.05) ............................ 77
Table 2-6: P value of comparisons of media effect on browning incidence of Corylus
colurna ‘Te Terra Red’ (=0.05) ........................................................................................... 77
Table 2-7: P value comparisons of media (2.5 mg BA/L + 0.05 mg IBA/L) effect on
number of shoots of Corylus colurna ‘Te Terra Red’ (=0.05) ................................. 77
Table 2-8: P value of comparisons of concentration of streptomycin included in
forcing solution effect on the contamination incidence (=0.05)........................... 77
Table 2-9: P value of comparisons of concentration of streptomycin included in
media effect on the contamination incidence (=0.05) .............................................. 77
1
Introduction
Salix matsudana is a member of the Salicaceae or the willow family, and the
genus Salix consists of around 300 species all over the world (Zomleter, 1994).
Under the common name of ‘Peking willow’, Salix matsudana is native to
northeastern China, and widely cultivated in several provinces in China (Wu et al.,
1994). As an ornamental tree, Salix matsudana has been introduced into North
America, Europe and Australia. In the U. S, there are several states that
commercially grow S. matsudana, including Colorado, Illinois, New York, Utah, Ohio,
Virginia (Emerine, 2012).
Salix matsudana is a medium-sized dioecious tree that can reach 20 to 40 feet in
height with similar width. The leaves are narrow, serrate margined, bright green in
summer and yellow-green in autumn. The stems are yellowish in youth, and turn
olive-green or brown when they become mature. It has showy catkins on male trees
but concealed flowers on female trees (Seiler et al, 2010).
Salix matsudana is fast growing but short lived. It is tolerant to all types of soil,
especially to salty ones, but prefers moist soil and grows well along water sources.
This tree is tolerant to many soil pH levels and very cold hardy, and full sun is
required to grow Salix matsudana. Compared to other species in the willow family
(such as Salix babylonica), it is more drought resistant (Gilman and Watson, 1994).
There are numerous pest and disease concerns about Salix matsudana. Some
cultivars are very susceptible to cankers; as a result several cultivars have been
2
selected for canker resistance. Because of the contorted or twisted branching effect
of some cultivars (e.g. ‘Tortuosa’), Salix matsudana is usually used as a bonsai plant
or its stems are harvested for floral arrangements. For other landscape uses, it is
also a good choice as a shade tree, lawn tree, park tree or for any wet sites (Wu et al.,
1994).
Corylus colurna is commonly called Turkish hazelnut, and is a member of the
genus Corylus, tribe Coryleae, family Betulaceae. (Cronquist, 1981). It is a deciduous
tree native to a broad area of southwest Asia and southeast Europe from China to
the Balkans (Kuhns, 1983).
The main use of hazelnut trees is the edible nut, which make it the second most
important nut tree in the world following the almond (Mehlenbacher, 1994). Turkey
ranks first in hazelnut production followed by Italy and the U.S. Oregon is the main
state in the U.S. to grow commercial hazelnut (Corylus avellana). In 2012, Oregon’s
production was 34,000 tons, valued at 63.4 million dollars (Clark and Coba, 2012).
In addition to the commercial hazel, some cultivars are also grown as
ornamental plants by horticulturists for their contorted stems and pendulous
growth habit, red or yellow leaf color, and cut leaf forms. C. colurna leaves are
rounded and hairy with double-serrate margins. This monoecious tree has visible
male catkins but extremely small female flowers concealed in buds. The small and
hard-shelled nuts make C. colurna of no value for commercial edible hazelnut, but
important in commercial hazelnut orchards because it does not sucker readily. This
characteristic makes it ideal as rootstock for grafting other desirable cultivars of
commercial hazelnuts such as C. avellana (Mehlenbacher, 1994).

3
The cultivation of C. colurna is easy because of its tolerance to heat, cold and
drought after establishment. It is also occasionally reported to be tolerant to
alkaline soil (Cerovic et al., 2007). Compared to other species (e.g. C. avellana) that
are susceptible to eastern filbert blight, C. colurna is resistant to serious pests and
other disease problems such as bacterial blight of hazelnut (Gilman and Watson,
1993). All these characteristics make C. colurna tolerant of urban conditions. It also
maintains a symmetrical crown that is attractive to landscape architects (Gilman
and Watson, 1993). Thus, C. colurna is planted in sidewalk cutouts, parking lot
islands and highway rights-of-way.
Cuttings and seeds are the common methods of propagation, however,
micropropagation of Salix matsudana or Corylus colurna could provide more
plantlets for rapid commercial scale-up in the nursery industry. It is well-known
that micropropagation is not limited by season, being available all year round. It
maintains the clonal characteristics, reduces the possibility of disease, and enables
easy distribution. Besides, the plantlets from micropropagation are also said to be
more uniform and practical than cuttings based on tests reported by Tormala and
Saarikko (1985).
Literature Review
When using the process of micropropagation, there are several aspects to be
considered before conducting the research. Choosing of explants, the initiation of
cultures, culture media (including the plant growth regulators added to the media)
4
and the problems during culture, such as contamination, browning, and
proliferation difficulty, may have great effect on the results.
1) Explant materials
Cloning in vitro of adult or mature woody plants is affected by characteristics
accompanying maturation such as reduced growth rate and the lack of rooting
ability. Maturation is an obstacle for a wider application of tissue culture technology
among woody species. Though many problems exist, researchers have successfully
multiplied mature trees by using materials such as shoots from the base of the tree,
or by special pre-treatment in vivo and/or in vitro culture. These methods to
improve propagation are prerequisite for possible cloning of adult trees and the
success in practice will mainly depend on the ability to rejuvenate them (Pierik,
1990).
The explants being used for tissue culture of Salix are usually the nodal
segments and the shoot tips. However, in some research, other parts of mother
plants were used as explants. Research on ovary culture of hybrid Salix was
conducted by Agrawal and Gebhardt (1994) for rapid mcropropagation. They
studied Salix fragilis X Salix lispoclados with Schenk and Hildebrandt (SH) semi-solid
medium (1972) with 0.2 mg 6-benzyladenine (BA) per liter for culturing the hybrid
seedlings.
Nodal cuttings of these seedlings were used for shoot multiplication in semi-
solid WPM (McCown and Lloyd, 1981) with 0.2 mg BA per liter and the shoot
proliferation was enhanced to 5-fold after transfer. SH medium without plant
growth regulators (PGR) was the best for rooting in vitro. Pereira et al. (2000)
5
reported that both proliferation and root induction was influenced by the source of
explant (Salix humboldtiana) used. MS medium supplemented with 2.3 M kinetin
enhanced shoot production while half-strength MS medium supplemented with 5.3
M NAA enhanced rooting.
The tissue culture of hazelnut might be accomplished by using the nodal
segments and shoot tips, however, as a recalcitrant species, using axillary buds and
embryo culture was commonly used in micropropagation of hazelnut. In the study
of Centeno et al. (1997), they used embryo tissue culture to evaluate the
endogenous indole-3acetic acid (IAA), abscisic acid (ABA) and cytokinins in Corulus
avellana L. cotyledons of different developmental stage and genetic source for their
somatic embryogenic capacity. They found that the N6-isopentenyl adenine (iP) type
and zeatin (Z) type cytokinin ratios were two good indexes of the embryogenic
competence of explants, suggesting the endogenous hormone balance is very
important in defining in vitro potential of hazelnut cotyledons.
According to a research conducted by Garton et al. (1983), organogenesis in
macro and micropropagules was affected by the clonal origin or genotype of the
plant material. They used rooted cuttings of ten willow clones that were treated
with 3 nutrient solutions where nitrogen, potassium and phosphorus were in
different concentrations. Ten weeks after treatments the willow cuttings were
placed in peat pellets under intermittent mist environment, and the lateral buds of
actively grown shoots were cultured in vitro on medium to promote shoot
proliferation. They found that effects of stock plant nutrition treatments were less
important than the clonal effects in micropropagation. Willow cuttings receiving the
6
highest concentration of N, P, and K had less root initiation rate than those cuttings
that received lowest concentration.
The time of taking explants from the field may have an effect on the
micropropagtion process. The carbohydrates, proteins, and growth substances in
the stock plants change as the temperature, day length, light intensity and water
availability changes, and different level of these substances can influence the explant
responding to the in vitro environment (Torres and Carlisi, 1986). Yu and Reed
(1993) used three cultivars of C. avellana grafted in the greenhouse, and they
collected the explants from March to July. It was found that explants collected in
March produce a greater number of successful initiations.
2) Initiation
Successful initiation in vitro is affected by the plant source. Older plants have
relatively low initiation rate and high contamination rate problems (Rodriguez et al.,
1989; Anderson, 1984), while juvenile plants or suckers have higher initiation rate
in vitro. The tissue culture of seedlings can be very successful (Anderson, 1984), and
shoots that are growing actively can acclimatize to in vitro condition easily.
To disinfest the plant material, the method of washing with 10% bleach
followed by rinsing in distilled water is often used (Neuner and Beiderbeck, 1993).
In a study by Tormala and Saarikko (1985), their disinfestation method was dipping
the explants in 70% ethanol for 30 seconds before soaking in 3% sodium
hypochlorite for 15-20 min and rinsing four times in sterile water.
3) Culture media
7
After Murashige and Skoog (1962) invented MS medium for plant tissue culture,
numerous media formulations have been developed by scientists. For certain
species and genotypes, the ratio of nutrients (including macronutrients and
micronutrients), the concentration of plant growth regulators and the type of
vitamins may vary in order to have successful tissue culture. MS medium, WPM
(McCown and Lloyd, 1981) and Driver & Kuniyuki Walnut (DKW) Medium (Driver
and Kuniyuki, 1984) are common formulations that have been used in hazelnut
tissue culture.
Plant growth regulators (PGRs) are another important factor to consider when
preparing the culture medium. PGR have been studied to determine their
combinations and concentrations to be used in media. N-6 benzyladenine (BA) and
zeatin are common cytokinins being studied for shoot proliferation, and indole
butyric acid (IBA) is usually used for root initiation and formation.
In the micropropagation of Salix species, the media usually used are MS medium
and WPM medium. Bhojwani (1980) studied a method for rapidly producing in vitro
multiplication of a hybrid willow. In his research, MS medium was used, and various
plant growth regulators such as benzylaminopurine (BAP), naphthaleneacetic acid
(NAA) and 2-isopentenyl adenine (2-iP) were tested. It was found that BAP at 0.1
mg/L + NAA at 0.2 mg/L was best for shoot multiplication, resulting in 4-fold
multiplication within 4 weeks. After being rooted on MS medium containing NAA for
10 days, the hybrid willows were transplanted to pots with over 90% success.
Similarly, Salix babylonica showed maximum proliferation on a modified MS
medium containing 1 mg BAP per liter. (Dhir et al., 1984) Incorporation of NAA or
8
gibberellic acid (GA) did not enhance shoot proliferation in the presence of
benzylaminopurine. Rooted plantlets were transferred successfully to pots and
maintained under high humidity conditions.
Paiva neto et al. (1998) conducted a study of in vitro induction of adventitious
roots of Salix humboldtiana. They used WPM supplemented with NAA and IBA to
culture the Salix leaf and nodal segments. Root hairs were only observed when NAA
was present. The bud growth was initiated in WPM containing 2.68 M NAA + 2.46
M IBA, while adventitious roots were found in other combinations of plant growth
regulators tested (4.92 M IBA; 2.68 M NAA + 4.92 M IBA; 5.37 M NAA + 4.92
M IBA; 5.37 M NAA).
Salix tetrasperma Roxb, which is commonly known as Indian willow, was
studied for in vitro regeneration as well (Khan et al., 2011). Similarly, shoot
induction on WPM containing 5 M 6-benzyladenine was the best for shoot number
and shoot length, whereas multiplication and elongation was better after
transferring to WPM containing 1 M BA + 0.5 M NAA. Half-strength WPM with 0.5
M IBA had the best results of rooting in vitro.
In the micropropagation of hazelnut, DKW medium, which was originally
developed for walnut, was more often used because of the common characters of
nut trees. Based on a two-stage system of micropropagation, Damiano et al. (2005)
developed an establishment stage basal medium, and a multiplication medium. They
found a medium that contains macro and minor nutrients from the formulation of
Perez-Tornero et al. (2000) in combination with the DKW organics was the best for
establishment. The MOLT medium developed by Damiano et al., which combines

