American Journal of Plant Sciences, 2012, 3, 1181-1186
http://dx.doi.org/10.4236/ajps.2012.39143 Published Online September 2012 (http://www.SciRP.org/journal/ajps) 1181
An Improved System for Shoot Regeneration from Stem
Explants of Lombardy Poplar (Populus nigra L. var. italica
Koehne)
Kamal Kanti Biswas1, Takeshi Mohri2, Satoshi Kogawara2, Yoshihiro Hase1, Issay Narumi1,
Yutaka Oono1
1Medical and Biotechnological Application Division, Japan Atomic Energy Agency (JAEA), Takasaki, Japan; 2Forestry and Forest
Products Research Institute (FFPRI), Tsukuba, Japan.
Received July 5th, 2012; revised July 30th, 2012; accepted August 10th, 2012
ABSTRACT
We developed a system for the regeneration of Lombardy poplar (Populus nigra L. var. italica ) shoo ts from internodal
stem explants. Using this system, shoots regenerated from 87% of the stem explants placed on Murashige and Skoog
(MS) medium supplemented with 0.1 mg/L indole-3-acetic acid and 0.5 mg/L benzylaminopurine without undergoing
callus formation. About 80% of the in vitro regenerated shoots developed roots on MS medium supplemented with 0.5
mg/L indole-3-butyric acid and 0.02 mg/L 1-naphthylacetic acid. Well-rooted seven-to eight-week-old regenerated
plants could be transferred to soil for further growth and the survival rate of such plants after three weeks was 88%. The
protocol presented here is simple and economical because it does not rely on pre-incubation in callus induction medium
or repeated subculture in shoot induction medium containing trans-zeatin, an expensive substance. The in vitro regen-
eration system presented here could be used for evaluation of radiation sensitivity for Lombardy poplar tissues.
Keywords: Lombardy Poplar; Shoot Regeneration; Stem Explants; Auxin and Benzylaminopurine; Radiation
Sensitivity
1. Introduction
Poplars are of growing scientific importance due to their
small genome size, short rotation cycle, ability to under-
go in vitro regeneration, and ease of transformation and
vegetative propagation. These important traits, coupled
with the existence of a library of full-length enriched
expressed sequence tags [1] and availability of the ge-
nome sequence of Populus tricocarpa, have established
poplar as a model system in molecular genetics [2-5] and
tree physiology [6-8] studies. Furthermore, these rapidly
growing plants, which have the potential to enhance
wood supplies for the plywood, hardboard, pulp, and
paper industries, represent a commercially important re-
source [9].
In vitro plant regeneration of Lombardy poplar [11] or
its close hybrids [10,12-14] was achieved from leaf or
stem explants. These methods consists of two steps of
tissue culture stages; first, stem or leaf explants were in-
cubated on the medium containing high concentration of
plant hormone auxin (in most case, it is called callus in-
ducing medium, CIM) for several days then transferred
to shoot induction medium (SIM) that dominantly con-
tains plant hormone cytokinin. The system is also avail-
able for Agrobacterium-mediated gene transformation
[10,11,15]. In an early study of Lombardy poplar regen-
eration by Mohri et al. [15], 0.5 mg/L 2,4-dichlorophe-
noxyacetic acid (2,4-D) was added to CIM for calli in-
duction. Then, four weeks old calli developed in CIM
were repeatedly subcultured in SIM containing 2 mg/L
trans-zeatin and 0.2 mg/L benzylaminopurine (BA), fol-
lowed by re-transfer to root induction medium (RIM). In
later by Nishiguchi et al. [11], much simple regeneration
method had been developed for Lombardy poplar rege-
neration, without callus formation. But, still they relied
on pre incubation of explants in CIM (0.5 mg/L 2,4-D
and 1 mg/L BA) for 6 days. There after, pre incubated
explants in CIM were subcultured in SIM (0.5 mg/L
zeatin and 0.1 mg/L BA) for several times, followed by
re-transfer to RIM. However, in our experience, we were
unable to perform efficient shoot regeneration with these
protocols. Thus, we tried to re-optimize shoot regenera-
tion protocol for Lombardy poplar by using different
types of auxins and cytokinins at various concentrations.
