Tree Physiology 26, 969–975
© 2006 Heron Publishing—Victoria, Canada
In vitro regeneration of Salix nigra from adventitious shoots
SATU LYYRA,1,2 AMPARO LIMA3 and SCOTT A. MERKLE1
1
Daniel B. Warnell School of Forest Resources, University of Georgia, Athens, GA 30602, USA
2
Corresponding author ([email protected])
3
Current address: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720–3102, USA
Received June 9, 2005; accepted September 2, 2005; published online April 3, 2006
Summary Black willow (Salix nigra Marsh.) is the largest
and only commercially important willow species in North
America. It is a candidate for phytoremediation of polluted
soils because it is fast-growing and thrives on floodplains
throughout eastern USA. Our objective was to develop a protocol for the in vitro regeneration of black willow plants that
could serve as target material for gene transformation. Unexpanded inflorescence explants were excised from dormant
buds collected from three source trees and cultured on woody
plant medium (WPM) supplemented with one of: (1) 0.1 mg
l – 1 thidiazuron (TDZ); (2) 0.5 mg l – 1 6-benzoaminopurine
(BAP); or (3) 1 mg l – 1 BAP. All plant growth regulator (PGR)
treatments induced direct adventitious bud formation from the
genotypes. The percentage of explants producing buds ranged
from 20 to 92%, depending on genotype and treatment. Although most of the TDZ-treated inflorescences produced buds,
these buds failed to elongate into shoots. Buds on explants
treated with BAP elongated into shoots that were easily rooted
in vitro and further established in potting mix in high humidity.
The PGR treatments significantly affected shoot regeneration
frequency (P < 0.01). The highest shoot regeneration frequency (36%) was achieved with Genotype 3 cultured on
0.5 mg l – 1 BAP. Mean number of shoots per explant varied
from one to five. The ability of black willow inflorescences to
produce adventitious shoots makes them potential targets for
Agrobacterium-mediated transformation with heavy-metal-resistant genes for phytoremediation.
Keywords: black willow, phytoremediation, thidiazuron (TDZ),
tissue culture.
Introduction
Black willow (Salix nigra Marsh.) is the largest and only commercially important willow species in North America. The
species is most common along rivers, but is also found in other
habitats where light and soil water availability are favorable
(Burns and Honkala 1990). Black willow does not tolerate
shade (Dionigi et al. 1985), but it is tolerant to flooding
(Hosner 1958, Dionigi et al. 1985) and is commonly found on
moderately acidic (lower pH limit is 4.5) to near neutral soils
(Dionigi et al. 1985).
Willow species are widely used for phytoremediation of
polluted soils (e.g., Perttu and Kowalik 1997, Vervaeke et al.
2003) because of their wide-spreading root systems and fast
growth. Willows can also survive and grow in hydroponic systems in the presence of heavy metals at above normal critical
concentrations (Punshon and Dickinson 1997). Black willow
has been found to accumulate both heavy metals (Punshon et
al. 2003a, 2003b) and organic contaminants (Nzengung et al.
1999). Because black willow is fast-growing and thrives on
floodplains throughout the eastern USA, it is a good candidate
for phytoremediation of polluted soils.
In vitro propagation from adventitious or axillary buds is a
useful technique for producing clonal plantlets. There are several reports of axillary shoot multiplication in willow species
(Bhojwani 1980, Chalupa 1983, Bergman et al. 1985, Neuner
and Beiderback 1993, Agrawal and Gebhardt 1994, AmoMarco and Lledo 1996). However, because the shoots arise
from preformed buds, this technique is unsuitable for gene
transformation. There are only two published reports on in vitro regeneration of Salix species from adventitious buds or somatic embryos (Grönroos et al. 1989, Stoehr et al. 1989), and
only Stoehr et al. (1989) were able to regenerate significant
numbers of plantlets. Stoehr et al. (1989) initiated callus cultures from leaf explants of mature Salix exigua (Nutt.) trees
cultured on medium with 6-benzoaminopurine (BAP) and
2,4-dichlorophenoxyacetic acid (2,4-D) and then induced adventitious shoot production from the callus by subculturing it
on medium with BAP. Grönroos et al. (1989) induced somatic
embryos from callus derived from pistils of Salix viminalis
(L.), cultured first on medium with 2,4-D and BAP and then on
regeneration medium with different combinations of plant
growth regulators (PGRs); however, the somatic embryos only
rarely developed into plants.