9
DKW and WPM, was successful in producing more and better shoots in the
multiplication stage. Lower salt concentrations (15-25% less) in MOLT medium
may have contributed to the results.
In Nas and Read (2004) research, a new culture medium called NRM was
developed based on the composition of C. avellana kernels. The modifications they
made were: a) The average of MS, DKW, and WPM nitrogen was applied because the
N level in kernels was too high to use in micropropagation, and copper levels were
set the same as MS since copper in kernels was high as well; b) 100 mg Sequestrene
138 Fe per liter was used as the iron source; c) the four compounds (Cu SO4, MgSO4,
MnSO4, ZnSO4) provide all the sulfur needed in the medium; d) MgSO4 and KH2PO4
and minor element concentrations are higher than MS, DKW or WPM; e) in MS and
WPM, the myo-inositol concentration is at 100 mg/L, and 1000 mg/L in DKW. NRM
modified their formula to have 200 mg myo-inositol /L. Hybrid of C. americana and
C. avellana explants were used in the research to test the NRM medium together
with MS, WPM, DKW, and NN (Nitsch and Nitsch, 1969). They concluded that
increasing Cu and myo-inositol were related to the increased shoot length up to
three fold and increased shoot number up to 93%. The explants cultured on medium
containing 2.25 mg 5CuSO4 /L+ 400 mg myo-inositol /L could grow 35-50 mm
shoots and about 5-7 axillary buds. Based on their research results, it was also
recommended that when developing a medium for plants that are difficult to
establish in vitro, 5-20% w/v mineral and organic contents of seeds can be
considered for use with moderate modification (Details shown in Appendix)
4) Problems
10
Vitrification was first described by Phillip and Matthews (1964), and Hackett
and Anderson (1967) in the 1960’s. The term “vitrifcation” was used to describe two
types of processes related to tissue culture. If the organ and tissue have abnormal
morphological appearance and physiological function, “vitrification” was used.
Another circumstance to use this term is to describe the transition from liquid to
solid state. Since it caused lots of confusion, Debergh et al. (1992) proposed to
replace it with the term “hyperhydricity”. In some of the research, the
hyperhydricity phenomenon was observed due to the composition of the culture
media, the quality of the culture explants, and the environmental conditions where
the culture containers were maintained. The so-called vitrified or vitreous plants
appear turgid or hyperhydric, watery at their surface, and hypolignified. Their
organs are somehow translucent, in some cases less green, and easily breakable
(Gaspar, 1991). The vitrification phenomenon was observed only on the
multiplication medium, and never happened during rooting (Gaspar, 1991).
Juvenility and phase change in woody plant species exert profound impacts on
plant morphology and the ability of explants to be successfully propagated in vitro,
and for many woody species, the ability to reproduce sexually is only reached after
many years of juvenile growth. Thus, it has been very difficult for many recalcitrant
woody species to have successful micropropgation in vitro (Read and Bavougian,
2013). Read and Bavougian reported that in vitro technologies were used to induce
rejuvenation, such as meristem culture, chemical treatments, pruning and hedging,
forcing new growth and taking advantage of epicormic buds, grafting and
micrografting, and somatic embryogenesis.

11
The rejuvenating of the bud break could be accomplished by hormone
treatments (Beck et al., 1998), serial grafting (Valdes et al., 2003) and in vitro
micrografting (Perrin et al., 1994). Known as Korean weeping willow, explants from
20-year-old Salix pseudosiogyne trees were cultured on WPM or WPM supplemented
with BA, zeatin and GA3 (Park et al., 2008). The results indicated that bud break on
BA supplemented media had a higher bud break rate. Various levels of cytokinins
(2-iP, zeatin, dihydrozeatinriboside) and ABA were tested as well. The levels of
zeatin-type cytokinins were higher than those of isopentenyladenin type of
cytokinins in the explants cultured on BA supplemented media. Necrosis was
observed upon subculture of explants to the fresh basal WPM.
Contamination is the most common of all problems, and bacteria and fungi are
the main contaminants. Microbial contamination and the toxic effects of over
disinfestation are problems with achieving viable explants (Hand, 2013). Surface
disinfestation is essential in controlling the contamination rate, unfortunately in
many cases the internal microbe cannot be killed after the disinfestation process,
especially if adult trees were used. The contamination being observed within 1 week
after culture are results of external bacteria and fungi, while contamination caused
by internal ones could be observed from the second week (Messeguer and Mele,
1983).
Field-grown unpruned mature hybrid C. americana X C. avellana hazelnut
genotypes were used as explants source by Nas (2004). Dormant twigs (35-45cm
long) were forced by immersing the basal end of the twigs in forcing solution (Yang
et al., 1986) containing 8-hydroxyquinoline citrate at 200 mg/L + 2% sucrose +

12
gibberellic acid (GA3) at 10 mg/L. Explant contamination rates varied from 30-90%
depending on genotype.
There are many protocols that can be used for surface disinfestation of the
hazelnut explants. The first step usually begins with tap water washing for half an
hour after the explants were cut (Yu and Reed, 1995). The explants can then be
swirled in vessels that contain 10% chlorine bleach including 5 drops of Tween-20
for 10-30 min, followed by rinsing in sterile distilled water for 2-3 times (Nas and
Read, 2004). Ethanol can also be applied in the disinfestation process by dipping the
material in it before using the bleach (Bacchetta et al., 2008; Nas, 2004).
Contamination could also be reduced by using plant material from the
greenhouse. In the study of vegetative propagation of difficult-to-root Salix caprea,
Liesbach and Naujoks (2004) found that the majority of shoot tips and nodal
segments from five clones died within one month because of bacterial
contamination, and only nursery-grown explants were able to grow in vitro. Three
clones failed to initiate in vitro, while the other two were able to be cultured via
repeated subculture on various media.
Epiphytic and endophytic organisms can cause serious losses to
micropropagation in vitro in every stage of growth (Cassells, 1991; Leifert et al.,
1995). There are difficulties in detecting bacterial contamination if they remain
inside the plant tissue, which could not be eliminated by surface disinfestation
(Debergh & Vanderschaghe, 1988). Thus, the antibiotic therapy should be
considered if less contamination is important. There are several groups of
antibiotics and therefore the type of antibiotics to be used in media should be

13
determined by the bacteria present in culture (Buckley et al., 1995). In general,
carbenicillin, cephalothin, gentamicin, polymyxin, figampicin, streptomycin, and
kanamycin are some possible choices in plant tissue culture and the concentration
ranges from 10 mg to 100 mg per liter depending on the sensitivity of the explants
(Buckley et al., 1995; Falkiner, 1988; Kneifel & Leonhardt, 1992).
As a response of plants to excision, explant browning is another common
problem in culture (Bonga & Aderkas, 1992). When tissues are suffering from
stresses such as mechanical injury and explants are being cut from the stock plant,
phenolic compounds are produced and tissues often secrete brown or black
pigments. Phenolics released into the medium can oxidize explants, prevent shoots
from growing and cause the death of plant material (Debergh & Read, 1991; Thorpe
et al., 1991). According to the study of Yu and Reed (1995), the tissue browning and
explant contamination could be observed 1 week after culture, and the tissue
browning was confined to the plant and did not stain the medium.
There are methods that can be applied to reduce the browning rate such as
storing the stock plants under low light/in the dark, soaking explants in water after
excision and sub-culturing the explants frequently (Bonga & Aderkas, 1992;
Compton & Preece, 1986;).

14
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19
Chapter 1: Salix matsudana
Introduction
In a Nebraska-Hungary cooperation program, several Hungarian genotypes,
including Salix matsudana ‘Golden Spiral’ were studied for their stress tolerance for
urban landscape. (Read and Schmidt, 1997) ‘Golden Spiral’ has twisted upright
golden branches and twigs with bright green leaves, which make it a unique bonsai
plant and decorative tree through the year. Cuttings and seeds are the common
methods of propagating, however, micropropagation of Salix matsudana could
provide more and healthier plantlets for rapid commercial scale-up in the nursery
industry. In addition, the plantlets from micropropagation were more uniform and
practical than cuttings in tests by Tormala and Saarikko (1985).
Literature Review
Salix matsudana can be rooted easily via cuttings, yet there are still some
obvious benefits that tissue culture techniques could provide, such as accelerating
breeding programs and reducing the risk of disease spread. (Bergman et al., 1985)
1) Explant materials
Pereira et al. (2000) reported that both proliferation and root induction were
influenced by the source of explant (Salix humboldtiana) used. MS medium

20
supplemented with 2.3 M kinetin enhanced shoot production while half-strength
MS medium supplemented with 5.3 M NAA enhanced rooting.
According to Liesbach & Naujoks (2004), the applicability of micropropagation
for selected Salix caprea donor trees was strongly depending on the genotype. In
addition, based on the study of Bergman et al. (1985), the tendency of axillary
shoots to develop on shoot cultures depend on the genotype, the type of shoot and
the number of previous subcultures. In their research, five willow species clones
were used to test the effect of BA on the shoots cultured in vitro. It was found that
0.5 M or 1 M BA was the optimum concentration for shoot multiplication, while
for shoot elongation, media containing 0-0.5 M BA were better for the growing of
the Salix caprea depending on the genotypes. If the BA concentration was higher
than 10 M, it would cause the browning and death of shoots, and if concentration
higher than 0.5 M, root initiation was inhibited almost completely. Besides, it was
found that prolonged culture in vitro could increase the rooting ability for the most
recalcitrant clones.
2) Culture Media
In micropropagation of Salix, in addition to MS medium and WPM medium,
many other media were also reported in several studies. Chalupa (1983) used the
Gresshoff and Doy (1972) medium at full concentration or half concentration, and it
stimulated best growth of willow shoots from axillary bud compared to other media.
The growth of shoots was most rapid if the medium contained no cytokinin and
auxin, or when IBA concentration was at 0.1-0.2 mg/L.