Here, we report a simple shoot regeneration protocol
from stem explants of Lombardy poplar that is charac-

An Improved System for Shoot Regeneration from Stem Explants of Lombardy Poplar
(Populus nigra L. var. italica Koehne)
1182
terized by unre qui si te of p re-incubation ste p i n CIM.
2. Materials and Methods
2.1. Plant Materials, Growth Condition and
Surface Sterilization
Lombardy poplar plants were propagated in our labora-
tory from cuttings of poplars obtained from the Forestry
and Forest Products Research Institu te, Tsukuba, Ibaraki,
Japan. Donor plants were grown in pots containing a 1:1
mixture of v ermiculite and Metro-Mix 3 50 (Scotts-Sierra
Horticultural Products Company, Marysville, OH) at
26˚C under fluorescent tubes (90 µE·m–2·s–1, 16-h pho-
toperiod). The relative humidity was kept at 70%. The
plants received weekly applications of Hyponex solution
(1:1000) and were watered daily.
In preliminary experiments, surface sterilized explants
(1 cm) from leaves, petioles, and stems were used to ex-
amine regeneration proficiency to a range of different
concentration of 2,4-D, indole-3-acetic acid (IAA),
naphthylacetic acid (NAA), thidiazuron (TDZ), zeatin
and BA, singly or in combination. But, efficient and re-
producible regeneration were obtained only with stem
explants in preliminary experiments. Thus, only inter-
nodal stem explants were used for remaining experiments
of this study. Intern odal stem explants were excised from
newly growing branches of 3- to 4-month-old donor
plants, cut to 1-cm segments, and divided vertically into
halves (Figure 1A). Surface microorganisms were re-
moved from the explants by thorough washing under
running water for 20 min, followed by treatment with
sodium hypo-chlorite solution (15%) for 20 min. The
explants were then washed three times for 20 min each
with sterile water.
2.2. Culturing Tissues and Shoot Regeneration
For plantlet regeneration, the surface-sterilized stem ex-
plants were cultured on Petri dishes containing Mura-
shige and Skoog (MS) basal medium [16] supplemented
with 30 g/L sucrose, 1 ml/L vitamin B5, 3 g/L Gelrite
(Wako, Osaka, Japan) and various concentrations of IAA,
NAA and/or cytokinin (BA) (Table 1). The pH of media
was adjusted to 5.8 prior to autoclaving at 121˚C for 20
min. The explant cu ltures were maintained at 26˚C under
16-h light and 8-h dark intervals in a growth chamber
and subcultured in every 2 weeks.
2.3. Root Induction
For root induction, regenerated shoots were separated
and cultured on MS medium supplemented with 30 g/L
sucrose, 1 ml/L vitamin B5, 2 g/L Gelrite, 4 g/L agar and
various concentrations of indole-3-butyric acid (IBA) and
Figure 1. Direct plant regeneration of Lombardy poplar. (A)
Vertically divided internodal stem segments were used as
starting materials; (B) Shoot regeneration on medium sup-
plemented with 0.1 mg/L IAA and 0.5 mg/L BA; (C) and (D)
Root development [side view (C) and bottom view (D)] in
seven-to-eight-week-old in vitro raised plants on MS me-
dium supplemented with 0.5 mg/L IBA, 0.02 mg/L NAA, 4
g/L agar and 2 g/L Gelrite; (E) Regenerated plant growing
in a soil mixture of vermiculite and Metro-Mix 350 (1:1); (F)
Well-established and hardened in vitro raised Populus plant
growing in soil mixture. Bars indicate 1 cm (A)-(E) and 2
cm (F).
Table 1. Direct shoot regeneration from internodal stem
explants of Lombardy poplar.
