全 文 ：鳞叶菊组织培养技术研究
李艳敏，孟月娥 *，赵秀山，王利民，王慧娟，张
和臣 （河南省农业科学院园艺研究所，河南
郑州 450002）
摘 要 [目的]探讨鳞叶菊组织培养技术。[方
法]以鳞叶菊带芽茎段为外植体，研究了不同
浓度的 6-BA 和 NAA 对增殖培养、IBA 和
NAA以及继代周期等对生根培养的影响。[结
果] 鳞叶菊的组培苗需要低浓度的植物激素，
继代周期显著影响其生根培养；鳞叶菊最佳的
增殖培养基为 MS+6-BA 0.5 mg/L+NAA 0.05
mg/L+蔗糖 30 g/L+琼脂 5.5 g/L，增殖系数可
达 4.25；继代周期为 28 d的组培苗生根最好，
在培养基为 1/2 MS+IBA 0.1 mg/L+NAA 0.05
mg/L+蔗糖 20 g/L+琼脂 5.5 g/L中，其生根率
为 95.9%，平均根数和根长分别达到 4.69条
和 1.68 cm。[结论]该研究为鳞叶菊无菌培养
体系的建立奠定了基础。
关键词 鳞叶菊；增殖；生根；继代周期
基金项目 河南省发改委项目（2060403）。
作者简介 李艳敏（1978-），女，河南汤阴人，
助理研究员，硕士，从事园林植物生物技术研
究，E-mail:minzili@126.com。*通讯作者，研究
员，硕士，从事园林植物生物技术研究，E-mail:
yysylhh@126.com。
收稿日期 2012-12-10
修回日期 2013-01-29
Study on Tissue Culture of Leucophyta brownii
‘Canal Rocks Form’
Yanmin LI, Yue’e MENG*, Xiushan ZHAO, Limin WANG, Huijuan WANG, Hechen Zhang
Horticulture Institute of He’nan Academy of Agricultural Sciences, Zhengzhou 450002, China
Supported by Project of Development and Reform Commission of He’nan Province
(2060403).
*Corresponding author. E-mail: yysylhh@126.com
Received: December 10, 2012 Accepted: January 29, 2013A
Agricultural Science & Technology, 2013, 14(2): 230-234
Copyright訫 2013, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
Abstract [Objective] This study aimed to establish a tissue culture technology for
Leucophyta brownii ‘Canal Rocks Form’. [Method] Leucophyta brownii ‘Canal Rocks
Form’ stems with axillary buds were selected as explants in vitro to investigate the
effects of different concentrations of 6-BA and NAA on proliferation culture and the
effects of IBA, NAA and subculture cycle on rooting culture. [Result] The results
showed that Leucophyta brownii ‘Canal Rocks Form’ plantlets need low concentra-
tions of phytohormones and the rooting culture was significantly affected by the sub-
culture cycle; the optimal medium for proliferation culture was MS+0.5 mg/L 6-BA+
0.05 mg/L NAA+30 g/L sucrose+5.5 g/L agar, with a proliferation coefficient of 4.25;
the plantlets with a subculture cycle of 28 d were the most suitable for rooting cul-
ture, with a rooting rate of 95.9% in 1/2 MS+0.1 mg/L IBA+0.05 mg/L NAA+20 g/L
sucrose+5.5 g/L agar, and the average number and length of roots were 4.69 and
1.68 cm, respectively. [Conclusion] This study laid the foundation for establishing
sterile culture system of Leucophyta brownii ‘Canal Rocks Form’.
Key words Leucophyta brownii ‘Canal Rocks Form’; Propagation; Rooting; Subcul-
ture cycle
L eucophyta brownii ‘CanalRocks Form’, belonging togenus Leucophyta of family
Compositae (Fig.2A), is a small ever-
green shrub endemic to Australia [1]
with silver-gray leaves which are re-
flective in the night. Leucophyta
brownii ‘Canal Rocks Form’ is the
first choice for silver plant during color
matching for plant modules, which can
also be used as a warning plant on the
road boundary. Leucophyta brownii
‘Canal Rocks Form’ grows in alkaline
or neutral sandy land, which is resis-
tant to drought and salinity, with a
good prospect of application in saline-
alkali land greening. Currently, no re-
search has been reported on Leuco-
phyta brownii ‘Canal Rocks Form’ re-
productive technology. Therefore, in
this study, tissue culture of Leucophy-
ta brownii ‘Canal Rocks Form’ was
explored, to investigate the effects of
plant hormones on the proliferation
and rooting and the effects of subcul-
ture cycle on the rooting of Leucophyta
brownii ‘Canal Rocks Form’, thereby
establishing a sterile culture system,
which provides technical guidance for
the industrialized seedling culture and
laid the foundation for the physiologi-
cal, molecular and breeding research-
es of Leucophyta brownii ‘Canal
Rocks Form’.