21
Callus induction and plantlet regeneration of Salix exigua clones were examined
by Stoehr et al. (1989). BAP and 2,4-D were used with basal media. They found that
the callus production was best on WPM, while after calli were subsequently cultured,
half-strength MS medium without PGR was able to grow roots successfully. The
results that the shoot primordia only developed at 0.1 mg BAP per liter in two
clones suggested that there was a clonal variation in organogenic responses.
Thidiazuron (TDZ) was reported to induce callus formation of various species
and in some cases the cell proliferation rate was higher with TDZ than with other
plant growth regulators (Murthy et al., 1998). Merkle et al. (1998) reported that
TDZ at both 0.1 mg/L and 0.01 mg/L could induce embryogenesis from sweetgum
inflorescence explants. However, there were also reports that TDZ-induced buds fail
to elongate (Meyer and van Staden, 1988) or shoots did not convert into complete
plantlets (Huetteman and Preece, 1993). In the study of in vitro regeneration of Salix
nigra from adventitious shoots (Lyyra et al., 2006), they found that 47-92% of
inflorescences treated with 0.1 mg TDZ per liter produced buds but these buds
failed to elongate into shoots. They also observed that buds in liquid medium for 6
weeks had faster shoot elongation than those on semi-solid basal WPM in GA-7
vessels, which was different from Agrawal and Gebhardt’s results.
3) Problems
It is often a problem to establish in vitro cultures from adult trees because of the
age affects (Park et al.,2008), and the aging is related to the reduced growth rate and
seasonal dormancy (Pierik, 1990). The rejuvenating of the bud break could be
accomplished by hormone treatments (Beck et al., 1998), serial grafting (Valdes et

22
al., 2003) and in vitro micrografting (Perrin et al., 1994). Known as Korean weeping
willow, explants from 20-year-old Salix pseudosiogyne trees were cultured on WPM
or WPM supplemented with BA, zeatin and GA3 (Park et al., 2008). The results
indicated that bud break on BA supplemented media had a higher bud break rate.
Various levels of cytokinins (2-iP, zeatin, dihydrozeatinriboside and ABA) were used
for testing the results. The levels of zeatin type cytokinins were higher than those of
isopentenyladenin type of cytokinins in the explants cultured on BA supplemented
media. Necrosis was observed upon subculture of explants to the fresh basal WPM.
In the ovary culture of hybrid salix research (Agrawal and Gebhardt, 1994), they
used liquid medium for micropropagation, and if the shoots were kept in the liquid
medium, they would become hyperhydric and had smaller and less expanded leaves.
Contamination is another concern. In the study of vegetative propagation of
difficult-to-root Salix caprea (Liesbach and Naujoks., 2004), they found that the
majority of shoot tips and nodal segments from five clones died within one month
because of bacteria contamination, with only greenhouse-grown explants left. Three
clones failed to initiate in vitro, while the other two were able to culture via
repeated subculture on various media.
According to the study of in vitro propagation of Salix caprea, Schenck-
Hildebrandt (1972) and Ahuja’s medium (1983) were used (Neuner and Beiderbeck,
1993), though no significant differences were observed among all media used. They
also reported very serious contamination issues. The contamination rates varied
from 6% to 69% depending on the explant source (e.g. clones or habitats), the time
of explants being collected and the concentration of NaOCl.

23
Objectives
Three trees of Salix matsudana ‘ Golden Spiral’ were used for the experiments
that were conducted to test whether NRM was superior to other media (MS, WPM
and DKW) in growing more shoots and roots, longer stems and greener leaves. The
concentrations and combinations of plant growth regulators (BA, IBA) for shoot and
root growth in vitro were examined. After shoot multiplication and root initiation
the ‘Golden Spiral’ plantlets were to be transplanted into soil.
Materials and Methods
1) Plant material
Salix matsudana ‘Golden Spiral’ branches with axillary buds were obtained from
the field located at the horticulture garden at the University of Nebraska-Lincoln.
Field grown branches with axillary buds collected in winter (January, February,
March) were cut from the mother plant, kept in plastic bags with wet paper towels
and stored at 4 C for2 weeks. Before storing in the cooler, the branches were
disinfested by rinsing with tap water for 5 min, followed by soaking in 10% sodium
hypochlorite (Bleach of Hyvee Co.) for 15 min. After removal from the cooler,
branches (about 10-15 cm long) that contained 3-5 buds were treated with forcing
solution in GA-7 containers at 24 ± 1C under cool white fluorescent 16 hours a day.
After bud break in the forcing solution, softwood cuttings were disinfested in a
laminar flow hood with 70% ethanol spray for 5 seconds, followed by swirling in
small glass jars that contained 10% sterilized sodium hypochlorite with 20 drops
24
Tween 20 (wetting agent) per liter for 10 or 20 min, and rinsed for 5 min in sterile
deionized water three times. The disinfested softwood plant materials were cut into
single node or shoot tip explants, and each explant was cultured in one shell vial
containing 5 ml of the test media.
2) Forcing solution:
After the field grown materials were disinfested, apical and basal ends of the
hardwood cuttings were given a fresh cut. Axillary bud outgrowth was then forced
by immersing the basal end of branches in 30 ml of forcing solution (Yang and Read,
1986) containing 200 mg 8-hydroxyquinoline citrate per liter and 2% sucrose per
liter. The basal end of each cutting was pruned off, and the solution replaced twice a
week. After the bud break, tissue cultures were initiated by culturing explants in
shell vials containing 5ml medium. The cultures were maintained at 241C under
cool white fluorescent light for 16 hours per day. One week after the culture
initiation, the contaminated cultures were excluded. The numbers of new shoots
and roots were recorded as well.
3) Medium:
For culture establishment, the following culture media were used:
1: MS: Murashige and Skoog salts and vitamins supplemented with PGR noted;
2: DKW: Driver and Kuniyuki salts and vitamins supplemented with PGR noted;
3: WPM: Woody Plant Medium salts and vitamins supplemented with PGR noted;
4: NRM: Nas and Read Medium use DKW basal salts, NRM vitamins, and 0.6 mg
Sequestrene 138 Fe per liter supplemented with PGR noted.

25
All of the above media were adjusted to a pH ranging from 5.5 to 5.8 and
autoclaved at 121 °C for 30 min. The basal salts were from Phyto Technology
Laboratories Co..
The following 4 combinations and concentrations of plant growth regulators
were included in the 4 media.
Experiment 1) 2.5 mg BA/L + 0.05 mg IBA/L in MS, WPM, DKW or NRM
Experiment 4) 0.05 mg IBA/L in MS, WPM, DKW or NRM
Experiment 1 was first conducted on June 23rd 2013, and repeated two times on Aug
13th 2013;
Experiment 2 was first conducted on Aug 13th 2013, and repeated two times on Sep
19th, 2013;
Experiment 3 was fist conducted on Sep 9th 2013, and repeated two times on Oct
31st 2013;
Experiment 4 was first conducted on Oct 31st 2013, and repeated two times on Nov
25th, 2013.
Experiment 1, 2, 3, and 4 were conducted to test the influence of 4 media in
combination with 4 concentrations and combinations of PGR on the explant growth.
The shoot number, the stem length and the leaf color were three indicators used to
evaluate the explant growth. After the Salix explants were cultured on media, the
number of shoots or roots were additively recorded every 10 days for 40 days, and
when the shoot number was recorded 40 days after culture, the stem length and leaf
26
color were measured. The Pantone Matching System (PMS) Color Chart was used
(Figure 7) to evaluate the leaf color. If the ‘Golden Spiral’ explants leaf color matched
green color numbers PMS 361, 362, 363, 368, 369, 375, and 376, it was considered
that these explants had healthy leaf color, and the number of healthy-colored
explants was counted.
Experiment 5 was conducted to use the two stage system (shoot multiplication
stage and root initiation stage) to culture the explants and transplant the cultures
into soil. Based on the results of experiment 1, 2, 3 and 4, two of the better media
out of four were selected. The combination and concentration of PGR that would
stimulate shoot production were included in the two media for shoot multiplication.
The shoot number was recorded every 10 days for 40 days. Forty days after culture,
whole explants were transferred to rooting medium. After the explants had roots,
they were transplanted into soil.
4) Experimental Design and Statistical Analysis
One single shoot tip or nodal shoot of Salix matsudana ‘Golden Spiral’ was
cultured in each shell vial which contained 5 ml of each medium. For the four types
of media, each medium had 5 shell vials to culture 5 explants (5 replicates). The
experimental unit was each shell vial, and there were 20 shell vials (4 media*5 shell
vials) at every combination and concentration of PGR (experiment 1, 2, 3, and 4),
There were three trials. Experiment 5 was conducted for both shoot multiplication
and root initiation, and it was conducted 3 times (3 trials). Since the shoot number
was recorded additively every 10 days, to simplify the statistical analysis and to
check whether there was interaction between time and treatment, the experiment
27
was considered to be a Complete Random Design (CRD) with repeated measures.
For the stem length that was measured 40 days after culture, the experiment was
considered to be a 4X4 Factorial Treatment Design. Statistical analysis of data was
performed using PROC GLIMMIX and MEANS procedures
(SAS institute, 2013).
Results and Discussion
In Experiment 1, when the plant growth regulator was at a concentration of 2.5
mg BA/L + 0.05 mg IBA/L (Figure 1-1, Table 1-1, and 1-2), WPM and MS medium
had more shoot number than that of DKW and NRM. The shoot number in NRM
declined as the experiment progressed, which indicated that there was an
interaction between time and media (P value=0.0003 at α=0.05 of trial 1, trial 2 and
3 had similar results) . The shoot tip explants did not survive in NRM, while the
segment nodes with one leaf had grown new shoots successfully. This is probably
due to the sensitivity of the shoot tips to the Sequestrene 138 iron in NRM. For 3
trials of Experiment a, the results were similar.
However in experiment 2, when the IBA concentration dropped to 0.01 mg/L,
with BA at 2.5 mg/L (Figure 1-2, Table 1-1, and1-2), different results were observed.
NRM was the most effective medium to have a greater shoot number 40 days after
culture, while the other three media had no significant difference among each other
and there was no interaction between time and media.
When the BA concentration was reduced to 2.0 mg/L while the IBA remained at
0.01 mg/L (Experiment 3, Figure 1-3, Table 1-1 and 1-2), there was no significant
28
difference between DKW and NRM in shoot number when considering the
experiment design to be CRD with repeated measures, and there was no interaction
between time and media. When comparing the mean number of shoots of four
media in experiment 1, 2 and 3 40 days after culture, the experiment was analyzed
as a factorial treatment design with SAS and it was found that shoot number in
experiment 2 was significantly greater than those in experiment 1 and 3. Thus, the
combination and concentration in experiment 2 (2.5 BA mg/L + 0.01 mg IBA/L) was
better than the other two experiments.
When the media contained both BA and IBA, only new shoots were observed
while new roots were not found. If only IBA was included in media (Experiment 4),
roots were able to initiate and grow (Figure 1-4b, Table 1-1, and 1-2), though the
shoot number (Figure 1-4a, Table 1-1 and 1-2) was significantly less than that of
experiment 1, 2, and 3 (P value=0.0327 at =0.05 of trial 1). After finishing
Experiment 4, another experiment was conducted to test three levels of IBA (0.05,
0.1 and 0.2 mg IBA/L), and the results showed that there was no significant
difference among these three levels (P value=0.8574 at =0.05 of trial 1), which was
different from the results of the study by Chalupa (1983). This is probably due to the
species or the media in use. Since higher concentration of IBA did not increase the
shoot number, 0.05 mg IBA/L was considered to be the best concentration for root
initiation.
In Experiment 1, 2, 3, and 4, abnormal growth of some explants was observed.
Even though the shoot number was as many as 15, the Salix explants were not able
to elongate, only to form dwarf explants with multiple tiny leaves (<1cm) in vitro
29
(Figure 1-5). Thus, in addition to comparing the number of new shoots to evaluate
the performance of the 4 media, stem length of ‘Golden Spiral’ was recorded as the
second indicator (Figure 1-6, Table 1-3, and 1-4). Explants cultured on NRM had
significantly longer stems compared to the other 3 media (Table 1-3, and 1-4), and
there was no significant difference among MS, WPM, or DKW. Forty days after
culture, the explants in NRM were able to grow to 4-6cm, while explants in MS, WPM
and DKW grew to 2-4cm (Figure 1-5).
In Experiment 1, 2, 3 and 4, the third indicator to evaluate explant growth was
leaf color and Pantone color matching system was used (Figure 1-7a). Fewer green
leaves, as a type of vitrification phenomenon, were very common among the
explants that grew on MS medium, WPM and DKW medium, while the healthy-
colored explant number on NRM was significantly greater than those on MS, WPM
and DKW (Figure 1-7b, no error bars in figure because of zeros in data, statistical
analysis was done with original data). Since the stem elongation and leaf color of
explants cultured in MS and WPM were considered to be not as desirable as the
explants cultured on DKW and NRM, for the Experiment 5, DKW and NRM were
used for shoot multiplication and root initiation media.
In Experiment 5 (Figure 1-8a, 1-8b, Table 1-5), there was no significant
difference between DKW and NRM in Trial 1 in growing new shoots (P
value=0.0602 at =0.05) at shoot multiplication stage (DKW and NRM containing
2.5 mg BA/L + 0.05 mg IBA/L), but in Trial 2 and 3 the difference were significant (P
value<0.0001 at =0.05 of trial 1) In the second stage (DKW and NRM containing
0.05 mg IBA/L, and there was also no significant difference in root number (P
30
value=0.2972 at =0.05) in Trial 1 either, but the difference 40 days after culture
were significant again. After transplanting the explants into soil, among 20 of the
explants, 17 of them were able to grow into strong and healthy plantlets with the
stem length ranged from 15cm to 20cm (Figure 1-9). The three plantlets that had
only 1 or 2 roots died when they were transplanted into soil, and this might be
related to their failure to acclimatize to the soil environment. Plantlets from the
NRM had the fastest growth rate (Table 1-6) compared to explants from other
media, and this was possibly due to the longer stems and more roots that were
found on the plantlets produced on NRM.
Conclusion
The NRM supplemented with 2.0 mg BA/L + 0.05 mg IBA/L was the best
medium for shoot multiplication of Salix mtsudana ‘Golden Spiral’. For the root
initiation stage, IBA at a concentration of 0.5 mg/L was the best choice. Explants
grown on NRM had healthy green leaf color and good stem elongation. Shoots
produced in vitro with at least 2 roots grown into plantlets and were successfully
transferred to soil.
31
Figure 1-1: Shoot number of Salix matsudana 'Golden Spiral' explants cultured
in vitro on 4 media containing 2.5mg BA/L + 0.05 mg IBA/L
9
8
WPM, 7.4
Mean number of shoots
7 MS, 7
6
(Trial 1)
5
4 DKW, 4
3
2
1
NRM, 1.4
0
3-Jul 12-Jul 21-Jul 30-Jul
10
9
MS, 8.4
8 WPM, 7.8
7
6
(Trial 2)
5
DKW, 4.6
4
3
2
NRM, 1.6
1
0
23-Aug 2-Sep 12-Sep 22-Sep
9
8
MS, 7.6
7
6 WPM, 7.4
(Trial 3)
5 DKW, 5.2
4
3
2
1 NRM, 1.2
0
*MS = Murashige and Skoog Medium (1962); WPM = Woody Plant Medium (Lloyd
and McCown, 1981); DKW = Driver and Kuniyuki Walnut Medium (1984); NRM =
Nas and Read Medium (2002);
*Error bars stand for the standard error.
32
in vitro on 4 media containing 2.5 mg BA/L + 0.01 mg IBA/L
14
12 NRM, 12
10
(Trial 1)
8
DKW, 7.6
6
WPM, 6.6
MS, 5.6
4
0
14
12
NRM, 11.6
10
DKW, 7
(Trial 2)
6 MS, 6
4 WPM, 6.2
2
0
19-Sep 28-Sep 7-Oct 16-Oct
14
12
10 NRM, 10.6
(Trial 3)
8 WPM, 6.4
6 DKW, 6
4
MS, 4.8
2
0
33
in vitro on 4 media containing 2.0 mg BA/L + 0.01 mg IBA/L
10
DKW, 9.4
NRM, 8.8
8
WPM, 6.8
(Trial 1)
4 MS, 4.4
0
10
NRM, 9.2
8
DKW, 7.6
(Trial 2)
6 WPM, 5.8
MS, 4.8
4
0
7-Nov 16-Nov 25-Nov 4-Dec
10
NRM, 9.2
8
DKW, 7.4
(Trial 3)
6 WPM, 5.8
4
MS, 3.6
2
0
34
Figure 1-4a: Shoot number of Salix matsudana 'Golden Spiral' explants