Treatment (mg/L)
IAANAA2,4-DBA
Shoot
induction (%)a
Number of
shoots per
explanta
0 0 0 0 0 0
0 0 0.5 0.5 0 0
0.1 0 0.5 0.5 0 0
0.1 0 0 0.1 46 ± 2.31 1.87 ± 0.11
0.1 0 0 0.2 51 ± 2.40 1.96 ± 0.07
0.1 0 0 0.5 87 ± 3.71 3.87 ± 0.09
0.2 0 0 1 87 ± 4.81 3.63 ± 0.08
0.5 0 0 0.5 45 ± 3.71 2.13 ± 0.12
0 0.1 0 0.5 79 ± 4.37 3.18 ± 0.10
0 0.2 0 0.5 83 ± 3.33 3.55 ± 0.07
0 0.5 0 0.5 39 ± 4.80 2.05 ± 0.07
aResults r epresent means ± standard errors (SEs) of at least three replicated
experiments. Each experiment contained 40 to 50 explants. Measurements
were taken after five weeks of culture.

An Improved System for Shoot Regeneration from Stem Explants of Lombardy Poplar
(Populus nigra L. var. italica Koehne) 1183
NAA. Again, the pH of the medium was adjusted to 5.8.
2.4. Irradiation of Tissues
For investigation of radiation dose response relationship
on viability test and growth proficiency of regenerated
shoots, stem explants were excised to 1 cm, surface ster-
ilized, divided vertically into halves and cultured on me-
dium with 0.1 mg/L IAA and 0.5 mg/L BA in Petri
dishes. One week after from culture, Petri dishes con-
taining stem segments were subjected for irrad iation with
a range of doses (0 Gy to 30 Gy). The explants were ex-
posed to 320 MeV carbon ions accelerated by the azi-
muthally varying field cyclotron [22,23] or gamma rays.
Gamma rays obtained from 60Co source maintaining at
Japan Atomic Energy Agency (Takasaki, Japan). Irradia-
tion time for gamma rays was fixed to 30 min. Shoots
above 1 cm in length at five weeks after irradiation were
counted as regenerated shoots. Regeneration ratio was
calculated with dividing the number of stem explants that
produce regenerated shoots by total number of stem ex-
plants irradiated. The regeneration ratio of non-irradiated
control stems was plotted as 100%.
3. Results and Discussion
3.1. Direct Shoot Regeneration
In preliminary experiments, we cultured explants from
leaves, petioles, and shoots to medium supplemented
with different concentrations of 2,4-D, IAA, NAA, TDZ,
zeatin and BA, singly or in combination (d ata not shown).
Leaf and petiole explants did not develop any robust calli
or shoots on any of the media mentioned above. On the
other hand, stem explants developed calli on medium
supplemented with 0.5 mg/L 2,4-D and 0.5 mg/L BA;
however, no shoots emerged from these calli until five
weeks of culture (Table 1). Interestingly, we found that
direct shoot regeneration occurred when stem explants
were cultured on medium supplemented with NAA and
IAA in combination with BA (Table 1 and Figure 1(B)).
Over 87% of the stem explants produced at least one
shoot on medium supplemented with 0.1 mg/L IAA and
0.5 mg/L BA. The average number of shoots per explant
was 3.87 ± 0.09 (Table 1). A further increase of IAA to
0.2 mg/L or 0.5 mg/L and BA to 1 mg/L failed to im-
prove the regeneration efficiency. Thus, we used 0.1
mg/L IAA and 0.5 mg/L BA for the direct regeneration
of Lombardy poplar in this study. Application of the
synthetic auxin analog NAA yielded similar results as
IAA. Over 83% of the explants induced shoots on me-
dium supplemented with 0.2 mg/L NAA and 0.5 mg/L
BA, and the average number of shoots per explant was
3.55 + 0.07 (Table 1). Once again, an increased NAA
concentration (0.5 mg/L) failed to improve the regenera-
tion efficiency (Table 1). In Eastern Co ttonwood (Popu-
lus deltoides Bartram ex Marsh.), addition of cytokinin
(1 mg/L zeatin) alone resulted in the most efficient shoot
regeneration from stem explants in woody plant medium
(WPM) [17]. However, we found that cytokinin alone
could not stimulate the gro wth of calli or shoots in Lom-
bardy poplar and an optimal combination of auxin and
cytokinin (BA) was required for direct shoot regeneration.