Materials and Methods
Materials
Leucophyta brownii ‘Canal Rocks
Form’ stems with axillary buds were
selected as experimental materials.
Methods
Leucophyta brownii ‘Canal Rocks
Form’ generated branches were cut
into about 1 cm long stems with buds,
washed with cleanser essence, rinsed
with running water for 30 min, and
then transferred to the super-clean
bench, sterilized with 75% alcohol for
30 s, sterilized with 1% NaClO for 10
min, rinsed with sterile water once,
and sterilized with 0.5% NaClO for 12
DOI:10.16175/j.cnki.1009-4229.2013.02.023
Agricultural Science & Technology2013
Table 2 Effects of different concentrations of 6-BA on proliferation culture of Leucophyta
brownii ‘Canal Rocks Form’
No. 6-BAmg/L
Propagation
coefficient
Seedling
height∥cm Growth status of plantlets
CK 0 2.00 a 1.33 Less differentiated buds，thin and delicate
branches, rooted plantlets
1 0.5 3.83 b 2.92 Robust growth of seedlings with more differ-
entiated buds
2 1.0 2.8 ab 1.62 Leaf hypertrophy, showing a trend of vitrifi-
cation
3 2.0 1.83 a 1.58 Most of the generated buds were vitrified and
abnormal
Different lowercase letters between different rows in the same column indicate significant
differences (P<0.05).
Table 1 IBA and NAA concentrations
Treatment IBA∥mg/L NAA∥mg/L
CK 0 0
1 0.1 0.10
2 0.5 0.10
3 1.0 0.10
4 0.1 0.05
5 0.1 0.10
6 0.1 0.30
7 0.1 0.50
min, rinsed with sterile water for 3 -4
times, and then inoculated to medium.
After cultured for 30 d, sterile
seedlings were obtained (Fig.2B) for
propagation and rooting experiments.
Effects of different concentrations
of 6-BA on proliferation culture
The concentrations of 6-BA were 0.5,
1.0 and 2.0 mg/L respectively, with
blank medium containing no hormone
as the control, to investigate the ef-
fects of 6-BA on propagation of Leu-
cophyta brownii ‘Canal Rocks Form’,
10 bottles were treated per treatment,
with three replications, and the prolif-
eration coefficient and seedling height
were recorded 30 d after inoculation.
Effects of different concentrations
of NAA on proliferation culture
MS was selected as basic medium
and supplemented with 0.5 mg/L 6-BA
and 0, 0.01, 0.05, 0.10 and 0.50 mg/L
NAA, to observe the growth status of
Leucophyta brownii ‘Canal Rocks
Form’ plantlets, 15 plantlets were
treated per treatment, and the prolifer-
ation coefficient and seedling height
were recorded 30 d after inoculation.
Effects of different concentrations
of auxins on rooting culture Stem
segments longer than 2 cm were se-
lected for rooting culture. 1/2MS was
selected as basic medium and sup-
plemented with different concentra-
tions of auxins, IBA and NAA con-
centrations were shown in Table 1,
medium addition of hormone was
used as the control, 12 bottles were
treated per treatment, with three repli-
cations, the rooting time was ob-
served and the rooting rate, root
number and root length were counted
45 d after inoculation.
Effects of subculture cycle on root-
ing culture Leucophyta brownii
‘Canal Rocks Form’ plantlets cul-
tured for 28, 30 and 33 d were respec-
tively inoculated to the rooting medi-
um (1/2MS+0.1 mg/L IBA+0.05 mg/L
NAA+20 g/L sugar+5.5 g/L agar), to in-
vestigate the effects of subculture cy-
cle on rooting. Rooting time was ob-
served every 1 d during the initial inoc-
ulation period, and the rooting rate,
root number and root length were
counted 45 d after inoculation.
