全 文 ：Research on Tissue Culture and Rapid Propagation
Technology of Superior Individuals of Lonicera
edulis Turcz
JIN Can，CAO Hou-nan * ，ZONG Cheng-wen，LI Wen-jian，AN Feng-yun
Department of Horticulture，Agricultural College of Yanbian University，Yanji 133002
Abstract ［Objective］This paper aimed to study the tissue culture and rapid propagation technology of superior individuals of Lonicera edulis
Turcz． ［Method］Several superior individuals of Lonicera edulis Turcz were used as materials for selecting the primary medium，subculture medi-
um，rooting medium and acclimatization substrate during the tissue culture and rapid propagation．［Result］6-BA was the optimal cytokinin for tis-
sue culture of Lonicera edulis Turcz，compared with ZT; modified MS +1．0 mg/L of 6-BA + 0． 2 mg/L of IBA was the optimal medium as prima-
ry and subculture medium，modified MS + 1．5 mg/L of IBA was the optimal medium for rooting of Lonicera edulis Turcz，the rooting rate had a-
chieved 100% after cultured for 30 d． The optimal substrate for transplanting plantlets of Lonicera edulis Turcz was composed of humus and per-
lite ( 1∶1，V/V) ，survival rate was as high as 95% after 30 d． ［Conclusion］This study provided basis for the rapid propagation of superior seed-
lings of Lonicera edulis Turcz，as well as the establishment of industrialized breeding technical system and the implementation of scale
production．
Key words Lonicera edulis Turcz; Tissue culture; Superior individual
Received: August 15，2011 Accepted: September 26，2011
Supported by the National Natural Science Fundation of China
( 30960231) ; Project of the State Forestry Administration ( 2005-06) ．
* Corresponding author． E-mail: hncao@ybu． edu． cn
Lonicera edulis Turcz，also known as Blue honeysuckle
and Lonicera caerulea L．，etc．［1］，is one of Caprifoliaceae
Lonicera plants and a deciduous shrub． Lonicera edulis Turcz
fruits are rich in several kinds of amino acids，vitamins，min-
eral elements，trace elements，flavonoids and other active
substances［2］，which can be eaten raw or processed into
many kinds of health products． Blue-black or purple fruits con-
tain edible red pigment［3］ and are good resources for extrac-
ting natural plant pigments． Lonicera edulis Turcz seeds con-
tain anthocyanin，and leaves contain aucubin［4］ which have
some medical value． In China，wild resources storage of Loni-
cera edulis Turcz is the maximum in Greater Khingan
Mountains， Lesser Khingan Mountains and Changbai
Mountains［5］． Although there are abundant wild resources of
Lonicera edulis Turcz in China，the uneven distribution as well
as the unplanned predatory picking by human had caused se-
vere damages to wild resources of Lonicera edulis Turcz，
sharply reduced reserves and recoverable amounts， and
gradually intensified the supply and demand gaps［6］． In this
study，several superior Individuals with large grains，high yield
and high-quality of Lonicera edulis Turcz from Changbai
Mountains were used as materials for researching the tissue
culture and rapid propagation techniques，in order to provide
basis for the rapid propagation of superior seedlings of Lonice-
ra edulis Turcz，as well as the establishment of industrialized
breeding technical system and the implementation of scale
production．
Materials and Methods
Materials
Branches of five superior individuals of Lonicera edulis
Turcz in dormancy stage were provided by Yanbian Forestry
Scientific Research Institute for hydroponic，and the 14 d-old
stems were used．
Method
Disinfection and primary culture of explants The sprou-
ted shoots were cut off the leaves and placed into sterilized jar
and washed with sterile water，disinfected with 75% ethanol
for 30 s and 0． 1% mercuric chloride for 5 min，washed fully
using sterile water for 4 －5 times，and then cut into 1． 0 －1． 5
cm of shoot tips and stems in a clean bench，which were inoc-
ulated into primary medium，in which the modified MS medium
were used as basal medium and hormone combinations were
shown in Table 1． The medium contained 30 g/L of sucrose，