This indicates that the optimal hormone concentration for
direct shoot regeneration in poplar is genotype specific.
Alternatively, the difference in response might be for the
reason of different basal medium, although there is no
major difference between the composition of WPM and
MS. Thus, for efficient regeneration, appropriate combi-
nation and concentration of growth regulators need to be
determined precisely.
3.2. Root Induction
To optimize root induction conditions, the directly re-
generated shoots were cultured on media con taining IBA
and NAA. In agreement with a report [1 1], we fo und that
a combination of 0.5 mg/L IBA and 0.02 mg/L NAA was
the most effective dose for root development in Lom-
bardy poplar (Figure 1(C) and 1(D)), although 0.5 mg/L
IBA alone could induce roots in 50% of the regenerated
shoots (Table 2). Doubling the concen tration of IBA and
NAA in the medium did not improve the root induction
efficiency (Table 2). The average number of roots per
shoot in the presence of 0.5 mg/L IBA and 0.02 mg/L
NAA was 5.2 ± 0.34 (Table 2).
We separated the well-rooted plants that were approxi-
mately seven weeks old from the culture medium, trans-
ferred them to pots containing a 1:1 mixture of vermicu-
lite and Metro-Mix 350 and incubated them at 26˚C un-
der a 16/8 h light/dark regimen. Figure 1(E) shows a re-
generated plant growing in soil mixture. Initially, the
potted plants were covered with clear plastic bags to pro-
tect them from any direct physical stress, and the plants
Table 2. Root induction in the regenerated shoots.
Treatment (mg/L)
IBA NAA
Root induction
(%)a Number of roots
per shoota
0 0 0 0
0.5 0 50.00 ± 4.33 3.6 ± 0.24
0 0.02 0 -
0.5 0.01 60.83 ± 4.41 3.67 ± 0.12
0.5 0.02 78.33 ± 3.00 5.2 ± 0.34
1 0.04 75.83 ± 3.63 5.1 ± 0.31
aResults represent means ± SEs of at least three replicated experiments. Each
experiment contained 40 to 50 regenerated shoots. Measurements were
taken after seven weeks of culture.

An Improved System for Shoot Regeneration from Stem Explants of Lombardy Poplar
(Populus nigra L. var. italica Koehne)
1184
were unwrapped after 7 - 10 days. The in vitro raised
poplar plants grew well and showed a high percentage of
survival rates (88%) at three weeks of growth in soil.
Healthy plants were transferred to larger pots with fresh
soil. Well-established and hardened plants were allowed
to grow (Figure 1(F)) in an incubator with weekly ap-
plications of Hyponex solution (1:1000).
3.3. Test of Radiation Sensitivity of Tissues
The direct shoot regeneration protocol developed in this
study can be used to estimate radiation sensitivity of the
tissues, which is initial requirement for a strategy of tissue-
culture-mediated screening mutants with ionizing radia-
tion. Figure 2 represents dose response relationship cur-
ves for carbon ion-beam (Figure 2(a)) and gamma-ray
(Figure 2(b)) irradiation on viability test and growth
proficiency of in vitro regenerated Lombardy poplar
shoots. No visible decrease in growth of regenerated
shoots was found up to th e irradiation dose of 2.5 Gy and
5 Gy for ion beams and gamma rays, respectively. Hence,
irradiation with these doses to stem explants of Lom-
bardy poplar hardly affects survival ability of regen erated
shoots. It is relevant to mention that now a day, ionizing
radiation is attracting increasing attention as a new
mutagen [22,23]. Various novel mutants of a wide vari-
ety of plants, which include Arabidopsis thaliana, Lotus
japonicus, tobacco, rice, verbena, rose, carnation, Tore-
nia, petunia, and Hinoki cypress, have been obtained via
ion-beam irradiation [24]. Similarly, soybean mutant
lacking lipoxygenases was identified by gamma-ray irra-
diation [25]. Thus, using this strategy, isolation of mu-
tants would be achievable in poplar, as well.
