全 文 ：珍稀植物猬实的离体培养及
快速繁殖
袁秀云，蒋素华，王默霏，崔波 * （郑州师范学
院生物工程研究所，河南郑州 450044）
摘 要 该研究采用珍稀植物猬实的茎节段
为外植体，通过丛生芽诱导建立了高效的离体
培养及快速繁殖体系。丛生芽诱导的最适培
养基是 MS+4.44 μM 6-BA+0.54 μM NAA，其
次是 MS+4.44 μM 6-BA +0.27 μM NAA；在
MS 基本培养基中添加 6-BA 或者 6-BA 与
IAA（1.71 μM）或 NAA（0.27，0.54 μM）结合能
促进丛生芽的增值，增值系数为 3.4-8.2，增值
系数最大的培养基是 MS+4.44 μM 6-BA+
0.27 μM NAA；对于生根，1/2MS培养基中添
加 IBA或者 IBA结合 2,4-D和 NAA能够获得
100%生根率，而最适合的浓度组合为 1/2MS+
1.48 μM IBA+1.08 μM NAA+0.05 μM 2，4-
D；生根苗移栽温室 100%成活。
关键词 猬实；珍稀植物；茎节段；离体繁殖；
植物生长调节剂
作者简介 袁秀云（1970-），女，河南郑州人，教
授，从事植物生物技术研究，E-mail: yuanxiu-
yun@163.com。*通讯作者。
收稿日期 2013-03-10
修回日期 2013-04-02
Efficient in vitro Propagation of an Endangered
Species Kolkwitzia amabilis Graebn
Xiuyun YUAN, Suhua JIANG, Mofei WANG, Bo CUI*
Institute of Bioengineering, Zhengzhou Normal University, Zhengzhou 450044, China
*Corresponding author. E-mail: yuanxiuyun@163.com
Received: March 10, 2013 Accepted: April 2, 2013A
Agricultural Science & Technology, 2013, 14(4): 564-567
Copyright訫 2013, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
Abstract This study developed an efficient in vitro cultivation and propagation sys-
tem for an endangered species Kolkwitzia amabilis using nodal segments as ex-
plants. Multiple shoots were induced through axillary bud formation. The highest fre-
quency of multiple shoot induction was achieved when the nodal segment explants
were incubated in Murashige and Skoog (MS) medium supplemented with 4.44 μM
6-benzyladenine (6-BA) in combination with 0.54 μM α-naphthaleneacetic acid (NAA),
followed by treatment with 4.44 μM 6-BA in combination with 0.27 μM NAA. Shoot
multiplication could be induced in MS medium supplemented with stand-alone 6-BA
or 6-BA in combination with indole-3-acetic acid (1.71 μM) or NAA (0.27 or 0.54 μM),
with 6-BA and either compound, exhibiting a stronger effect on shoot multiplication.
The optimum combination of plant growth regulators for shoot multiplication was 4.44
μM 6-BA with 0.27 μM NAA. The maximum rooting percentage was obtained in a
half-strength MS medium supplemented with indole-3-butyric acid alone and in com-
bination with NAA and 2,4-dichlorophenoxyacetic acid, but the best combination of
plant growth regulators for rooting was 1.48 μM indole-3-butyric acid with 1.08 μM
NAA and 0.05 μM 2,4-dichlorophenoxyacetic acid. The rooted shoots were trans-
ferred to a greenhouse with a success rate of 100%.
Key words Kolkwitzia amabilis; Endangered species; Nodal segment; In vitro propa-
gation; Plant growth regulator
K olkwitzia amabilis is a decidu-ous leaf shrub from the Capri-foliaceae family first discov-
ered in 1901 by Richard Kolkwitz, a
German botanist. It has been listed as
a rare and endangered plant, and
classified under national protection
category 3 in China[1]. K. amabilis is an
ancient relic of plant species of a sin-
gle family in flora from the northern re-
gion of China, and considered to be an
isolated branch in Caprifoliaceae. It is
distributed patchily and discontinuous-
ly in slopes and forest margins across
six provincial areas including Shaanxi,
Shanxi, Henan, Hubei, Gansu, and
Anhui. Research of K. amabilis is of
important scientific value in the study
of the phylogeny of Caprifoliaceae,
ancient geography, as well as the oc-
currence of northern flora and the re-
lation with nearby floristic regions.