cultured in vitro on 4 media containing 0.05 mg IBA/L
7
6 NRM, 5.2
5 DKW, 5
(Trial 1)
4 MS, 5
WPM, 4.2
3
0
7 NRM, 6.2
6 WPM, 5.4
5 MS, 5.2
(Trial 2)
4 DKW, 5
3
0
5-Dec 15-Dec 25-Dec 1-Jan
7
NRM, 6.6
6
WPM, 5.4
5
DKW, 4.8
(Triala 3)
4
MS, 3.8
3
0
35
Figure 1-4b: Root number of Salix matsudana 'Golden Spiral' explants cultured
in vitro on 4 media containg 0.05mg IBA /L
7
Mean number of roots
6
5 NRM, 5.2
(Trial 1)
4
MS, 3.4
3
DKW, 2.8
2 WPM, 2.8
1
0
7
5 NRM, 5
(Trial 2)
4
MS, 3
3
DKW, 2.6
2 WPM, 2.6
1
0
7
6
5 NRM, 5
(Trial 3)
4
DKW, 3.8
MS, 3.4
3
2 WPM, 3
1
0
36
Figure 1-5: Growth comparison of Salix matsudana ‘Golden Spiral’ explants

cultured in vitro
*Top: Explants grown on Nas and Read Medium (NRM), Driver and Kuniyuki Walnut
medium(DKW), Woody Plant Medium (WPM) and Murashige and Skoog Medium
(MS) containing 2.0 mg BA/L + 0.05 mg IBA/L (left to right). The yellowish leaf color
and abnormal growth can be seen on WPM and MS. The elongation of the explants
and the roots can be seen on DKW and NRM.
*Bottom: Light leaf color and abnormal growth of salix matsudana ‘Golden Spiral’ on
MS medium
37
Figure 1-6: Stem length of Salix matsudana 'Golden Spiral' explants cultured in
vitro on 4 media containing various PGR
6
Mean stem length: cm
A(2.5 mg BA/L+0.05 mg
5 IBA/L)
B(2.5 mg BA/L + 0.01mg
(Trial 1)
4
IBA/L)
3
C(2.0 mg BA/L + 0.01mg
2 IBA/L)
D(0.05mg IBA/L)
1
0
MS WPM DKW NRM
6
A(2.5 mg BA/L+0.05 mg
5 IBA/L)
B(2.5 mg BA/L + 0.01mg
(Trial 2)
4
IBA/L)
3
C(2.0 mg BA/L + 0.01mg
2 IBA/L)
D(0.05mg IBA/L)
1
0
MS WPM DKW NRM
6
A(2.5 mg BA/L+0.05 mg
5 IBA/L)
B(2.5 mg BA/L + 0.01mg
(Trial 3)
4
IBA/L)
3
C(2.0 mg BA/L + 0.01mg
2 IBA/L)
D(0.05mg IBA/L)
1
0
MS WPM DKW NRM
38
Figure 1-7a: Pantone Matching System Color Chart for comparing healthy-
colored explants of Salix matsudana ‘Golden Spiral’ cultured in vitro
--Salix
matsudana
*Green color number PMS 361, 362, 363, 368, 369, 375 and 376 were considered to
be healthy leaf color.

39
Figure 1-7b: Number of healthy-colored explants of Salix matsudana 'Golden

Spiral' cultured in vitro on 4 media containing various PGR
5
Number of healthy-colored
4
explants (Trial 1)
2.5 mg BA/L+0.05mg IBA/L

3
2.5mg BA/L+0.01 mg IBA/L
2 2.0 mgBA/L+0.01 mg IBA/L
0.05 mg IBA/L
1
0
MS WPM DKW NRM
5
4
explants (Trial 2)

3
0.05 mg IBA/L
1
0
MS WPM DKW NRM
5
4
explants (Trial 3)

3
0.05 mg IBA/L
1
0
MS WPM DKW NRM
Nas and Read Medium (2002); PGR stands for plant growth regulators
Error bars stand for the standard error.
40
Figure 1-8a: Shoot number of Salix matsudana 'Golden Spiral' explants

cultured on DKW and NRM containing 2.5 mg BA/L+ 0.01 mg IBA/L for shoot
root initiation
14
12
NRM, 11.2
10
DKW, 8.7
(Trial 1)
0
14
12 NRM, 11.9
10
(Trial 2)
6 DKW, 6.5
0
14
12
NRM, 11.1
10
(Trial 3)
8
6 DKW, 6.2
4
2
0
Nas and Read Medium (2002);*Error bars stand for the standard error.
41
Figure 1-8b: Root number of Salix matsudana 'Golden Spiral' explants cultured
on DKW and NRM containing 2.5 mg BA/L+ 0.01 mg IBA/L for shoot
root initiation
4 NRM, 4.1
Mean number of new
3
roots (Trial 1)
DKW, 2.7
2
0
27-Sep 7-Oct 17-Oct 27-Oct
-1
-2
6
5
4 NRM, 4.1
3
(Trial 2)
2 DKW, 2.2
1
0
-1
-2
5
4 NRM, 4.2
3
(Trial 3)
DKW, 2.4
2
0
-1
Nas and Read Medium (2002);*Error bars stand for the standard error.
42
Figure 1-9: 1 month after Salix matsudana ‘Golden Spiral’ explants

transplanted from Nas&Read Medium containing 0.05 mg IBA/L to soil
*Top: The explants that had roots observed in vitro on the 4 media (mostly from
Nas&Read Medium )were selected to transplant into soil and they were able to
survive and grow fast in soil.
*Bottom: The Salix matsudana ‘Golden Spiral’ that was able to elongate and initiate
roots in NRM and transplanted into soil. It grew to 26 cm within 1 month.
43
Table 1-1: Mean shoot number±SE of Salix matsudana ‘Golden Spiral’ in 4

media containing PGRs in Experiment 1,2,3, and 4(Figure 1-1, 1-2, 1-3, and 1-4)
2.5 mg 2.5 mg 2.0 mg BA/L 0.05 mg

BA/L+0.05 BA/L+0.01 +0.01 mg IBA/L 0.05 mg
mg IBA/L mg IBA/L IBA/L (shoots, IBA/L(roots,
(Figure1-1) (Figure1-2) (Figure1-3) Figure1-4a) Figure1-4b)
MS 7.00±2.28 5.60±0.55 4.40±2.51 5.00±1.00 3.40±1.81
WPM 7.40±2.70 6.60±2.61 6.80±1.76 4.20±0.84 2.80±1.10
DKW 4.00±1.30 7.60±1.95 9.40±4.15 5.00±1.22 2.80±2.59
NRM 1.40±2.19 12.00±2.35 8.80±2.95 5.20±0.83 5.20±2.59
*In this table, the data were collected the final time recording the number of new
shoots (roots) for each set of experiment.
Table 1-2: P value of comparisons of treatment (media) differences Least

Squares Means (LSM) at =0.05 in experiment 1, 2, 3, and 4
2.5 mg 2.5 mg 2.0 mg 0.05 mg 0.05 mg

BA/L+0.05 BA/L+0.01 BA/L+0.01 IBA/L IBA/L
mg IBA/L mg IBA/L mg IBA/L (Figure1- (Figure1-
Comparisons (Figure1-1) (Figure1-2) (Figure1-3) 4a) 4b)
MS and WPM 0.4250 1.0000 0.1393 0.6933 1.0000
MS and DKW 0.1128 0.3255 0.0043 0.4905 0.8258
MS and NRM 0.0024 <0.0001 0.0396 0.4309 0.0010
WPM and DKW 0.0185 0.3255 0.1524 0.2797 0.8258
WPM and NRM 0.0002 <0.0001 0.472 0.2386 0.0010
DKW and NRM 0.1284 0.0003 0.472 0.9214 0.0020
Table 1-3: Mean shoot length ± SE of Salix matsudana ‘Golden Spiral’ in 4

media containing PGRs in Experiment 1, 2, 3, and 4 (Figure 1-6)
2.5 mg 2.5 mg 2.0 mg