Because of the lack of studies on in vitro regeneration of
black willow, our objective was to regenerate black willow
trees by dissecting inflorescences from dormant buds and culturing them on woody plant medium (WPM) with three PGR
treatments (Lloyd and McCown 1980). The long-term goal
was to develop a protocol for the in vitro regeneration of black
willow plants that could serve as target material for gene transformation.
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LYYRA, LIMA AND MERKLE
Materials and methods
Plant material
Twigs (10–20 cm long) with dormant buds were collected
from the uppermost branches of three mature black willow
trees growing in Athens, GA, in early February 2004. To the
best of our knowledge, these trees were wild genotypes native
to the Athens area and thus, their potential value as clones is
unknown. The twigs were placed in plastic bags with wet paper towels and stored at 4 °C until used. In the laboratory, dormant buds were excised from the twigs and surface sterilized
in a laminar flow cabinet according to the following procedure: 70% ethanol (4 min), 20% Roccal II 1096 (National Laboratories, L&F Products, active ingredient 10% alkyl dimethyl
benzyl ammonium chloride; 5 min), 20% Clorox with five
drops of Tween 20/100 ml (15 min), sterile water (3 min),
0.01 M HCl (3 min) followed by three rinses of sterile water
(3 min each) and 0.5% (w/v) Captan 50 (Micro Flo, Lakeland,
FL; 5 min). The Captan treatment was followed by three additional sterile water rinses (3 min each). Following surface sterilization, inflorescences were excised from the dormant buds
with the aid of a dissecting microscope (see Figure 1A).
Culture initiation
Preliminary studies showed that 0.01 mg l – 1 thidiazuron
(TDZ) in WPM induced bud initiation and shoot production
(Merkle and Lima, unpublished results). Based on these studies and our experience with other woody species, we tested the
effects of the plant growth regulators TDZ and BAP on bud
initiation. After dissection, inflorescences were cultured in
plastic petri plates (60 × 15 mm) containing semi-solid WPM
with MS iron (Murashige and Skoog 1962) and vitamins of
Gresshoff and Doy (1972), supplemented with one of: (1)
0.1 mg l – 1 TDZ; (2) 0.5 mg l – 1 BAP; or (3) 1 mg l – 1 BAP. Media also contained 20 g l – 1 sucrose and were gelled with 3 g l – 1
Phytagel (Sigma). The pH of the medium was adjusted to 5.7
with 0.1 M NaOH or 0.1 M HCl before autoclaving at 121 °C
for 25 min. All PGRs were added to the medium before
autoclaving. Fifteen inflorescences were cultured per genotype and per treatment. Cultures were maintained in the dark at
25 °C and transferred to fresh medium monthly. The occurrence and type of callus and any adventitious bud formation
were recorded monthly.
Shoot elongation
Four and a half months following culture initiation, all surviving explants, whether or not they had produced adventitious
buds, were transferred to one of two shoot elongation media.
Treatment 1: inflorescences (33) with mainly multiple adventitious buds or showing some bud elongation, or both, were
transferred to GA-7 vessels (Magenta Corp.) containing
100 ml of semi-solid WPM without PGRs and incubated under
Figure 1. (A) Black willow dormant bud and dissected inflorescence. (B–F) Black willow inflorescences cultured on woody plant medium
(WPM) with (B) 1 mg l – 1 6-benzoaminopurine (BAP) (Genotype 3) 1 month following initiation; (C) 1 mg l – 1 BAP (Genotype 2) 2 months following initiation; (D) 0.1 mg l – 1 thidiazuron (TDZ) (Genotype 3) 2 months following initiation; (E) 0.1 mg l – 1 TDZ (Genotype 3) 3 months following initiation; and (F) 0.5 mg l – 1 BAP (Genotype 3) 4.5 months following initiation. Bars = 0.5 mm.
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IN VITRO REGENERATION OF SALIX NIGRA
cool white fluorescent lights (40 µmol m – 2 s – 1) in a 16-h
photoperiod at 25 °C. Treatment 2: the remaining surviving inflorescences (65), some of which had produced at least one adventitious bud, were transferred to 50-ml flasks containing
10 ml of liquid WPM and one-tenth the concentration of PGRs
of the original callus initiation medium (0.01 mg l – 1 TDZ,
0.05 mg l – 1 BAP or 0.1 mg l – 1 BAP). The flasks were incubated in the dark on a gyratory shaker (75 rpm) at 25 °C. Of the
original 135 inflorescence explants, 37 were discarded because of contamination or necrosis, leaving 98 explants. The
surviving explants available for transfer to the elongation
treatments from each treatment and genotype were:
10–15 explants per genotype from the 0.1 mg l – 1 TDZ treatment; 8–11 explants per genotype from the 0.5 mg l – 1 BAP
treatment; and 10–11 explants per genotype from 1 mg l – 1
BAP treatment.