K. amabilis has a graceful outline
owing to its cespitose and compact
stem and the blooming flowers are
densely clustered with a beautiful color
during the early summer. Its inferior o-
vary can form dense burrs like a small
hedgehog, and it is an excellent gar-
den plant due to its structure and resis-
tance to cold and drought. As a high -
er-value ornamental, it can be used
for linear planting and clump planting
in lawns, corners, artificial rockwork,
and pavilions, as well as for potting or
cutting flowers.
However, the population of K.
amabilis keeps decreasing gradually
and studies on this species are limited
to its seed characteristics and cultiva-
tion techniques. Gao et al. [2] reported
that the endangered species are char-
acterized by their inferior seed quality,
low seed setting rate, and difficulty
with propagating sexually. Seedlings
of K. amabilis are not found in its natu-
ral habitat, and its propagation relies
mainly on natural root sprouting[3]. The
DOI:10.16175/j.cnki.1009-4229.2013.04.001
Agricultural Science & Technology2013
Table 1 Effects of PGRs at different concentrations and combinations on shoot induction
from nodal segments in K. amabilis
Plant growth regulators∥μM Observed results Inductionpercentages∥%
1.11 6-BA+2.26 2,4-D No shoots; a few callus, necrosis 35.23
1.11 6-BA+4.52 2,4-D No shoots; a few callus, necrosis 50.46
4.44 6-BA+0.27 NAA No Callus; multiple shoots 85.05
4.44 6-BA+0.54 NAA No Callus; multiple shoots 100
Table 2 Shoot multiplication in different media
Plant growth regulators∥μM Multiplication Coefficient
4.44 6-BA 3.40
4.44 6-BA+1.71 IAA 7.78
4.44 6-BA+0.27 NAA 8.22
2.22 6-BA+0.54 NAA 6.43
1.11 6-BA+0.27 NAA 0
community of K. amabilis belongs to
xeric shrublands and woodlands. Be-
cause of its simple composition and
poor diversity of species, the com-
munity succession of K. amabilis is at
the primary stage with little stability [2].
K. amabilis has been found to have a
low fruiting rate, and the seeds have a
longer dormancy resulting in seed
propagation impossibility deteriorated
by hard pericarp.
Currently, cutting, offshoot, and
layering are used to produce plant ma-
terials from K. amabilis. These meth-
ods are marked by such disadvan-
tages as their seasonal restrictions,
long cycle, and low breeding coeffi-
cient[4]. In view of protection of endan-
gered species, scientific research, and
landscape application needs, it is cru-
cial to establish an efficient propaga-
tion and plant regeneration system.
Although clonal propagation of
endangered species just appears re-
cently, more attention has been at-
tached to it[5]. Use of biotechnology has
been proven to be a simple and effi-
cient measure to conserve and rein-
troduce rare and endangered plants
back into their natural environments[6-7].
Cultivation in vitro is widely used to
produce plant materials from ornamen-
tal[8], medicinal[9-10], and cutting flower[11]
species and particularly meaningful in
cases in which seeds are not avail-
able[12]. To the best of our knowledge,
studies on the successful regeneration
of K. amabilis have not been reported
to date. Yi et al. [13] not only found that
6-benzyladenine (6-BA; 1-3 mg/L) in
combination with α-naphthaleneacetic
acid (NAA; 0.1-0.2 mg/L) could induce
callus tissue and organ differentiation
using hypocotyls of K. amabilis as ex-
plants and that 0.2 mg/L NAA or in-
dole-3-butyric acid (IBA) could induce
the rooting of rootless seedlings. Fur-
thermore, it is demonstrated the feasi-
bility of rapid propagation through in-
ducing adventitious buds from callus
tissue. However, a rapid propagation
system in vitro could not be estab-
lished accordingly. The first step in us-
ing hypocotyls as explants is to germi-
nate seeds and form aseptic
seedlings. Due to the special method
of removing seed dormancy and the
low germination rate of the seeds, us-
ing callus induction from aseptic
seedlings is not suitable for rapid
propagation. The primary aim of the
present study was to develop a reliable
and efficient in vitro propagation
method for K. amabilis.