with pH at 5． 5． Each individual and each media were com-
bined and totally 20 flasks of medium were inoculated． In or-
der to reduce the material loss caused by contamination，one
single explant was inoculated in each single flask． The prolif-
eration multiples were counted after 30 d．
The tissue culture were under conditions of temperature
at 25 ℃，light intensity of 1 500 －2 000 lx for 12 h /d．
Subculture of Lonicera edulis Turcz
Selection of the optimal hormone combination The
effects of different ratio of 6-BA and IBA on subculture of Loni-
cera edulis Turcz were explored． Plantlets obtained through
primary culture were inoculated into the medium，in which the
modified MS medium were used as basal medium and the
hormone combinations were shown in Table 2． Each individual
and each media were combined and totally 20 flasks of medi-
um were inoculated． The proliferation multiples were counted
after 30 d．
Selection of the optimal basal medium concentration
Plantlets for subculture were inoculated into the medium，as
shown in Table 3． Each individual and each media were com-
bined and totally 20 flasks of medium were inoculated． The
proliferation multiples were counted after 30 d．
Rooting culture of Lonicera edulis Turcz Plantlets ob-
tained through subculture were inoculated into the rooting me-
Agricultural Science ＆ Technology，2011，12( 11) : 1585 －1588
Copyright 2011，Information Institute of HAAS． All rights reserved． Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2011.11.038
dium，in which the modified MS medium were used as basal
medium and hormone combinations were shown in Table 4．
Each individual and each media were combined and totally 20
flasks of medium were inoculated． The proliferation multiples
were counted after 30 d．
Acclimatization of plantlets Flasks with rooted plantlets
were transferred to room temperature conditions，and the
caps were taken off． The flasks were placed for 1 － 2 d，for
the seedling hardening，after that，the plantlets were taken
out and washed away the medium in the roots then planted in-
to the sand and irrigated with water． Plastic cloths were used
to cover the plantlets，with timely irrigation． After sand culture
for 1 －2 d，the plastic cloths were gradually removed for venti-
lation and sand culture for 7 －10 d．
After sand culture，plantlets were transplanted into sub-
strates with different combinations as shown in Table 5，20
plantlets were transplanted into each substrates，and the sur-
vival rate were counted after 30 d．
Results and Analysis
Effects of 6-BA and ZT on the primary culture of Lonicera
edulis Turcz
Shoot tips and stems of Lonicera edulis Turcz were inoc-
ulated in the medium containing 6-BA，differentiation was ob-
served after 5 d，proliferation of plantlets was significant after
14 d，and differentiated plantlets were verdant and in good
condition with relatively high proliferation multiple after 30 d
( Fig． 1) ． Differentiation of shoot tips and stems medium con-
taining ZT was observed about 14 d after inoculation，differen-
tiated plantlets were less in poor condition with relatively low
multiplication after 30 d． As can be seen from Table 1，effects
of 6-BA and ZT on proliferation multiples of Lonicera edulis
Turcz were significantly different．
Table 1 Effects of different hormone combinations on proliferation
multiples of Lonicera edulis Turcz
Number of
medium
Hormone combinations∥mg/L
6-BA ZT IBA
Proliferation
multiples
1 0． 5 0． 2 5． 05 b
2 1． 0 0． 2 5． 98 a
3 0． 5 0． 2 2． 47 d
4 1． 0 0． 2 2． 62 c
Different lowercase letters indicated significant differences．
Fig． 1 Primary cultured plantlets of Lonicera edulis Turcz
Effects of different ratio of 6-BA and IBA on the subcul-
ture of Lonicera edulis Turcz
As can be seen from Table 2，when the concentration of
6-BA was altered，the proliferation multiples of plantlets obvi-
ously changed． To be specific，proliferation effects were rela-
tively better when the concentration of 6-BA was 1． 0 mg/L，
after subculture for 30 d，plantlets in modified MS +1．0 mg/L
of 6-BA + 0．2 mg/L of IBA had the highest proliferation multi-