Culture conditions MS was select-
ed as basic medium and supplement-
ed with different concentrations and
types of cytokinins and auxins for pro-
liferation culture and rooting culture.
Medium was supplemented with 20 -
30 g/L sucrose and 5-6 g/L agar. The
temperature of culture room was (25±
2)℃, illumination time was 14 h/d, and
light intensity was 58 μmol/(m2·s)[2-7].
Data processing The experimental
data were analyzed using DPS v7.05
software.
Results and Analysis
Effects of different concentrations
of 6-BA on proliferation culture of
Leucophyta brownii ‘Canal Rocks
Form’
During the proliferation culture,
6-BA is the most commonly used plant
growth regulator, a nd different plants
require different concentrations of
6 -BA. As can be seen from Table 2, in
CK without addition of any hormone,
Leucophyta brownii ‘Canal Rocks
Form’ seedlings had a small number
of differentiated buds and thin and del-
icate branches with rooting; after sup-
plemented with low concentrations of
6-BA, proliferation and height of
seedlings were promoted, showing ro-
bust growth with a large number of dif-
ferentiated buds; however, when 6-BA
concentration was higher than 1 mg/L,
the promotion effects of 6-BA on pro-
liferation and height of seedlings were
reduced, showing a trend of vitrifica-
tion. The maximum proliferation coef-
ficient was 3.83 when 6-BA concen-
tration was 0.5 mg/L, followed by 2.8
when 6-BA concentration was 1 mg/L,
with no significant difference; when
6-BA concentration further increased
to 2 mg/L, the proliferation coefficient
was reduced to 1.83, showing signifi-
cant differences compared with the
other treatments and no significant dif-
ference compared with the control.
The seedling height reached the high-
est of 2.92 cm when 6-BA concentra-
tion was 0.5 mg/L, which was 1.62 cm
when 6-BA concentration was 1 mg/L;
however, when 6-BA concentration fur-
ther increased to 2 mg/L, the seedling
height was reduced to 1.58 cm, while
that in the control group was only 1.33
cm. Therefore, based on comprehen-
sive consideration of the proliferation
coefficient and seedling height of Leu-
cophyta brownii ‘Canal Rocks Form’
plantlets, the optimal concentration of
6-BA was determined as 0.5 mg/L.
Effects of different concentrations
of NAA on proliferation culture of
Leucophyta brownii ‘Canal Rocks
Form’
NAA has certain effects on prolif-
eration culture of Leucophyta brownii
‘Canal Rocks Form’. As can be seen
from Table 3, Leucophyta brownii
‘Canal Rocks Form’ seedlings grew
well when NAA concentration was 0-
0.05 mg/L (Fig.2C), seedlings grew
weaker with large callus when NAA
concentration was higher than 0.1
mg/L, and some seedlings died. The
maximum proliferation coefficient was
4.25 when NAA concentration was
0.05 mg/L, followed by 3.43 when NAA
concentration was 0.01 mg/L; when
231
Agricultural Science & Technology 2013
Table 5 Effects of subculture cycle on rooting culture of Leucophyta brownii ‘Canal Rocks
Form’
No. Subculturecycle∥d
Rooting
rate∥%
Root
number
Root
length∥cm Rooting time
A 28 95.9 a 4.69 a 1.68 a 8 d after inoculation
B 30 81.5 ab 4.38 ab 0.84 b 9 d after inoculation
C 33 44.2 b 2.24 b 0.82 b 9 d after inoculation
Different lowercase letters between different rows in the same column indicate significant
differences (P<0.05).