Rooting, plantlet production and axillary shoot
multiplication
After 6 weeks in shoot elongation media, adventitious bud and
shoot formation were recorded and the percentages were calculated based on the initial number of explants. The explants
in Treatment 1 were transferred to fresh medium for further
elongation. When shoots reached 1 cm, they were excised
from the clusters and individually transferred to fresh medium
of the same composition in GA-7 vessels for rooting. Similarly, elongated shoots from explants in Treatment 2 were
transferred to semi-solid basal WPM in GA-7 vessels for rooting. Because the adventitious buds on explants treated originally with TDZ did not elongate, these buds were transferred
to fresh liquid medium with either 0.01 or 0.001 mg l – 1 TDZ
(dark, 75 rpm) or no TDZ (light, 75 rpm) for another 4 weeks.
We also transferred some bud clusters from TDZ-treated explants to GA-7 vessels containing either semi-solid basal
WPM with 5 g l – 1 activated charcoal or semi-solid basal DKW
(Driver and Kuniyuki 1984) with 1 mg l –1 zeatin in an attempt
to induce these buds to elongate.
Once rooted, some plantlets were transferred to peat-based
potting mix (Fafard No. 3) in a humidifying chamber set to
provide a 16-h photoperiod at 90 µmol m – 2 s – 1 of photosynthetically active radiation and 26 °C. Plantlets, which were
watered with tap water and Hoagland’s solution (Hoagland
and Arnon 1950) once a week, were gradually acclimatized to
lower humidity and after two to three months were transferred
to the greenhouse. Instead of being transferred to ex vitro conditions, some plantlets were used for axillary shoot multiplication by dividing them into approximately 1-cm-long nodal
segments and culturing them on semi-solid DKW medium
supplemented with 1 mg l – 1 zeatin in GA-7 vessels with 16-h
photoperiod at 25 °C. New axillary shoots were transferred to
basal DKW medium in GA-7 vessels for rooting.
Statistical analysis
Effects of genotype and culture initiation treatment and their
interactions on bud and shoot formation as well as the effects
of shoot elongation treatments on shoot formation were subjected to two-way analysis of variance (ANOVA) (GLM pro-
971
cedure; SAS Institute Inc. 1997). Interactions between the culture initiation and shoot elongation treatments were also subjected to two-way ANOVA. Mean comparisons of the effects
of genotype or treatments on bud and shoot formation were
made with Duncan’s multiple range test (SAS Institute Inc.
1997).
Results and discussion
Callus proliferation was observed within 3 weeks following
culture initiation, developing first at the base of the inflorescences (Figure 1B). Initially, white, spongy callus appeared,
but it later turned to hard yellow or watery brown callus. None
of these calli were morphogenic. In all genotypes, all of the
PGR treatments induced adventitious bud formation. Pale yellow buds appeared directly on the inflorescences (Figure 1D),
most of which had darkened and appeared to be otherwise
dead (Figures 1C and 1E). Similarly, Merkle et al. (1998) reported that embryos of sweetgum (Liquidambar styraciflua
L.) arose directly from the tissues of staminate inflorescences
that had turned black and appeared to be dead. However, the
direct production of adventitious buds from explants in our
study differs from studies with other willow species where adventitious buds (Stoehr et al. 1989) and somatic embryos
(Grönroos et al. 1989) were derived from the explants via an
intervening callus.
Plant growth regulator treatment and genotype significantly
affected the frequency of adventitious bud formation with the
highest frequency (92%) induced by TDZ treatment with Genotype 3 (P < 0.05; Figure 2A). The interaction between treatment and genotype was not significant (P = 0.6). Both treatment and genotype significantly affected the number of buds
produced per explant (P < 0.01), with the highest number of
buds per explant induced by the TDZ treatment (Figure 3). For
this variable, the interaction between treatment and genotype
was not significant (P = 0.2).
At 4.5 months following initiation, some BAP-treated adventitious buds began to elongate in the dark (Figure 1F), and
after 6 weeks in the shoot elongation treatments, buds on
explants treated initially with either of the BAP concentrations
elongated into shoots (Figures 2B and 4B). Percentages of
explants producing shoots ranged from 7 to 36% (Figure 2B).