BA/L+0.05 BA/L+0.01 BA/L+0.01 0.05 mg
mg IBA/L mg IBA/L mg IBA/L IBA/L
MS 3.20±0.84 3.60±1.14 2.80±0.84 2.60±1.14
WPM 3.40±0.89 2.50±1.00 3.00±1.22 2.00±0.71
DKW 2.00±1.58 2.10±0.74 3.00±1.00 2.40±0.89
NRM 4.50±1.32 5.40±0.89 4.60±1.14 3.50±1.12
*MS = Murashige and Skoog Medium (1962);

*WPM = Woody Plant Medium (Lloyd and McCown, 1981);
*DKW = Driver and Kuniyuki Walnut Medium (1984);
*NRM = Nas and Read Medium (2002);
44
Table 1-4: Simple effect comparisons of medium*PGR Least Squares Means of

stem length in Experiment 1, 2, 3, and 4 (Figure 1-6)
Simple
Effect Standard
Level pgr _pgr Estimate Error DF t Value Pr > |t|
medium a 1 2 3.6000 1.6729 59 2.15 0.0355
medium a 1 3 4.8000 1.6729 59 2.87 0.0057
medium a 1 4 4.2000 1.6729 59 2.51 0.0148
medium a 2 3 1.2000 1.6729 59 0.72 0.4760
medium a 2 4 0.6000 1.6729 59 0.36 0.7211
medium a 3 4 -0.6000 1.6729 59 -0.36 0.7211
medium b 1 2 0.8000 1.6729 59 0.48 0.6343
medium b 1 3 0.6000 1.6729 59 0.36 0.7211
medium b 1 4 3.2000 1.6729 59 1.91 0.0606
medium b 2 3 -0.2000 1.6729 59 -0.12 0.9052
medium b 2 4 2.4000 1.6729 59 1.43 0.1567
medium b 3 4 2.6000 1.6729 59 1.55 0.1255
medium c 1 2 -3.6000 1.6729 59 -2.15 0.0355
medium c 1 3 -5.4000 1.6729 59 -3.23 0.0020
medium c 1 4 -1.0000 1.6729 59 -0.60 0.5523
medium c 2 3 -1.8000 1.6729 59 -1.08 0.2863
medium c 2 4 2.6000 1.6729 59 1.55 0.1255
medium c 3 4 4.4000 1.6729 59 2.63 0.0109
medium d 1 2 -10.6000 1.6729 59 -6.34 <.0001
medium d 1 3 -7.6000 1.7744 59 -4.28 <.0001
medium d 2 3 3.0000 1.7744 59 1.69 0.0962
Simple
Effect Standard
Level media media Estimate Error DF t Value Pr > |t|
pgr 1 a b 1.8000 1.6729 59 1.08 0.2863
pgr 1 a c 5.2000 1.6729 59 3.11 0.0029
pgr 1 a d 7.8000 1.6729 59 4.66 <.0001
pgr 1 b c 3.4000 1.6729 59 2.03 0.0466
pgr 1 b d 6.0000 1.6729 59 3.59 0.0007
pgr 1 c d 2.6000 1.6729 59 1.55 0.1255
pgr 2 a b -1.0000 1.6729 59 -0.60 0.5523
pgr 2 a c -2.0000 1.6729 59 -1.20 0.2367
pgr 2 a d -6.4000 1.6729 59 -3.83 0.0003
pgr 2 b c -1.0000 1.6729 59 -0.60 0.5523
pgr 2 b d -5.4000 1.6729 59 -3.23 0.0020
pgr 2 c d -4.4000 1.6729 59 -2.63 0.0109
pgr 3 a b -2.4000 1.6729 59 -1.43 0.1567
pgr 3 a c -5.0000 1.6729 59 -2.99 0.0041
pgr 3 a d -4.6000 1.7744 59 -2.59 0.0120
pgr 3 b c -2.6000 1.6729 59 -1.55 0.1255
pgr 3 b d -2.2000 1.7744 59 -1.24 0.2199
pgr 3 c d 0.4000 1.7744 59 0.23 0.8224
pgr 4 a b 0.8000 1.6729 59 0.48 0.6343
pgr 4 a c -755E-17 1.6729 59 -0.00 1.0000
pgr 4 b c -0.8000 1.6729 59 -0.48 0.6343
*medium a stands for MS, medium b stands for WPM, medium c stands for DKW, and
medium d stands for NRM; *pgr 1 stands for 2.5mg BA/L + 0.05 mg IBA/L, pgr2
stands for 2.5 mg BA/L + 0.01 mg IBA/L, pgr 3 stands for 2.0 mg BA/L + 0.01 mg
IBA/L, and pgr 4 stands for 0.05 mg IBA/L
45
Table 1-5 Mean shoot or root number ± SE of Salix matsudana ‘Golden Spiral’
on DKW and NRM for shoot multiplication and root initiation in Experiment 5
(Figure 1-6a, 1-6b)
Mean shoot Mean root

number ± SE number ± SE
DKW 8.7±1.64 2.7±0.84
NRM 11.2±0.83 4.1±1.30
Table 1-6: Growth rate of Salix matsudana ‘Golden Spiral’ after transplanted
into soil
MS WPM DKW NRM

Growth rate
(cm/Day) 0.9 1.2 1.1 1.8

46
Literature Cited for Chapter 1:
Ahuja, M. R. 1983. Somatic cell differentiation and rapid clonal propagation of aspen.
Silvae Genet. 32:131-135.
Agrawal, D.C. and K. Gebhardt. .1994. Rapid micropropagation of hybrid willow

(Salix) established by ovary culture. Journal of Plant Physiology. 143(6):763-765.
Beck, L. B., R. Dunlop, and J. Staden. 1998. Rejuvenation and micropropagation of

adult Acacia mearnsii. Plant Growth Regul 26: 149-153.
Bergman L, von Arnold S & Eriksson T. 1985. Effects of N6-benzyladenine on shoots

of five willow clones (Salix ssp.) cultured in vitro. Plant Cell Tiss. Org. Cult. 4: 135–
144.
Chalupa. V. 1983. In vitro Propagation of Willows (Salix spp.), European Mountain-

ash (Sorbus aucuparia L.) and Black Locust (Robinia pseudoacacia L.). Biologia
Plantarum. 25(4): 305-307.

Gresshoff, P. M. and C. H. Doy. 1972. Development and differentiation of hapliud

Lycoperisicon esculentum (tomato). Planta 107:161-170.
Huetteman, C. A. and J. E. Preece. 1993. TDZ: a potent cytokinin for woody plant
tissue. Plant Cell Tissue Organ Cult. 33: 105-119.
Liesebach, M., G. Naujoks. 2004. Approaches on vegetative propagation of difficult-

to-root Salix caprea. Plant Cell. Tissue and Organ Culture. 79: 239-247.
Lyyra, S., A. Lima and S. A. Merkle. 2006. In vitro regeneration of Salix nigra from
adventitious shoots. Tree Physiology. 26, 969-975.
Merkle, S. A., A. N. Kimberly, P. J. Battle and R. L. Bailey. 1998. Somatic

embryogenesis and plantlet regeneration from immature and mature tissues of
sweetgum (Liquidambar styraciflua). Plant Sci. 132: 169-178.
Meyer, H. J. and J. van Staden. 1988. In vitro multiplication of Ixia flexuosa.

HortScience. 23:1070-1071.
47
Murthy, B. N. S., S. J. Murch and P. K. Saxena. 1998. Thidiazuron: A potent regulator

of in vitro plant morphogenesis. In Vitro Cell. Dev. Biol. Plant 34: 267-275.
Neuner H & Beiderbeck R (1993) In vitro propagation of Salix caprea L. by single

node explants. Silvae Genet. 42: 308–310.
Park. S. Y., Y.W. Kim, H.K. Moon, H.N. Murthy, Y.H. Choi, and H. M Cho. 2008.
Micropropagation of Salix pseudolasiogyne from nodal explants. Plant Cell Tiss
Organ Cult. 93: 341-346.
Pereira, A.M.S., B. W. Bertoni, R.M. Moraes, and S.C. Franca. 2000. Micropropagation
of Salix humboldtiana Hild. Revista Brasileria de Plantas Medicinais. 2(2): 17-21.
Perrin Y., L. Lardet, F. Enjalric, and M. P. Carron. 1994. Rejeunissement de clones

matures d’Hevea brasiliensis (Miill. Arg.) par microgreffage in vitro. Can J Plant Sci
74:623-630.
Pierik, R. L. M. 1990. Rejuvenation and micropropagation. Pp, 91-101. In: Nijkamp, H.

J. J., L. H. V. Van Der Plas, and J. Van Aajtrik (ed). Prgress in Plant Celular and
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48
Chapter 2: Corylus colurna
Introduction
Red/purple Turkish Hazel is the common name of Corylus colurna ‘Te Terra
Red’. This small tree, or large shrub is the plant material being used in this research.
‘Te Terra Red’ trees are available in many nurseries in the UK, whereas in the US,
especially in Nebraska, there are very few nurseries that can afford this decorative
tree. However, its purple leaves that appear in spring, and they totally fade to green
foliage exhibited by autumn, make it desirable for landscape purposes. It is thought
that ‘ Te Terra Red’, a unique hybrid (C. avellana X C. colurna) that also has red-
purple catkins is certainly a worthy candidate for landscape use in the United States
(Dirr. 2011)
Hazelnut trees are traditionally produced by rooting suckers, layering, cuttings
and grafting (Rodriguez et al., 1989) and the common method of propagating C.
colurna is by seed. However, traditional systems of propagation of Corylus species
are not sufficient to obtain a great number of plants from a tree selected in breeding
programs (Messeguer & Mele, 1987). In contrast, it is hypothesized that
micropropagation can produce hazelnut trees rapidly for commercial scale-up in
nurseries. Though the micropropogation of hazelnut is challenging, as for many
other woody species, using shoot multiplication from single buds or embryos as
explants in tissue culture is a rapid way of asexual propagation of hazelnut (Perez et
al., 1987).
49
Literature review
1) Explant collecting time and size
The time of taking explants from the field may have an effect on the success of
the the micropropagtion process. The carbohydrate, proteins, and growth
substances in the stock plants change as the temperature, day length, light intensity
and water availability changes, and different levels of these substances can influence
the explant responce to the in vitro environment (Torres, 1986). Yu and Reed (1995)
used three cultivars of C. avellana grafted in the greenhouse, and they collected the
explants from March to July. It was found that explants collected in March produced
a greater number of successful shoots and roots in vitro. Fifteen cultivars of C.
avellana were tested by Bacchetta et al. (2005) on the initiation time. The explants
collected in the spring produced better growth in vitro. However, according to
Messeguer and Mele’s (1983) research, the results indicate that plant material taken
in autumn produce a greater number of shoots than those in spring. In addition, it
was reported that explants with a diameter of > 4mm had a better chance of
establishment (Diaz Sala et al., 1994;), and explants less than 1.5mm diameter did
not grow (Messeguer and Mele, 1983).
2) Initiation of bud break and disinfestation
Forcing solution systems can be used to facilitate bud break (Yang et al., 1986).
The forcing solution with 8-hydroxylquinoline citrate at 200mg/L + 2% sucrose +
gibberellic acid (GA) at 10mg/L was used to stimulate bud break. Bud break occurs
50
by immersing the basal end of branches in forcing solution (Yang et al., 1986)
Explant contamination rates varied from 30-90% depending on genotype.
There are many protocols that can be used for surface disinfestation of the
hazelnut explants. The first step usually begins with tap water washing for half an
hour after the explants are cut (Yu and Reed, 1995). The explants can then be
swirled in vessels that contain 10% chlorine bleach, including 5 drops of Tween-20,
for 10-30 min, followed by rinsing in sterile distilled water for 2-3 times (Nas and
Read, 2004). Ethanol can also be applied in the disinfestation process by dipping the
material in it before using the bleach (Bacchetta et al., 2008; Nas, 2004). This
method has drawbacks because it can stimulate phenolic formation, which can
cause browning of explants (Bassil et al., 1991). Hydrogen peroxide (H2O2) is another
powerful oxidant that can be used at 3-10% (v/v) for 1-30 min prior to sterile water
in the disinfestation process, either in combination with other disinfestants or alone
(Smith, 2012).
3) Culture media
A modified MS medium (half-strength macro and micronutrients) was promoted
by Messeguer and Mele (1983) for culture of hazelnut in vitro. Another modified MS
medium specific for hazelnut developed by Anderson (1984) has NaH2PO4 instead of
KH2 PO4 as the potassium source, and has 75% less NH4NO3 and KNO3. He found that
compared to MS and WPM media, the shoot proliferation in his modified medium
was better. It was reported that replacing FeEDTA with 200 mg Sequestrene 138
iron EDDHA per liter in the MS medium can result in healthier and greener plantlets
(Al Kai et al., 1984).