Table 4 Effects of different concentration ratios of IBA and NAA on rooting culture of
Leucophyta brownii ‘Canal Rocks Form’
Treatment IBA∥mg/L NAA∥mg/L Rooting rate∥% Rooting time
CK 0 0 25 22 d after inoculation
1 0.1 0.1 40.5 10 d after inoculation
2 0.5 25.8 10 d after inoculation
3 1.0 20.6 10 d after inoculation
4 0.1 0.05 65.6 8 d after inoculation
5 0.1 47.2 11 d after inoculation
6 0.3 36.8 12 d after inoculation
7 0.5 27.5 12 d after inoculation
Table 3 Effects of different concentrations of NAA on proliferation culture of Leucophyta
brownii ‘Canal Rocks Form’
No. NAA∥mg/L
Propagation
coefficient
Seedling
height∥cm Growth status of plantlets
CK 0 2.4 1.98 Robust growth
1 0.01 3.43 2.57 Robust growth, more differentiated buds
2 0.05 4.25 1.69 Robust growth, more differentiated buds
3 0.10 3.00 1.82 Weak growth, large callus, some seedlings
died
4 0.50 2.43 1.11 Large callus, some seedlings died, with
vitrification phenomenon
NAA concentration further increased
to 0.5 mg/L, the proliferation coefficient
was reduced to 2.43, which was con-
sistent with that in the control group of
2.4. The seedling height reached the
highest of 2.57 cm when NAA con-
centration was 0.01 mg/L, and that in
the control group was 1.98 cm; when
NAA concentration was higher than
0.01 mg/L, the seedling height showed
a downward trend. Overall, low con-
centration of NAA has promotion ef-
fects on proliferation and growth of
Leucophyta brownii ‘Canal Rocks
Form’. Comprehensively considering
the proliferation coefficient and
seedling height, the optimal concen-
tration of NAA was determined as
0.01-0.05 mg/L.
Effects of different concentration
ratios of auxins on rooting culture
of Leucophyta brownii ‘Canal
Rocks Form’
Auxin has great effects on rooting
of Leucophyta brownii ‘Canal Rocks
Form’, which not only affects the root-
ing rate, root length and root number,
but also greatly affects the rooting
time. As can be seen from Table 4, in
CK medium without addition of auxins,
Leucophyta brownii ‘Canal Rocks
Form’ plantlets began rooting 22 d af-
ter inoculation, while the rooting time
was significantly advanced after the
supplement of auxins, and the
plantlets began rooting 8 -12 d after
inoculation. When NAA concentration
was 0.1 mg/L and IBA concentration
ranged between 0.1-1 mg/L, the root-
ing rate showed a downward trend
with the increase of IBA concentration,
which reached the highest of 40.5%
when IBA concentration was 0.1 mg/L;
when IBA concentration was 0.1 mg/L
and NAA concentration ranged be-
tween 0.05-0.5 mg/L, the rooting rate
also showed a downward trend with
the increase of NAA concentration,
which reached the highest of 65.6%
when NAA concentration was 0.05
mg/L.
As shown in Fig.1, after supple-
mented with auxins, the root number
and root length of treatment groups
were both higher than those of the
control group, in medium No.1-3, NAA
concentration maintained unchanged,
the root number showed a downward
trend with the increase of IBA con-
centration, which reached the maxi-
mum of 4.9 when IBA concentration
was 0.1 mg/L, while the root length in-
creased first and then decreased,
which reached the maximum of 1.5 cm
when IBA concentration was 0.5 mg/L,
followed by 1.16 cm when IBA con-
centration was 0.1 mg/L; in medium
No.4-7, IBA concentration maintained
unchanged, the root number increased
first and then decreased with the in-
crease of NAA concentration, which
reached the maximum of 4.89 when
NAA concentration was 0.1 mg/L, fol-
lowed by 4.39 when NAA concentra-
tion was 0.05 mg/L, while the root
length showed a downward trend,
which reached the maximum of 1.21
cm when NAA concentration was 0.05
mg/L. In conclusion, the optimal auxin
dosage for rooting of Leucophyta
brownii ‘Canal Rocks Form’ was de-
termined as 0.1 mg/L IBA and 0.05
mg/L NAA.
Effects of subculture cycle on root-
ing culture of Leucophyta brownii
‘Canal Rocks Form’
Subculture cycle has relatively
great effects on rooting of Leucophyta
brownii ‘Canal Rocks Form’, which
greatly affects the rooting rate, root
number and root length but shows no
significant effect on rooting time. As
can be seen from Table 5, plantlets
with a subculture cycle of 28 d began
rooting on the 8th d after inoculation,
while those with subculture cycles of
30 and 33 d began rooting on the 9th d
after inoculation. The rooting rate
reached the highest of 95.9% with a
subculture cycle of 28 d, which was
significantly higher than that of plan-
tlets with a subculture cycle of 33 d
(44.2%) and showed no significant dif-
ference from that of plantlets with a
subculture cycle of 30 d (81.5%). The
root number of plantlets with subcul-
ture cycles of 28 and 30 d was 4.69
232
Agricultural Science & Technology2013
Fig.1 Effects of different concentration ratios of IBA and NAA on root number and root
length of Leucophyta brownii ‘Canal Rocks Form’
and 4.38 respectively, showing no sig-
nificant difference; the root number of
plantlets with a subculture cycle of 33
d was the minimum of only 2.24, which
was significantly lower than that of
plantlets with a subculture cycle of 28
d. The root length of plantlets with a
subculture cycle of 28 d reached the
maximum of 1.68 cm, which was
significantly higher than that of
plantlets with subculture cycles of
30 d and 33 d. Therefore, Leucophyta
brownii ‘Canal Rocks Form’ plantlets
with a subculture cycle of 28 d were
appropriate for rooting (Fig.2D).