ple which had achieved 6． 11，and the plantlets were growing
luxuriantly; when the concentration of 6-BA was higher or low-
er than 1． 0 mg/L，the proliferation multiples of plantlets were
both reduced，and the plantlets were very weak and growing
slim．
Table 2 Effects of different hormone combinations on the subculture
of Lonicera edulis Turcz
Medium
No．
Hormone combinations∥mg/L
6-BA IBA
Proliferation
multiples
1 0． 5 0． 1 3． 92 g
2 0． 5 0． 2 3． 86 h
3 0． 5 0． 3 3． 57 i
4 1． 0 0． 1 5． 45 c
5 1． 0 0． 2 6． 11 a
6 1． 0 0． 3 5． 64 b
7 1． 5 0． 1 4． 09 f
8 1． 5 0． 2 4． 23 e
9 1． 5 0． 3 4． 48 d
Different lowercase letters indicated significant differences
Effects of different basal medium concentrations on the
subculture of Lonicera edulis Turcz
After 3 － 4 times of subculture，tip blight and weakened
growth were observed in plantlets of Lonicera edulis Turcz． In
order to improve this phenomenon，basal medium selection
for subculture of Lonicera edulis Turcz was carried out in this
research． As can be seen from Fig． 2，after transplanted in
3 /2 modified MS medium ( 1．0 mg/L of 6-BA + 0．2 mg/L of
IBA) for subculture，the yellow leaf tips turned green，and the
plantlets were growing luxuriantly; as shown in Table 3，prolif-
eration multiples of plantlets were relatively higher in 3 /2 mod-
ified MS medium．
Table 3 Effects of different basal medium concentrations on subcul-
ture of Lonicera edulis Turcz
Basal
medium *
Proliferation
multiples Growth condition after 30 d
Modified MS
medium
1．54 c light green leaves，fine plantlets
3 /2 modified
MS medium
2．03 a light green leaves，strong plantlets
2 modified
MS medium
1．85 b light green leaves，delicate plantlets
* : contained 1． 00 mg/L of 6-BA + 0． 2 mg/L of IBA; Different low-
ercase letters indicated significant differences．
Fig． 2 Subcultured plantlets of Lonicera edulis Turcz
6851 Agricultural Science ＆ Technology Vol． 12，No．11，2011
Rooting culture of Lonicera edulis Turcz
During the process of primary culture of Lonicera edulis
Turcz，some plantlets of Lonicera edulis Turcz appeared not
only proliferation and growth but also rooting culturing in pri-
mary medium containing ZT，6-BA and IBA for over 30 d．
Therefore，in this study，the effects of ZT，6-BA and IBA on
the rooting of plantlets of Lonicera edulis Turcz were explored．
As can be seen from Table 4，roots were able to differentiate
in each rooting medium，but there were significant differences
among them． In the modifiedMS + 1．5 mg/L of IBA，the pla-
ntlets started rooting after 7 d of the inoculation，which had
generated plenty of roots after 10 d，and the rooting rate had
achieved 100% after 30 d． The experiment indicated that the
plantlets in No． 5 rooting medium had differentiated relatively
long and strong roots ( Fig． 3) ，while plantlets in No． 3 and 4
rooting medium had differentiated relatively short roots with
very fine lateral roots．
Table 4 Effects of different hormone combinations on rooting rates
of Lonicera edulis Turcz
Number of
medium
Hormone combinations∥mg/L
6-BA ZT IBA
Rooting
rate∥%
1 0．05 － 1． 5 10． 1 e
2 0． 10 － 1． 5 12． 3 d
3 － 0． 05 1． 5 45． 5 b
4 － 0． 10 1． 5 20． 4 c
5 － － 1． 5 100． 0 a
Different lowercase letters indicated significant differences．
Fig． 3 Root of plantlets in No． 5 rooting medium
Acclimatization of plantlet
As shown in Table 5，survival rate of the plantlets trans-
planted to No．4 substrate was the highest，and the growth of
plantlets was shown in Fig． 4． The experiment suggested that
during the sand culture of Lonicera edulis Turcz，humidity
maintaining was relatively critical，and the plantlets grew
strong after timely irrigation，which was beneficial for rooting．
Humus was nutrient-rich and had a relatively better ability to
retain water and nutrients，while perlite had large gaps，which
increased the ventilation of the soil surface，so that substrates
composed of humus and perlite were conducive to the survival
of transplanted plantlets．
Table 5 Effects of different substrates on survival rates of transplan-
ted plantlets
Number of