Materials and Methods
Plant material
The branches of K. amabilis were
obtained from Henan Agricultural Uni-
versity in China. The leaves were re-
moved from the branches, and the
stems were cut short, thoroughly
washed with running tap water for 3-4
h, surface-sterilized with 70% (v/v) al-
cohol for 1-2 min and 0.1% (m/v) mer-
curic chloride for 8-10 min, and then
rinsed with sterile distilled water five
times.
Shoot induction
The stems as explants were cut
into nodal segments at 2-3 cm in leng-
th and inoculated immediately to pre-
vent the cut edges from drying. The
effects of plant growth regulators at
different concentrations and combina-
tions (PGRs) on shoot induction from
the nodal segments were explored.
6-BA (1.11 or 4.44 μM) in combination
with 2,4-Dichlorophenoxyacetic acid
(2,4-D; 4.52 or 9.04 μM) or NAA (0.27
or 0.54 μM) was added to Murashige
and Skoog (1962) (MS) medium so-
lidified with 3%(w/v) sucrose and 0.80%
(w/v) agar (Table 1). Shoot induction
percentages were calculated for the
number of explants exhibiting visible
shoots after one month in culture di-
vided by the total number of explants
tested.
Shoot multiplication
Single shoots were isolated from
multiple shoot clusters and implanted
into MS basal medium to promote
shoot growth. The MS basal medium
was supplemented with 6-BA, NAA,
and indole-3-acetic acid (IAA) at differ-
ent concentrations, either alone or in
combination. Subsequently, 6-BA (4.44,
2.22, and 1.11 μM) was used alone or
in combination with 1.71 μM IAA or
with 0.27 or 0.54 μM NAA (Table 2).
Shoot multiplication coefficients were
then calculated for the number of visi-
ble shoots after one month in culture
divided by total number of implanted
single shoots.
Rooting and acclimatization
Regenerated shoots (7-8 cm in
length) were excised from the parent
culture and transferred to half-strength
MS solid medium with different con-
centrations of 6-BA (2.22 μM), NAA
(1.08 or 1.62 μM), IBA (1.48 or 2.47
μM), and 2,4-D (0.05 μM), either alone
or in combination, for rooting (Table 3).
Shoots that formed roots were accli-
matized in an open culture vessel un-
der the culture condition for one week,
removed there from culture vessels,
washed gently with running tap water,
and planted in 5 cm diameter pots con-
taining the mixture of peat and ver-
miculite (2∶1, v/v). The plantlets were
kept in the greenhouse for growth.
All culture media were adjusted to
pH 5.8-6.0 using 0.1 N HCl or 0.1 N
NaOH before autoclaving at 121 ℃ for
20 min. The explants were then cul-
tured under light/dark for 12 h respec-
565
Agricultural Science & Technology 2013
a, Shoot induction; b, Shoot multiplication; c, A few calluses were induced at the cut sur-
faces from the base of the shoots; and the leaves that contacted the medium enlarged and
developed a few calluses as well in 1/2MS with 1.08 μM NAA and in that with 2.22 μM 6-BA
combined with 1.62 μM NAA after 3 weeks; d, and e Rooted shoots resulting from 1/2MS
supplemented with 1.08 μM NAA combined with 1.48 μM IBA and 0.05 μM 2;4-D after 6
weeks; f, Differentiation of calluses from the stem segments and leaves of shoots from 1/
2MS supplemented with 2.22 μM 6-BA and 1.62 μM NAA.