Conclusions and Discus-
sions
In this study, Leucophyta brownii
‘Canal Rocks Form’ stems with axil-
lary buds were selected as explants in
vitro for proliferation culture and root-
ing culture using different concentra-
tions and types of phytohormones.
Phytohormones play important roles in
the process of plant tissue culture.
During the process of proliferation cul-
ture of Leucophyta brownii ‘Canal
Rocks Form’, low concentrations of
6-BA and NAA lead to the robust
growth and the increased number of
differentiated buds; on the contrary,
excessively high concentrations of
phytohormones will increase vitrifica-
tion and inhibit the height growth,
which is consistent with the researches
of Spiraea salicifolia L., Gerbera jam -
esonii Bolus and Ajania purpurea [8-10].
However, several varieties of Aster-
aceae plants need high concentrations
of 6-BA and NAA during the process of
proliferation culture. For instance,
Wedelia chinensis requires 2.0 mg/L
6-BA for adventitious bud induction [11];
1.0 mg/L 6-BA and 0.1-0.2 mg/L NAA
should be supplemented in the medi-
um for proliferation culture of Stevia
rebaudiana Bertoni[12-15], indicating that
different plant varieties have different
levels of sensitivity and tolerance to
phytohormones.
Results of this study showed that
Leucophyta brownii ‘Canal Rocks
Form’ can generate roots even in
blank medium without addition of any
hormone, which indicates that Leuco-
phyta brownii ‘Canal Rocks Form’ is
easy to generate roots and has low
demand of auxin. Subsequent experi-
ments further confirm that during the
rooting culture of Leucophyta brownii
‘Canal Rocks Form’, the induction
time is as long as 22 d in medium
without addition of auxin, and the root-
ing rate is only 25% ; after supple-
mented with auxins, the induction time
is significantly shortened to 8 -12 d,
and the rooting rate, root number and
rot length are also increased. When
IBA concentration is higher than 0.1
mg/L and NAA concentration is higher
than 0.05 mg/L, auxins show inhibitory
effects on rooting rate, root number
and rot length of Leucophyta brownii
‘Canal Rocks Form’, which is con-
sistent with previous studies[16-18], dur-
ing the rooting process of Gerbera
jamesonii Bolus, the rooting rate and
root number are low in hormone-free
medium, while IBA and NAA can both
lead to better rooting effects within the
range of 0.01-0.1 mg/L.
Subculture cycle is an important
research content of plant tissue cul-
ture, the growth condition of seedlings
greatly affects the proliferation and
rooting culture. Some studies have
shown that subculture cycle affects the
subculture breeding ability of Anubias
nana plantlets, excessively long inter-
val in subculture cycle leads to aging
of tender stems and slow growth at
late stages[19]. Subculture cycle greatly
affects the rooting of Leucophyta
brownii ‘Canal Rocks Form’, the root-
ing rate, root number and root length
of plantlets with a subculture cycle of
28 d were all significantly higher than
those of plantlets with a subculture cy-
cle of 33 d, which might be related to
the physiological conditions of the
A, Leucophyta brownii ‘Canal Rocks Form’; B, Axillary bud generation of Leucophyta
brownii ‘Canal Rocks Form’; C, Proliferation culture of Leucophyta brownii ‘Canal Rocks
Form’; D, Rooting culture of Leucophyta brownii ‘Canal Rocks Form’.
Fig.2 Tissue culture of Leucophyta brownii ‘Canal Rocks Form’
233
Agricultural Science & Technology 2013
Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WU
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