51
Messeguer and Mele (1983) concluded that 2.85 M zeatin performed better than
BA for shoot elongations of C. avellava. Yu and Reed (1993) found that BA
concentration at 6.66-13.3M on modified DKW medium had the best results in
growing shoots from‘ Tonda Gentile Romana’ hazelnut explants. Four cytokinins
(kinetin, iso-pentenyladenine, zeatin and BA) were tested by Thomson and Deering
(2011) with the ‘Knoxfield2’ medium, which is a medium similar to the medium
used in the studies of Anderson (1984) and Al Kai et al. (1984). The results showed
that 22.2 M BA was the best for shoot initiation.
The basal immersion of shoots for 15s in immersion solutions containing IBA (5
mM) plus polyamines produced a rapid 100% rooting response. Enhancement of
rooting was observed when micro shoots were immersed in IBA solution, then
rooted in media containing polyamines. (Diaz-Sala et al., 1994; Berros et al., 1995;
Yu & Reed, 1995;) According to Bassil et al. (1991), root formation occurred on all
shoots dipped in 1mM IBA for 15 s followed by a culture period of four weeks on
auxin-free medium. Perez et al. (1986) reported that IBA promoted embryo
initiation, and they also concluded that the addition of BA and IBA in various
concentration and combinations in two consecutive cultures facilitated embryonic
induction in the primary explants. Based on Gonzalez (1990) research, half-strength
K(h) (Cheng, 1975) medium containing 50 M IBA + 4.5 M Kin could increase the
number of roots developed. However, Perez et al. (1987) also reported that the
shoots treated with 3.0 g IBA per liter did not stimulate root development after 20
days, but they observed high amount of callus on bases instead.

52
Epiphytic and endophytic organisms can cause serious losses to
micropropagation in vitro in every stage of growth (Cassells, 1991; Leifert et al.,
1995). There are difficulties in detecting bacterial contamination if they remain
inside the plant tissue, which could not be eliminated by surface disinfestation
(Debergh & Vanderschaghe, 1988). Thus, the antibiotic therapy should be
considered if less contamination is desired. There are several groups of antibiotics
that can reduce the contamination rate and therefore the type of antibiotics to be
used in media should be determined by the bacteria present in culture (Buckley et
al., 1995). In general, carbenicillin, cephalothin, gentamicin, polymyxin, figampicin,
streptomycin, and kanamycin are some possible choices in plant tissue culture and
the concentration ranges from 10 mg to 100 mg per liter depending on the
sensitivity of the explants (Buckley et al., 1995; Falkiner, 1988; Kneifel & Leonhardt,
1992).
4) Problems
There are several problems that might happen after culturing the explants in
vitro, including contamination, browning, necrosis, to name just a few.
According to several researchers, keeping the mother plants in the greenhouse
made it possible to reduce the contamination rate to a great degree ( Messeguer and
Mele, 1983; Yu and Reed, 1995; Bacchetta et al., 2008). Yu and Reed (1995)
suggested that the higher contamination rate of explants collected in June was
related to the rainfall of the season.
As a response of plants to excision, explant browning is another common
problem in culture (Bonga & Aderkas, 1992). When tissues are suffering from
53
stresses such as mechanical injury and explants being isolated from the stock plant,
metabolism of phenolic compounds is produced and tissues often secrete brown or
black pigments. Phenolic compounds that are released into the medium can oxidize
explants, prevent shoots from growing and cause the death of plant material
(Debergh & Read, 1991; Thorpe et al., 1991). According to the study of Yu and Reed
(1995), the tissue browning and explant contamination could be observed 1 week
after culture, and the tissue browning was confined to the plant and did not stain the
medium.
There are methods that can be applied to reduce the browning rate such as
storing the stock plants under low light or in the dark, soaking explants in water
after excision and sub-culturing the explants frequently (Bonga & Aderkas, 1992;
Compton & Preece, 1986;). Frequent sub-culturing is relatively costly and needs lots
of labor. In addition, it also limits the conditioning function of the medium by
accumulation of excretion products that may stimulate growth (Bonga & Aderkas,
1992). The browning is also related to the disinfestation agents being used since
these agents can kill the axillary meristems by easily entering the stems from the
scars of the leaf bases and the surrounding tissues (Debergh & Read, 1991). Ethanol
is powerful in killing the microorganism, however, it also contributes to tissue
browning at the same time.
Objectives
Two hazelnut trees of C. colurna ‘Te Terra Red’ were used for the experiments
that were conducted to test whether NRM was superior to other media (MS, WPM
54
and DKW) for growing explants and producing the roots. The concentrations and
combinations of plant growth regulators (BA, IBA) for shoot and root growth were
examined. As the research progressed, the explants were lost because of the
contamination so that there was not sufficient number of explants to continue the
experiment. Thus, the most effective disinfestation methods and the media or
forcing solution supplemented with antibiotics (streptomycin, kanamycin) to
control contamination were the focus of this study.
Materials and Methods
1) Plant material
Hazelnut branches with axillary buds were obtained from the field located at
horticulture garden at the University of Nebraska-Lincoln. There are two ‘Te Terra
Red’ trees in the garden in total, and both of them are years old. They are adult trees
about 4-5m in height and 3m in width. Bud break usually happens at the end of April,
and early in May the wormholes on leaves are formed. In autumn, the leaves start to
fall at the end of October. Explants were collected from January to December
throughout the year.
2) Surface disinfestation
The field grown branches with axillary buds collected in winter (November,
December, January, February, March) were cut from the mother plant, kept in
plastic bags with wet paper towels and stored at 4 C for 2 weeks, 1 or 2 month
(Experiment 1). Before storing in the cooler, the branches were disinfested by
rinsing with tap water for 20, 40 or 60 min (Experiment 2) followed by soaking in
55
10% sodium hypochlorite (Hyvee Co.) for 20, 30, 40min(Experiment 3). After
removal from the cooler, branches (about 10-15 cm long) containing 3-5 buds were
treated with forcing solution in GA-7 container at 24 ± 1C under cool white
fluorescent light 16 hours a day.
After bud break in the forcing solution, softwood cuttings were disinfested in a
laminar flow hood with 70% ethanol spray for time for 30 s 90 s or 5 min
(Experiment 4), followed by swirling in small glass jars containing 10% sterilized
sodium hypochlorite with 20 drops Tween-20 (wetting agent) per liter for 10 or 20
min, and rinsed for 5 min in sterile deionized water three times.
In the first year, the disinfested softwood plant materials were cut into a single
node or shoot tip explants, and each explant was cultured individually in shell vials
that contained 5 ml media separately. In the second winter, instead of culturing the
shoots after bud break, the axillary buds were disinfested with the same method
and then cultured directly in to the shell vials in order to reduce the contamination
incidence (Experiment 5).
3) Forcing solution:
After the field grown materials were disinfested, apical and basal ends of the
hardwood cuttings were given a fresh cut. Axillary bud outgrowth was then forced
by immersing the basal end of branches in forcing solution (Read &Yang, 1987)
containing 200 mg 8-hydroxyquinoline citrate per liter and 2 % sucrose per liter.
The basal end of each cutting was pruned off, and the solution replaced twice a week.
56
In the first year of research, the hazelnut plant materials collected in summer
and autumn were not kept in cooler since they were actively growing, and forcing
solution was not used. In the second year, the materials collected in summer and
autumn were treated with forcing solution supplemented with streptomycin at
concentrations of 0 mg/L, 20 mg/L and 40 mg/L to control the explant
contamination in vitro (Experiment 6).
After the bud break, tissue cultures were initiated by culturing explant in shell
vials containing 5ml medium. The cultures were maintained at 241C under cool
white fluorescent light for 16 hours per day. One week after the culture, the
contaminated explants were excluded and the contamination incidence was
calculated. 1 month after discarding contaminated cultures, the number of browning
explants was counted and browning incidence was calculated.
4) Medium:
For culture establishment (Experiment 7), the following culture media were
used:
1: MS: Murashige and Shoog salts and vitamins supplemented with noted PGR.
2: DKW: Driver and Kuniyuki salts and vitamins supplemented with noted PGR
3: WPM: Woody Plant Medium salts and vitamins supplemented with noted PGR
4: NRM: Nas and Read Medium use DKW basal salts, NRM vitamins, and 0.6mg/L
Sequestrene 138 Fe supplemented with noted PGR.
All of the above media were adjusted to a pH ranging from 5.5 to 5.8 and
autoclaved at 121 °C for half an hour. The basal salts were from Phyto Technology
Laboratories Co. (2013).