Culture initiation treatment significantly affected this variable
(P < 0.01), with 0.5 mg l – 1 BAP resulting in the highest mean
shoot regeneration frequency (24%). Although the effect of
genotype was not significant (P = 0.2), the interaction between treatment and genotype was significant (P < 0.05), with
highest shoot regeneration frequency (36%) observed in the
0.5 mg l – 1 BAP and Genotype 3 combination. The number of
shoots per explant varied from 1 to 5 and was significantly affected by treatment and by the interaction between treatment
and genotype (P < 0.01; Figure 3), but not by genotype. Similarly, Stoehr et al. (1989) reported that BAP induced adventitious shoot regeneration from S. exigua leaf explants and
several studies have shown that BAP affects axillary shoot
multiplication in other willow species (Bhojwani 1980, Cha-
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LYYRA, LIMA AND MERKLE
Figure 3. Mean numbers of buds and shoots formed per explant from
all tested genotypes (SN 1–SN 3 combined) grown initially on woody
plant medium with thidiazuron (TDZ; 0.1 mg l – 1), 6-benzoaminopurine (BAP I; 0.5 mg l – 1) or BAP II (1 mg l – 1 BAP). Bud numbers
were recorded following culture initiation, and shoot numbers were
recorded following 6 weeks of culture in shoot elongation media (n =
40–43). Statistical significance (P < 0.01) is indicated with different
letters. Bars represent standard error.
Figure 2. Adventitious bud (A) and shoot (B) formation frequencies
(percentage of explants forming buds or shoots) of three black willow
genotypes (SN 1–SN 3) treated with thidiazuron (TDZ; 0.1 mg l – 1),
6-benzoaminopurine (BAP I; 0.5 mg l – 1) or BAP II (1 mg l – 1 BAP).
The statistical analysis of these data is presented in the text.
lupa 1983, Bergman et al. 1985, Neuner and Beiderback 1993,
Agrawal and Gebhardt 1994, Amo-Marco and Lledo 1996).
Although 47–92% of the inflorescences treated with 0.1 mg
l – 1 TDZ produced buds (Figure 2A), these buds failed to elongate into shoots (Figure 2B). We obtained some evidence that
0.1 mg l – 1 TDZ inhibited shoot elongation of the buds. None
of the additional treatments tested (liquid WPM medium with
0.01 mg l – 1 TDZ, 0.001 mg l – 1 TDZ or no TDZ, semi-solid
WPM medium with 5 mg l – 1 charcoal or semi-solid DKW medium with 1 mg l – 1 zeatin) induced shoot elongation (Figure 4A). In previous work in our laboratory, 0.01 mg l – 1 TDZ
resulted in some shoot regeneration from S. nigra (Merkle and
Lima, unpublished data). Both 0.1 and 0.01 mg l – 1 TDZ induced embryogenesis from sweetgum inflorescence explants
(Merkle et al. 1998). Thidiazuron has been shown to induce
callus formation of various species and in some cases the cell
proliferation rate was higher with TDZ than with other growth
regulators (Murthy et al. 1998). However, others have reported
that TDZ-induced buds fail to elongate (Meyer and van Staden
1988), as we found, and that TDZ-induced shoots do not convert into complete plantlets (Huetteman and Preece 1993, Lu
1993). These failures might be a result of a non-optimal concentration of TDZ in the media or its prolonged persistence in
cultured tissues (Murthy et al. 1998). It has been recom-
mended that the lowest effective TDZ concentration should be
used and that explants should be kept on TDZ-supplemented
medium for the shortest time required for each species. Lu
(1993) recommended that TDZ exposure of tissue should be a
maximum of 8 weeks. We did not test a culture initiation treatment without PGRs. Given the strong adventitious shooting
response to the lowest cytokinin treatment tested, it is possible
that inflorescence explants could produce adventitious shoots
even in the absence of PGRs.
Shoot regeneration frequency and number of shoots per
explant did not differ significantly between the two shoot elongation treatments (P = 0.5). The interaction between the culture initiation and shoot elongation treatments was not significant for either shoot regeneration frequency (P = 0.9) or
number of shoots elongating per explant (P = 0.3). However,
culturing the buds in liquid medium (WPM with one tenth the
concentration of PGRs used in the semi-solid callus initiation medium) for 6 weeks resulted in faster shoot elongation
than on semi-solid basal WPM in GA-7 vessels. Some of the
elongated shoots also produced roots in liquid medium (Figure 4B). Although culture on semi-solid medium induced
shoot elongation, the elongated shoots required transfer to
fresh medium before they rooted. In liquid culture, the number
of adventitious shoots regenerated from one explant ranged
from 1 to 5, whereas only 1–2 shoots per explant regenerated
on semi-solid medium. Shoots from both liquid and semisolid cultures were easily rooted in basal WPM medium (Figure 4C) and continued growth following transfer to potting
mix in the humidifying chamber. The survival percentage in
the humidifying chamber was 94%. Following hardening off,
all plants transferred to the greenhouse survived (Figure 4D).