substrates Substrates Survival rate∥%
1 pine needle mulch∶sand =1∶1 70 d
2 pine needle mulch∶perlite =1∶1 80 c
3 humus∶sand =1∶1 85 b
4 humus∶perlite =1∶1 95 a
Different lowercase letters indicated significant differences．
Fig． 4 Plantlets transplanted in substrates ( humus∶perlite =1∶1)
Conclusion and Discussion
Plantlets of Lonicera edulis Turcz grew well in modified
MS +1．0 mg/L of 6-BA +0．2 mg/L of IBA，the monthly multi-
plication had achieved 6． 11． Tip blight phenomenon was ob-
served in some plantlets after 3 －4 times of subculture，which
was relatively significant in new plantlets． Therefore，3 /2
modified MS medium was suitable to be used as basal medi-
um for subculture． Plantlets could be subcultured in 3 /2 modi-
fied MS medium for once after 3 － 4 times of subculture，
which could not only save costs but also be beneficial for the
7851JIN Can et al． Research on Tissue Culture and Rapid Propagation Technology of Superior Individuals of Lonicera edulis Turcz
rapid propagation of Lonicera edulis Turcz．
During the large-scale production，it can be found that
the hormones accumulated inside plantlets with the increase of
subculture times，which was easy to form callus，thus weak-
ened the growth of plantlets and generated many invalid plant-
lets． Therefore，hormone concentrations should be appropri-
ately reduced with the increasing times of subculture．
Zhang Qi-chang，et al．，［7］ had reported that the optimal
basal medium for rooting of Lonicera edulis Turcz was 1 /4
MS，in which the rooting rate had achieved 96． 7%，that was
significantly different with the results of this research． Rooting
effects in 1 /4 modified MS medium，1 /2 modified MS medium
and modifiedMS medium were compared in this research，re-
sults of which indicated that the plantlets of Lonicera edulis
Turcz were hard to generate roots in 1 /4 and 1 /2 modified MS
medium but grew well in modified MS medium．
Survival rate of transplanted plantlets is the key factor de-
termining the efficiency of large-scale production． It can be
found during the later large-scale production that the survival
rates of plantlets of Lonicera edulis Turcz varied significant in
greenhouses and in field． The survival rates of robust trans-
planted plantlets could almost achieve 100% in fields，which
were much higher than that in greenhouse，in addition，the re-
juvenation period was remarkably shortened，plantlets genera-
ted a large number of new roots after transplanted in sand for
5 －7 d，which were applied for nutritional bowl transplanting
and were able to adapt to natural light after 10 －15 d．
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Responsible editor: FAN Xiao-hui Responsible proofreader:
檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
WU Xiao-yan
蓝靛果优良单株组培快繁技术的研究( 摘要)
金 灿，曹后男* ，宗成文，李文剑，安丰云 ( 延边大学农学院园艺系，吉林延吉 133002)
［目的］对蓝靛果优良单株组培快繁技术进行研究。
［方法］以蓝靛果几个优良单株为材料，对其组培快繁中的初代、继代和生根培养基及移栽基质进行筛选。
［结果］与 ZT相比，6-BA是较适合蓝靛果组培的细胞分裂素;改良 MS +6-BA 1． 0 mg /L + IBA 0． 2 mg /L是较适合蓝靛果的初代和继代培养
基;改良 MS + IBA 1． 5 mg /L是较适合蓝靛果生根的培养基，生根培养 30 d，生根率达 100% ;较适合蓝靛果组培苗移栽的基质类型是腐殖土
与珍珠岩体积比为 1∶1的基质，移栽 30 d后的成活率达 95%。
［结论］为快速繁育蓝靛果优质苗木，建立工厂化育苗技术体系，进而实现规模化生产提供依据。
关键词 蓝靛果;组织培养; 优良单株
基金项目 国家自然基金( 30960231) ;国家林业局项目( 2005-06) 。
作者简介 金灿( 1985 － ) ，女，黑龙江建三江人，在读硕士，从事果树遗传育种研究，E-mail: jincan162@ hotmail． com。* 通讯作者，教授，博士，硕士生
导师，从事果树遗传育种研究，E-mail: hncao@ ybu． edu． cn。
收稿日期 2011-08-15 修回日期
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2011-09-26
( From page 1573)
地衣芽孢杆菌 16S-ITS标记及其特异性分析( 摘要)
陈 冲1* ，佟建明2，张潞生3 ( 1．山西省农业科学院果树研究所，山西太谷 030815; 2．中国农业科学院北京畜牧兽医研究所，北京 100094;
3．中国农业大学农学与生物技术学院，北京 100094)
［目的］建立地衣芽孢杆菌的分子鉴定方法。
［方法］通过对地衣芽孢杆菌 TS-01的 16S和 ITS序列进行克隆测序和差异性分析，以该区序列为靶序列设计地衣芽孢菌 TS-01的特异性引
物，并用该引物扩增全部受试菌种。
［结果］从 ITS和 16Sr DNA区间设计了地衣芽孢杆菌种特异性引物，特异性引物 PCR的最佳退火温度为 67． 2 ℃，循环数为 24 个;地衣芽孢
杆菌 TS-01可扩增出 1条 905 bp的标记片段，其他受试菌株均为阴性，从而证明试验得到了在种水平上对该菌种进行准确鉴定的特异
16S-ITS标记。
［结论］地衣芽孢杆菌 TS-01分子鉴定方法的建立，为地衣芽孢杆菌的分子诊断奠定了基础。
关键词 地衣芽孢杆菌; 特异引物; 16S; ITS
作者简介 陈冲( 1980 － ) ，男，山西晋中人，助教，从事果树分子生物学工作。* 通讯作者。
收稿日期 2011-09-20 修回日期 2011-09-25
8851 Agricultural Science ＆ Technology Vol． 12，No．11，2011