57
The following plant growth regulators were contained in the 4 media.
a) 2.5 mg BA/L + 0.05 mg IBA/L in MS, WPM, DKW or NRM
b) 2.0 mg BA/L + 0.01 mg IBA/L in MS, WPM, DKW or NRM
c) 2.0 mg BA/L+ 0.01 mg IBA/L in MS, WPM, DKW or NRM
d) 0.05 mg IBA/L in MS, WPM, DKW or NRM
After the culture, the number of new shoot was recorded every 15 days for 55
days, and after the explants had root initiation, they were transplanted into soil.
In the fist year, the antibiotics were not included in media, and in the second
year, antibiotics were included in MS medium at 0 mg streptomycin/L, 20 mg
streptomycin/L, 40 mg streptomycin/L and 20 mg streptomycin/L + 20 mg
kanamycin/L (Experiment 8). The contamination incidence was calculated 1 week
after culture, and the browning incidence was calculated 1 month after discarding
the contaminated cultures.
4) Experimental Design
One single shoot tip or nodal shoot of Corylus colurna ‘Te Terra Red’ was
cultured in each shell vial containing 5 ml of each medium. For the four types of
media, each medium had 5 shell vials to culture 5 explants (5 replicates). The
experimental unit was each shell vial, and there were 20 shell vials (4 media*5 shell
vials) at every combination and concentration of PGR, and they were conducted
three times (3 trials). For the experiments that tested the effects of culture on the
contamination and browning of explants, the experiments were considered to be
completely random design (CRD). For experiment 7, since the shoot number was
recorded every 10 days, to simplify the statistical analysis and to check whether
58
there was interaction between time and treatment, the experiment was considered
to be a Complete Random Design (CRD) with repeated measures. The Statistical
analysis of data was performed using SAS PROC GLIMMIX and MEANS procedures
(SAS institute, 2013).
Results and Discussion
During the first week after tissue culture, 521 explants out of 602 cultures were
lost because of the contamination of the external or internal pathogen or the tissue
browning. The contamination and the browning incidence of the explants 10 days
after culture were related to the disinfestation method.
In experiment 1, the branches were kept in a cooler for a different time period.
It was found that storing branches for 1 month had significant less contamination
incidence than that of 0 week or 2 weeks of storage, but not significant different
from that of 2 months (Figure 2-1,Table 2-1. Data that are shown by incidence in all
figures, and the statistical analysis was done by original data). The results indicated
that storing the branches in cooler for 1 month was effective in reducing the
contamination incidence.
In experiment 2, tap water was used to rinse the branches and it was found that
a longer time of tap water rinsing did not help decrease the contamination incidence
of the plant materials, but did increase the browning incidence (Figure 2-2, Table 2-
2). Thus, 20 min tap water rinsing was sufficient for the branches.
In experiment 3 and 4, the data showed that the longer the time the 10 % bleach
or 70% ethanol spray was used, the lower the contamination incidence (Figure 2-3
59
and 2-4, Table 2-3 and 2-4), though the browning incidence increased. Soaking in
bleach for 20 min was not significantly different from 0 min, while 30 min made
significantly reduce the contamination incidence. Ethanol spray for 90 s was
significantly better in controlling the contamination, and extending time to 5 min
was not effective for further controlling. Thus, soaking in bleach for 30 min and
spraying with 70% ethanol for 90s were better choice.
Using the axillary buds taken in winter to culture in the media directly had a
significantly lower contamination incidence in experiment 5 (Figure 2-5) The shoots
after bud break had hairy leaves and stems, which could carry more pathogens and
being difficult to disinfest (Figure 2-6 top. The oval shaped buds with relatively
smooth surface were easier to disinfest. Milky fluids were observed at the bottom of
the buds in culture, and all of the buds with this phenomenon did not break bud
(Figure 2-6 bottom).
Other reasons related to the contamination and browning were also studied.
There were two ‘Te Terra Red’ trees available to be used for tissue culture, and one
of them (tree B) was observed to have many leaves with wormholes, which indicate
that they might be more infected with internal pathogens (Figure 2-7, Table 2-5).
The contamination incidence of the explants from the less healthy tree was 87.3%
(352 out of 403 buds were contaminated), which was significantly higher than
contamination rate of 60.9% (241 out of 396) of the other tree A.
The medium contributed to the browning incidence as well (Figure 2-8, Table 2-
6). MS medium had the lowest browning rate (76.4%), which is significantly
different from DKW and NRM, but not significantly different from WPM. NRM
60
seemed to have the highest 93.2% browning rate, being significantly different from
MS and WPM, but not significantly different from DKW. This might be related to the
similarity of the formula between DKW and NRM, and the micronutrient differences
or the Sequestrene 138 Iron in the NRM.
After discarding the contaminated and brown explants, the surviving explants
(Figure 2-9) with new shoots were transferred to the media containing varied
concentrations of plant growth regulators. The four media also stimulated bud
break differently, and the shoot elongation in the media was not showed in MS or
WPM.
However, the growth of these buds was slow and very few of them had shoot
elongation, and if only IBA was added in the media, the shoots all died without any
root initiation. When the concentration of BA was 2.5 mg/L and IBA was 0.05 mg/L,
MS and WPM were able to stimulate as many as 5 leaves in vitro, while NRM had 12
leaves at most, being significantly higher than MS and WPM (Figure 2-10, Table 2-7).
The bottom leaves of explants (from buds) became dried out and all explants died in
the media supplemented with BA at 2.0 mg/L and IBA at 0.01 mg/L or with IBA at
0.05 mg/L. The death of the explants was usually observed after 2-3 month after the
tissue culture initiation, and this may be related to the depletion of plant growth
regulators. If the explants were transferred from the shell vials to GA-7 vessels, they
were able to survive longer in vitro (grow as long as 4 month).
‘Te Terra Red’ leaves are red after bud break in the field, however, in the
experiments conducted in lab, all the new shoots turned to green shortly after bud
break in vitro (Figure 2-9). The growth of ‘Te Terra Red’ is relatively slow and the
61
data were collected every 15 days. Even so, the elongation of the explants was
seldom observed 2 months after culture. If the branches were kept in cooler for 0
weeks or 2 weeks, the buds were not found to have any good multiplication or
elongation. The 1 months cold storage mimic the long winter in Nebraska and help
the buds to finish the dormant period, and perhaps meanwhile reduce the amount of
pathogens.
The acclimatization of the hazelnut explants was not successful (Figure 2-11).
After 4 month of culture, the explants of ‘Te Terra Red’ that have at least 3 roots
were transplanted into soil, and the plantlets died with fungus spores covered all
over the stems and leaves. This might be due to the unsterilized soil that had
pathogen and the plantlets were not strong enough to survive in the soil. Similarly,
the explants of ‘Yam Hill’ could not be transplanted into soil successfully. However,
if the plantlets were transferred to bigger GA-7 vessels before transplant to soil, it
helped to increase the possibility of survival. The ‘Yam Hill’ plantlets subcultured on
MS and NRM produced an average of 4 roots and were transferred into soil
successfully.
In the second year, the antibiotics were used in the media in an attempt to
further control contamination (Table 2-8). If the branches were treated with the
forcing solution supplemented with 20 mg streptomycin/L (Figure 2-12), the
contamination incidence dropped compared to those that were not supplemented
with streptomycin. 40 mg streptomycin/L was not more effective than 20 mg
streptomycin /L in reducing the contamination rate (P value=0.5706).

62
When the media included the antibiotics, the contamination incidence also
declined (Figure 2-12, Table 2-9). 20 mg streptomycin/L was effective in reducing
the contamination incidence significantly, but was not significantly from 40 mg/L.
20 mg Streptomycin/L+20 mg Kanamycin/L had a similar effect of reducing
contamination, while making no difference from 20 mg Streptomycin/L alone.
However, it should be noticed that different types of antibiotics included in media
can slow down the selection of resistant strains.
Conclusion
In order to have successful tissue culture of hazelnut, taking the branches in
winter and storing in cooler for 1 month is recommended. In order to reduce the
contamination rate, rinsing with tap water for 20 min, followed by soaking in 10%
bleach for 30 min and spraying with 70% ethanol for 90s should be effective for
surface disinfestation. If the plant material is harvested in summer, then the
branches can be treated with forcing solution that includes 20 mg streptomycin/L,
and the single nodes or shoots can be cultured onto the media. Another suggestion
is to use axillary buds, rather than softwood cuttings which can be cultured directly
into shell vials containing NRM medium supplemented with 20 mg streptomycin/L
+ 20 mg kanamycin/L, and the plant growth regulators BA at 2.5 mg/L and IBA at
0.05 mg/L. After the bud breaks and 3-4 leaves are forced, whole explants could be
transferred into bigger containers such as GA-7 vessels. The explants will probably
need to be subcultured for several times until at least 3-4 roots are formed before
transplanting into soil.

63
Figure 2-1: Effects of cooler storing time of branches on the contamination and
browning incidence for Corylus colurna ‘Te Terra Red’
100% 89.20%
87.90% 87.30%
92.30% 80.10%
80% 88.70%
Borwning incidence
Contamination and
76.50% 77.30%
60%
(Trial 1)
contamination
40% browning
20%
0%
0 week 2 weeks 1 month 2 month
100% 88.90%
86.40% 88.20%
90% 79.10%
80% 91.50%
browning incidence
Contamination and
85.40%
70% 74.80% 78.20%
60%
(Trial 2)
50% contamination
40% browning
30%
20%
10%
0%
100%
86.50% 88.90% 88.20%
93.00% 79.60%
80%
browning incidence
86.50%
Contamination and
75.90% 77.10%
60%
(Trial 3)
contamination
40% browning
20%
0%
*The contamination incidence was calculated as the number of contaminated

explants/ number of explants cultured X 100%. After discarding the contaminated
explants, browning incidence = number of browning explants/ number of explants
that were not contaminated X 100%
64
Figure 2-2: Effects of tap water rinsing time of branches on the contamination
and browning incidence for Corylus colurna ‘Te Terra Red’
100% 94.50%
81.20% 82.40% 82.50%
80% 88.30%
browning incidence
86.50%
Contamination and
85.90%
78.30%
60%
(Trial 1)
contamination
40% browning
20%
0%
0 min 20 min 40 min 60 min
100% 93.70%
82.40% 83.10% 81.40%
browning incidence
Contamination and
80% 87.90% 87.20% 86.40%

79.10%
(Trial 2)
60%
contamination
40% browning
20%
0%
100% 91.50%
82.80% 84.10% 83.10%
80% 88.30%
browning incidence
Contamination and
86.20% 84.40%
76.50%
60%
(Trial 3)
contamination
40% browning
20%
0%

65
Figure 2-3: Effects of 10% bleach soaking time of branches on the

100% 98.80%
91.30%
86.40%
78.30%
80% 89.90%
browning incidence
Contamination and
82.50% 84.10% 85.20%
60%
(Tiral 1)
contamination
40% browning
20%
0%
100% 97.60%
90.30%
86.10%
90.20% 77.80%
80%
browning incidence
Contamination and
81.40% 83.90% 84.30%
60%
(Trial 2)
contamination
40% browning
20%
0%
100% 96.90%
90.20% 87.30%
78.10%
80% 88.30%
browning incidence
85.10%
Contamination and
81.70% 84.90%
60%
(Trial 3)
contamination
40% browning
20%
0%

66
Figure 2-4: Effects of 70% ethanol spray time of branches on the

100% 92.50% 89.70%

96.30% 81.20% 81.10%
80% 89.90%
browning incidence
Contamination and
79.50% 81.40%
60%
(Trial 1)
contamination
40% browning
20%
0%
0s 30s 90s 5min
100% 93.30% 90.80%

87.20%
96.70% 80.90%
80% 90.10%
browning incidence
Contamination and
80.00% 80.20%
60%
(Trial 2)
contamination
40% browning
20%
0%
0s 30s 90s 5min
100% 94.50%
88.60% 86.50%
95.90% 83.20%
80% 88.60%
browning incidence
Contamination and
85.10%
79.20%
60%
(Trial 3)
contamination
40% browning
20%
0%
0s 30s 90s 5min

67
Figure 2-5: Effects of explant materials (softwood cutting/buds) on the

100% 93.50% 90.30%

84.40%
80% 72.40%
browning incidence
Contamination and
60%
(Trial 1)
contamination
40% browning
20%
0%
softwood buds
100% 94.10% 91.20%

85.50%
80% 73.20%
browning incidence
Contamination and
(Trial 2)
60%
contamination
40% browning
20%
0%
softwood buds
100% 95.00% 92.10%

84.90%
80% 73.00%
browning incidence
Contamination and
(Trial 3)
60%
contamination
40% browning
20%
0%
softwood buds

68
Figure 2-6: Contamination and browning of Corylus colurna ‘Te Terra Red’
buds cultured in vitro
*Top: 20 days after bud break in vitro, the Corylus colurna explants being
contaminated by internal pathogens.
*Bottom: Tissue browning found 15 days after axillary buds were cultured in vitro
69
Figure 2-7: Corylus colurna 'Te Terra Red' explant contamination and
browning incidence of two mother plants
100%
90% 87.30%
80% 77.30%
75.00%
Contamination and Browning incidence
70%
60.90%
60%
50% contamination
browning
40%
30%
20%
10%
0%
A tree B tree
*The two mother plants were from the horticulture garden at University of
Nebraska-Lincoln. The branches from the mother plant were collected all the year
round. A tree was the taller tree with relatively healthy leaves in summer, while tree
B was observed to have most leaves with wormholes in summer.
* Error bars stand for the standard error.
70
Figure 2-8: Browning rate of Corylus colurna 'Te Terra Red' explants cultured
on 4 media containing various PGR
100.0%
93.20%
86.30%
80.0% 78.10%
76.40%
Browining incidence
60.0%
40.0%
20.0%
0.0%
MS WPM DKW NRM
*MS stands for Murashige and Skoog Medium (1962); WPM stands for Woody Plant
Medium (Lloyd and McCown, 1981); DKW stands for Driver and Kuniyuki Walnut
Medium (1984); NRM stands for Nas and Read Medium (2004);
*PGR stands for plant growth regulators
71
Figure 2-9: Corylus colurna ‘Te Terra Red’ explants (axillary buds) cultured in
vitro that were able to grow shoots successfully
*The explants in the third shell vial in top picture showed that right after bud break
the leaf color was red, while 10 days after bud break most of the leaves turned green.
72
Figure 2-10: Corylus colurna 'Te Terra Red' explants cultured on 4 media
containing 2.5mg BA/L+0.05mg IBA/L
6
NRM, 5.8
5
DKW, 3.4
3 WPM, 2.6
MS, 2.6
0
Feb-14th Feb-28th Mar-14th Mar-28th