Axillary shoot multiplication from stem segments harvested
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IN VITRO REGENERATION OF SALIX NIGRA
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Figure 4. (A) Regenerated
buds after shaking black willow inflorescence in flask for
6 weeks in shoot elongation
medium (woody plant medium (WPM) containing
0.01 mg l – 1 thidiazuron
(TDZ)) in the dark. Bar =
1 mm. (B) Regenerated shoots
with roots after shaking black
willow inflorescence in flask
for 6 weeks in shoot elongation medium (WPM medium
containing 0.05 mg l – 1 6-benzoaminopurine (BAP)) in the
dark. (C) Regenerated black
willow shoots in WPM medium 6 months following initiation. (D) Regenerated black
willow plantlets in the greenhouse 11 months following
initiation. (E) Black willow
axillary shoot multiplication
on Driver and Kuniyuki medium (DKW) with 1 mg l – 1
zeatin.
from the adventitious shoots and cultured on DKW medium
with 1 mg l – 1 zeatin was also successful (Figure 4E) and regenerated shoots rooted easily in basal DKW medium.
Our observation of vegetative bud development on inflorescences corroborates previous reports that cultured floral buds
can revert to a vegetative stage (Rastogi and Sawhney 1989).
Floral or inflorescence tissues have been widely employed
to induce somatic embryogenesis (Srivastava and Steinhauer
1981, Grönroos et al. 1989, Jörgensen 1989, Gingas 1991,
Kiss et al. 1992, Lopez-Baez et al. 1993, Teixeira et al. 1994,
Alemanno et al. 1996, Merkle et al. 1998) or adventitious buds
from mature trees (Bawa and Stettler 1972, Srivastava and
Steinhauer 1981, Bonga 1984, Bonga and von Aderkas 1988,
Chung et al. 1993, Loutfi and Chlyah 1998). Bawa and Stettler
(1972) regenerated shoots of Populus trichocarpa Torrey &
A. Gray from female inflorescences. Chung et al. (1993) induced organogenesis from immature flower buds of Populus deltoides Bartr. ex. Marsh. and Populus maximowiczii
A. Henry followed by successful adventitious shoot regeneration. Loutfi and Chlyah (1998) accomplished this with inflorescences of date palm (Phoenix dactylifera L.). The reproductive structures of conifers can also produce adventitious
buds, as shown by Bonga (1984) and Bonga and von Aderkas
(1988), who induced adventitious buds both directly and indirectly via callus from strobili of Larix decidua Mill.
We initiated willow cultures from inflorescences excised
from dormant buds collected in February. Others have reported that floral tissues have a temporary morphogenetic
competence at a specific stage early in their development
(Bawa and Stettler 1972, Bonga and von Aderkas 1988, Loutfi
and Chlyah 1998). In our previous work, inflorescences from
buds collected as early as January and as late as March produced adventitious buds at moderate frequencies (Merkle and
Lima, unpublished data), whereas material collected in November was characterized by a low regeneration frequency
(Lyyra, unpublished data). Thus, it appears that the window of
morphogenetic competence for black willow inflorescences
extends over several months.
In conclusion, we succeeded in regenerating adventitious
shoots of black willow. Our work is the second report describing the production of adventitious shoots in the Salix genus.
Our results indicate the potential to regenerate black willow
target material for gene transfer; however, regeneration frequency needs to be increased. To date, there have been no
published reports on the regeneration of transgenic willows.
Vahala et al. (1989, 1993) produced transformed callus of different willow species following co-cultivation of stem segments with Agrobacterium, but the callus was non-morphogenic. Xing and Maynard (1995) reported production of three
transgenic shining willow trees (Salix lucida Muhl.) following
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LYYRA, LIMA AND MERKLE
co-cultivation of stem segments with Agrobacterium. However, the transformed shoots were chimeric. With further optimization, the ability of black willow inflorescences to produce
adventitious shoots may make them suitable targets for Agrobacterium-mediated transformation with heavy metal resistance genes for phytoremediation.
Acknowledgments
We thank Paul Montello and Gisele Andrade for help in the laboratory. The work was funded by the Academy of Finland and by
McIntire-Stennis funds allocated to the Daniel B. Warnell School of
Forest Resources, University of Georgia.
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