Fig.1 In vitro propagation of Kolkwitzia amabilis
Table 3 Effects of various media on rooting of excised shoots of K. amabilis cultured in 1/2
MS
Plant growth regulators∥μM Rootingpercentages∥%
Number and length( cm) of root
3 weeks 6 weeks
2.47 IBA 100 5 roots, 2 cm 5 roots, 12 cm
1.62 NAA 0 - -
2.22 6-BA+1.62 NAA 0 - -
1.08 NAA+1.48 IBA+0.05 2,4-D 100 10 roots, 1 cm 40 roots, 10 cm
tively, namely photoperiod, using cool
white lamps at a photon flux density of
40 -60 μmol/(m2·s), with temperature
of (25±2)℃. It is notable that cultures
were observed weekly.
Results
Shoot induction
Multiple shoot clusters were initi-
ated from the nodal segments in the
MS basal medium supplemented with
4.44 μM 6-BA in combination with NAA
(0.27 or 0.54 μM) (Fig.1a). The induc-
tion percentages of multiple shoot
clusters were 100% in the MS basal
medium supplemented with 4.44 μM
6-BA in combination with 0.54 μM NAA
and 85.02% when 0.27 μM NAA was
used with 4.44 μM 6-BA. However, in
the MS basal medium containing 1.11
μM 6-BA and 2,4-D (2.26 or 4.52 μM),
the explants enlarged and few calluses
produced at the cut surfaces in all
media within 15-26 days of inocula-
tion. No shoots formed from the cal-
luses, and the explants turned yellow
and necrotic, with induction percent-
ages at 35.23% and 50.46% in the MS
basal medium, supplemented with
1.11 μM 6-BA and 2.26 μM 2,4-D, and
with 1.11 μM 6-BA and 4.52 μM 2,4-D,
respectively. Meanwhile, multiple
shoot clusters did not emerge from the
nodal segments in these media (Table
1).
Shoot multiplication
After the explants were cultured
for 35 days, clumps with multiple
shoots were divided into single shoots
and then transferred to multiplication
media, where they would develop into
multiple shoots, which could proliferate
by 3.4 to 8.22-fold with a 30-day sub-
culture in medium containing 4.44 μM
6-BA alone or in combination with 1.71
μM IAA or with 0.27 or 0.54 μM NAA.
When 2.22 μM 6-BA was combined
with 0.54 μM NAA, the multiplication
coefficient was 6.43. However, the
combination of 1.11 μM 6-BA and 0.27
μM NAA could not induce the multipli-
cation of a single shoot (Table 2). Con-
currently, some shoots developed ear-
lier could grow 7-8 cm high (Fig.1b).
Rooting and acclimatization
Individual plants separated from
multiple shoots were cultured in vari-
ous half-strength MS media supple-
mented with different PGRs. No ad-
ventitious roots were differentiated in
medium with 1.08 μM NAA and in that
with 2.22 μM 6-BA combined with 1.62
μM NAA within 3 weeks. A few callus-
es were induced at the cut surfaces
from the base of the shoots, and the
leaves that contacted the medium en-
larged and developed a few calluses
as well (Fig.1c). However, differentiat-
ed adventitious roots were induced
within three weeks in medium supple-
mented with 2.47 μM IBA and in that
supplemented with 1.08 μM NAA
combined with 1.48 μM IBA and 0.05
μM 2,4-D (Table 3). In terms of the
rooting percentages, two treatments
seemed to be effective with a rate of
100%. However, shoots with 2.47 μM
IBA had significantly fewer roots than
those with 1.08 μM NAA combined
with 1.48 μM IBA and 0.05 μM 2,4-D.