Error bars stand for the standard error.
73
Figure 2-11: Corylus colurna ‘Te Terra Red’ explants that were able to initiate
roots but failed to grow in soil.
*The top picture showed that there were two root initiations, though the leaves
were tiny. The middle and bottom picture showed that after transplanting into soil,
the hazelnut explants were not able to grow but died
74
Figure 2-12: Effects of forcing solution supplemented with streptomycin on

the contamination and browning incidence for Corylus colurna ‘Te Terra Red’
100% 94.40% 91.10%

90.10%
81.90%
78.20% 75.90%
80%
browning incidence
Contamination and
60%
(Trial 1)
contamination
40% browning
20%
0%
0 mg/L 20 mg/L 40 mg/L
100% 89.90% 90.30%

82.20% 85.60%
77.50% 76.80%
80%
browning incidence
Contamination and
60%
(Trial 2)
contamination
40% browning
20%
0%
100% 91.20%
88.50% 86.30%
83.20%
80% 76.10% 75.00%
browning incidence
Contamination and
60%
(Trial 3)
contamination
40% browning
20%
0%

75
Figure 2-13: Effects of media supplemented with streptomycin on the

100% 89.60% 87.60%

85.90% 82.30% 82.60%
browning incidence 76.90% 75.70% 76.20%
Contamination and
80%
(Trial 1)
60%
40% contamination
browning
20%
0%
0 mg 20 mg 40 mg 20 mg
stre/L stre/L stre/L stre/L+20
mg kana/L
100% 90.00% 88.40%

84.70% 82.90% 83.40%
77.30%
browning incidence
76.30%
Contamination and
80% 72.40%
(Trial 2)
60%
40% contamination
browning
20%
0%
0 mg 20 mg 40 mg 20 mg
mg kana/L
100% 87.90%
77.50%
browning incidence
74.90% 75.20%
Contamination and
80% 86.50% 86.10%

81.00% 82.30%
(Tirial 3)
60%
40% contamination
browning
20%
0%
0 mg 20 mg 40 mg 20 mg
mg kana/L
* Stre=Streptomycin, Kana=Kanamycin. *The contamination incidence was

calculated as the number of contaminated explants/ number of explants cultured X
100%. After discarding the contaminated explants, browning incidence = number of
browning explants/ number of explants that were not contaminated X 100%
76
Table 2-1: P value comparisons of cooler storing time effect on contamination

and browning incidence for Corylus colurna ‘Te Terra Red’ (=0.05)
Comparisons P value
0 week and 2 weeks 0.0845
0 week and 1 month 0.0487
0 week and 2 months 0.0145
2 weeks and 1 month 0.0425
2 weeks and 2 months 0.0233
1 month and 2 months 0.0927
Table 2-2: P value comparisons of tap water rinsing time effect on the
(=0.05)
Comparisons P value
0 and 20 min 0.0136
0 and 40 min 0.0085
0 and 60 min 0.0097
20 and 40 min 0.0583
20 and 60 min 0.0621
40 and 60 min 0.0701
Table 2-3: P value comparisons of 10% bleach soaking time effect on the
(=0.05)
Comparisons P value
0 and 20 min 0.1753
0 and 30 min 0.0489
0 and 40 min 0.0082
20 and 30 min 0.0522
20 and 40 min 0.0421
30 and 40 min 0.0691
Table 2-4: P value comparisons of 70% ethanol spray time effect on the
(=0.05)
Comparison P value
0 and 30s 0.0572
0 and 90s 0.0329
0 and 5 min 0.0289
30s and 90s 0.0487
30s and 5 min 0.0214
90s and 5 min 0.0627
77
Table 2-5: P value comparisons mother plants effect on contamination and

browning incidence of Corylus colurna ‘Te Terra Red’ (=0.05)
P value
Contamination <0.0001
Browning 0.6443
Table 2-6: P value of comparisons of media effect on browning incidence of
Corylus colurna ‘Te Terra Red’ (=0.05)
Comparison p value
MS and WPM 0.6703
MS and DKW 0.0259
MS and NRM 0.0010
WPM and DKW 0.0570
WPM and NRM 0.0022
DKW and NRM 0.1019
Table 2-7: P value comparisons of media (2.5 mg BA/L + 0.05 mg IBA/L) effect
on number of shoots of Corylus colurna ‘Te Terra Red’ (=0.05)
Comparison P value
MS and WPM 1.0000
MS and DKW 0.5619
MS and NRM 0.0408
WPM and DKW 0.5619
WPM and NRM 0.0408
DKW and NRM 0.1383
Table 2-8: P value of comparisons of concentration of streptomycin included
in forcing solution effect on the contamination incidence (=0.05)
Comparisons p value
0 mg/L and 20 mg/L 0.0079
0 mg/L and 40 mg/L 0.0008
20 mg/L and 40 mg/L 0.3045
Table 2-9: P value of comparisons of concentration of streptomycin included
in media effect on the contamination incidence (=0.05)
Comparisons (Stre=Streptomycin,
Kana=Kanamycin) p value
0 mg stre/L and 20 mg stre/L 0.0254
0 mg stre/L and 20 mg stre/L + 20 mg kana/L 0.0074
78
Literature Cited for Chapter 2
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81
Appendix
The composition of NRM in comparison with MS, DKW, WPM and NN media
NRM MS DKW WPM NN
Macrominerals (mg l−1)
NH4NO3 530 1650 1416 400 720
Ca(NO3)2·4H2O 700 – 1960 556 –
CaCl2·2H2O 90 440 147 96 202
MgSO4·7H2O 1600 370 740 370 185
KNO3 550 1900 – – 950
KH2PO4 1300 170 259 170 68
K2SO4 – – 1560 990 –
Microminerals (mg l−1)
H3BO3 6.2 6.2 4.8 6.2 10
CuSO4·5H2O 2.5 0.025 0.25 0.25 0.025
MnSO4·H2O 20 16.9 33.5 22.3 18.9
Na2MoO4·2H2Oa 2.5 0.25 0.39 0.25 0.25
ZnSO4·7H2O 8.8 8.6 – 8.6 10
Zn(NO3)2·6H2O – – 17 – –
Sequestrene 138 Fe 100 – – – –
FeSO4·7H2O – 27.8 33.4 27.8 2.78
Na2·EDTA – 37.3 44.7 37.3 37.3
KI – 0.83 – – –
CoCl2·6H2O – 0.025 – – –
Vitamins (mg l−1)
Thiamine (B1) 0.60 0.1 2.0 1.0 0.5
Riboflavin (B2) 0.21 – – – –
Nicotinic acid (B3) 1.15 0.5 2.0 0.5 5.0
Pyrodoxine (B6) 0.60 0.5 – 0.5 0.5
α-Tocopherol (E) 20.0 – – – –
Vitamin C (ascorbic acid) 1.0 – – – –
Glycine 0.85 2.0 2.0 2.0 2.0
Folic acid – – – – 0.5
Biotin – – – – 0.05
Myo-inositol 200 100 1000 100 100
Sucrose (g l−1) 30 30 30 20 20
Agar (g l−1) 5–6 10 – 6 8
Gelrite (g l−1) – – 2 – –
＊Table from M.N. Nas and P.E. Read, 2004. A hypothesis for the development of a defined
tissue culture medium of a higher plants and micropropagation of hazelnuts. Scientia
Horticulturae.

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EFFECTS OF CULTURE MEDIA AND PLANT

Follow this and additional works at: http://digitalcommons.unl.edu/agronhortdiss

REGULATORS ON MICROPROPAGATION OF WILLOW (Salix

matsudana ‘Golden Spiral’) AND HAZELNUT (Corylus colurna

‘Te Terra Red)

Presented to the Faculty of

The Graduate College at the University of Nebraska

In partial Fulfillment of Requirements

For the Degree of Master of Science

Under the supervision of Professor Paul E. Read

REGULATORS ON MICROPROPAGATION OF WILLOW (Salix

matsudana) AND HAZELNUT (Corylus colurna)

University of Nebraska, 2014

Advisor: Paul E. Read

contamination rate and grew slowly on the various media. By improving

contamination rate and browning rate can be decreased greatly.

THIS THESIS IS DEDICATED TO MY PARENTS, GUANGZHI SHI AND LIJING CHENG

My sincere gratitude goes to my adviser, Dr. Paul Read, who provided me an

I can ever asked for.

My great appreciation is also extended to my committee members Dr. Ellen

extending my study experience.

words can say.

Under the common name of ‘Peking willow’, Salix matsudana is native to

Virginia (Emerine, 2012).

Salix matsudana is a medium-sized dioecious tree that can reach 20 to 40 feet in

but concealed flowers on female trees (Seiler et al, 2010).

Corylus colurna is commonly called Turkish hazelnut, and is a member of the

genus Corylus, tribe Coryleae, family Betulaceae. (Cronquist, 1981). It is a deciduous

the Balkans (Kuhns, 1983).

In addition to the commercial hazel, some cultivars are also grown as

ornamental plants by horticulturists for their contorted stems and pendulous

characteristic makes it ideal as rootstock for grafting other desirable cultivars of

commercial hazelnuts such as C. avellana (Mehlenbacher, 1994).

drought after establishment. It is also occasionally reported to be tolerant to

maintains a symmetrical crown that is attractive to landscape architects (Gilman

islands and highway rights-of-way.

Cuttings and seeds are the common methods of propagation, however,

micropropagation of Salix matsudana or Corylus colurna could provide more

plantlets for rapid commercial scale-up in the nursery industry. It is well-known

When using the process of micropropagation, there are several aspects to be

considered before conducting the research. Choosing of explants, the initiation of

and the problems during culture, such as contamination, browning, and

proliferation difficulty, may have great effect on the results.

Cloning in vitro of adult or mature woody plants is affected by characteristics

ability. Maturation is an obstacle for a wider application of tissue culture technology

or by special pre-treatment in vivo and/or in vitro culture. These methods to

conducted by Agrawal and Gebhardt (1994) for rapid mcropropagation. They

proliferation was enhanced to 5-fold after transfer. SH medium without plant

explant (Salix humboldtiana) used. MS medium supplemented with 2.3 M kinetin

enhanced shoot production while half-strength MS medium supplemented with 5.3

M NAA enhanced rooting.

The tissue culture of hazelnut might be accomplished by using the nodal

embryo culture was commonly used in micropropagation of hazelnut. In the study

competence of explants, suggesting the endogenous hormone balance is very

important in defining in vitro potential of hazelnut cotyledons.

According to a research conducted by Garton et al. (1983), organogenesis in

with 3 nutrient solutions where nitrogen, potassium and phosphorus were in

actively grown shoots were cultured in vitro on medium to promote shoot

that received lowest concentration.

micropropagtion process. The carbohydrates, proteins, and growth substances in

March produce a greater number of successful initiations.