4. Conclusion
We found that shoot regeneration from Lombardy poplar
stem segments can be achieved by a simple procedure, in
which pre-culture in CIM is not necessary. Similar pro-
cedure is reported in Eastern Cottonwood (P. de ltoides)
[17-19] and male Himalayan poplar (P. ciliata) [20]. No
callus induction in this method might be another advan-
tage, because it is well known that plants regenerated via
a callus phase may differ from the mother plant due to
somaclonal variations [21]. The present protocol is also
economical, because it results high frequency (87%) di-
rect shoot regeneration without trans-zeatin, an expen-
sive subst ance that have used i n t he previous protocols of
Table 3. Comparison of regeneration systems in Lombary poplar (Populus nigra cv. italica) and related species.
Genetic
background
No. of media
used for shoot
regeneration*
Name of
media Growth regulators (mg/L) Explant tissue typeIncubation period (week) Reference
SIM IAA (0.1) + BA (0.5)
Populus nigra L.
var italica Koehne 1 RIM IBA (0.5) + NAA (0.02) Stem 8
Present study
CIM 2,4-D (0.5)
SIM t-zeatin (2.0) + BA (0.2)
Populus nigra L.
var italica Koehne 2
RIM IBA (0.5) + NAA (0.02)
Stem 10 [15]
CIM 2,4-D (0.5) + BA (1.0)
SIM t-zeatin (0.5) + BA (0.1)
Populus nigra L.
var italica Koehne 2
RIM IBA (0.5) + NAA (0.02)
Stem 13 [11]
CIM NAA (0.2) + BA (0.5)
SIM NAA (0.05) + BA (0.5) Populus nigra L. 2
RIM None
Leaf 13~ [10]
CIM NAA (0.5) + BA (1.0)
SIM NAA (0.25) + BA (0.6)
Populus nigra
× P. pyramidales 2
RIM NAA (0.1) +BA (0.1)
Leaf INT [14]
CIM IAA (0.1) + BA (0.5)
SIM NAA (0.01) + BA (0.5)
Populus nigra var thevestina
× (P. diversifolia +
P. tomentosa) 2
RIM IAA (0.8)
Leaf INT [12]
CIM IBA (0.1) + BA (0.5)
SIM IBA (0.1) + BA (1.0)
Populus nigra var thevestina 2
RIM IBA (1.0)
Leaf INT [13]
*RIM is not included. INT: Information not traceable.

An Improved System for Shoot Regeneration from Stem Explants of Lombardy Poplar
(Populus nigra L. var. italica Koehne) 1185
0
25
50
75
100
0510 15 20 25 30 35
Irradiation dose (Gy)
(a)
Regeneration ratio (% of control)
0
25
50
75
100
0510 15 20 25 30 35
Irradiation dose (Gy)
(b)
Regeneration ratio (% of control)
Figure 2. Dose response relationship curves for carbon ion-
beam (a) and gamma-ray (b) irradiation on regeneration
ratio of in vitro raised Lombardy poplar. Shoots above 1 cm
in length at five weeks after irradiation were counted as
regenerated shoots. Regeneration ratio was calculated with
dividing the number of stem explants that produce regen-
erated shoots by total number of stem explants irradiated.
The regeneration ratio of non-irradiated control stems was
plotted as 100%.
Lombardy poplar regeneration (Table 3) [11,15,17]. Our
method is able to produce and perpetuate a large number
of disease-free Lombardy poplar plants, and will thus
benefit physiological and genetic studies of hardwood
plants by providing a constant supply of competent and
efficient plant materials.
5. Acknowledgements
The authors wish to express their gratitude to Dr. Kenji
Shinohara, FFPRI, Tsukuba for providing Populus nigra
plants.
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