Roots initiated from the shoot base
and approximately 5 roots (2 cm in
height) in the medium with 2.47 μM
IBA were lengthened, so that they
were approximately 12 cm long after 6
weeks. Moreover, no new roots were
found. In the medium with 1.08 μM
NAA, 1.48 μM IBA, and 0.05 μM 2,4-D,
approximately 10 roots at 1 cm long
were observ ed after the shoots were
cultured for 6 weeks, the number of
roots increased to approximately 40,
and the length was 10 cm (Fig.1d and
e). Plantlets with well-developed shoots
and roots in the medium with 1.08 μM
NAA, 1.48 μM IBA, and 0.05 μM 2,4-D
were acclimatized in an open culture
vessel under the culture condition for
one week and then subsequently re-
moved. The rooted shoots were finally
transferred to plastic pots and placed
in the greenhouse for further develop-
ment and growth. It is notable that the
success rate is as high as 100%.
Discussion
The importance of in vitro culture
of plant tissues in the conservation of
endemic[14-15], rare and endangered[16]
566
Agricultural Science & Technology2013
Responsible editor: Xiaoxue WANG Responsible proofreader: Xiaoyan WU
species has grown in recent years [17].
In vitro culture has also been used in
genetic transformation using callus in-
duced as the recipient [18]. In this study,
we investigated in vitro cultivation and
rapid clonal propagation of K. amabilis
using nodal segments as explants for
the first time to establish its conserva-
tion or application in landscaping.
Direct shoot regeneration from
nodal segments of Piper longum has
been reported[11]. It is a simple and rapid
propagation method in plants. In our
study, 6-BA combined with NAA could
induce shoot clusters from nodal seg-
ments, and the effects of 0.54 μM NAA
were better than those of 0.27 μM.
However, 6-BA combined with 2,4-D
could induce a few calluses at the cut
surfaces of stems and no shoots
formed from the nodal segments. In
general, PGRs can induce axillary
buds to form shoots, the activity or
dormancy of the axillary bud is con-
trolled by endogenous levels of regu-
lators of plant, and a single axillary
shoot is formed from either a single
bud or an overlapping buds (some
species) in the following year. Howev-
er, when balance of PGRs is broken in
plants, the multiple shoots may be in-
duced by an exogenous hormone.
Thus, a good balance supplied to the
culture medium and endogenous lev-
els of regulators is crucial for in vitro
cultivation. In K. amabilis, the combi-
nation of 4.44 μM 6-BA and 0.54 μM
NAA was determined to be good for
shoot induction.
From observations, 6-BA either
alone or in combination with IAA or
NAA played a role in promoting the
proliferation of shoots in K. amabilis.
The multiplication coefficient of shoots
lowered as the concentration of 6-BA
decreased and, to a certain extent
(1.11 μM), with decreasing NAA con-
centration (0.27 μM). Shoot multipli-
cation was not observed. These indi-
cate that cytokinin plays a key role in
shoot multiplication of K. amabilis and
6-BA has a stronger effect on shoot
multiplication than NAA.
Furthermore, it is observed that
IBA (2.47 μM), instead of NAA (1.62
μM), either alone or in combination
with 6-BA (2.22 μM) could induce root-
ing and had a higher effect owing to
larger number of roots. However, IBA
(1.48 μM) combined with NAA (1.08
μM) and 2,4-D (0.05 μM) had a
stronger effect not only on the number
but also on the length of roots. These
suggest that IBA plays a crucial role in
inducing rooting and IBA in combina-
tion with NAA and 2,4-D may poten-
tially play the most significant role.
Rooting analysis gave confusing
results in that a few calluses were in-
duced at the cut surfaces from the
base of the shoots and leaves, which
contacted a half-strength medium with
2.22 μM 6-BA and 1.62 μM NAA, con-
sidering that such concentration and
combination of PGRs may be able to
induce multiple shoot clusters or shoot
multiplication if added in MS medium.
These findings show that PGRs with
the same concentration and combina-
tion can have different effects in me-
dia. We also attempted to achieve
plant regeneration through the differ-
entiation of calluses from the stem
segments and leaves of shoots (Fig.
1f). However, no such differentiation
was detected in some media and the
calluses turned yellow and finally
necrotic, suggesting that the optimum
medium for differentiation remains to
be determined.
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