全 文 ：书Discussion on the Regeneration Technology of
Gazania rigens L． Leaves
LI Ye-feng，SHENG Ya-dan，WANG Cai-chen，WANG Ning，LU Xiao-ping *
Gold Mantis School of Architecture and Urban Environment，Soochow University，Suzhou 215123
Abstract ［Objective］The research aimed to study the regeneration technology of Cazania rigens L． leaves and screen out the optimum medi-
um formula for the regeneration of Cazania rigens L． leaves． ［Method］Using Japan imported C． rigens leaves as materials，the orthogonal test
was made for the callus and adventitious buds induction in MS medium with different kinds and concentrations of hormones． The optimum medi-
um formula for the regeneration of C． rigens leaves were screened out． ［Result］On the medium of MS + 0． 8 －1． 0 mg/L TDZ + 0． 05 －1． 0
mg/L NAA，compact type and bright green calli were formed． When the leaves were inoculated on the medium of MS + 0． 5 －1． 0 mg/L TDZ +
0． 05 －1． 0 mg/L NAA，many adventitious shoots can be induced and the induction rate reached 100%． When strong adventitious shoots with the
height of 2． 0 －3． 0 cm were transplanted into the medium of 1 /2 MS +0．1 mg/L NAA，the rooting situations were good and the rooting rate was
100%． ［Conclusion］The research provided a new way for the rapid propagation of C． rigens and laid the foundation for the genetic transforma-
tion and new varieties breeding of C． rigens．
Key words Cazania rigens L．;Callus;Plantlet regeneration;TDZ
Received:March 11，2010 Accepted:May 25，2010
Supported by the Extra-curricular Academic Research Found of 12th
Batch of Students in Soochow University (KY2010114A)． Science
and Technology Support (Agriculture)Project of Suzhou Province
(SNG0908)．
* Corresponding author． E-mail:szlxp@yahoo． com． cn
Cazania rigens L． is a variety of perennial herbaceous
plants of genus Cazania of family Compositae［1］． It is origina-
ted in South Africa，likes warm and sunny，it is a kind of good
flower flowering plants for park and garden (suitable for par-
terre and flower border) ，as well as flowers arranging． The
corolla of C． rigens is colorful，and the petals are bright，
which was flowering at morning and closing in evening，the li-
fetime of a simple flower can last as long as 10 days，in addi-
tion，it has capacity of tolerance to drought，heat and lean-
ness． Therefore，after short-term domesticated，different vari-
eties (lines)of C． rigens can be directly applied in the colori-
zation of city square，streets，public green space and court-
yard． Because the C． rigens has some advantages such as
evergreen in four seasons，flowering in three seasons and
strong antireversion force，as well as both of the leaf and flow-
er can be saw and enjoyed，it is a kind of new optimal ground-
cover plants that can be used for growing in ground and pot-
ting． The cultivation technology of C． rigens has been repor-
ted in China for a long time，but it is mostly used in the potting
and needed sowing every year，the use of it in the landscape
application has not been reported yet． Combined with city
construction planning，the planting and embellishing of season
flowering plants in the vacant area of street tree cave，espe-
cially the planting of C． rigens to street tree cave will have an
important significance to change the simple color of cover
plants and enrich the diversity of cover plants，as well as to
effective utilize the street tree cave for the vegetation green-
ing． In order to accelerate the breeding process of seedlings
of C． rigens and establish the regeneration technology system
of C． rigens leaves，the C． rigens leaves were used as exper-
imental materials in this study to screen out the optimum me-
dium formula for the regeneration of C． rigens leaves by the
orthogonal test of adding different types and concentrations of
hormone，as well as to provide a new way for the rapid propa-
gation of C． rigens and lay the foundation for the genetic
transformation and new varieties bredding of C． rigens．
Materials and Methods
Materials
C． rigens was used as experimental material，which was
introduced by Kyushu University of Japan;plant growth regu-
lator Thidiazuron (TDZ) was purchased from Shanghai
ShengGong company;6-BA and NAA were purchased from
Hangzhou Haotian biotechnology company．
Methods
Explants disinfection The C． rigens plants that grown
strong were selected，the leaves were washed by tap water
for 1 h，and the surface disinfection was according to the fol-
lowing process on the superclean bench:dipped with 70% of
alcohol for 30 s，disinfected with 0． 1% of corrosive sublimate
for 8 min，and then washed with sterile water for three to five
times． After disinfection，the leaves were cut into explants (1
cm)on the sterile filter paper．
Cultivation conditions MS medium was used as the basic
medium，and different kinds and concentrations of hormone
was added;the sugar content in medium was 30% and the
agar content was 7． 0%，with the pH of 5． 6 －5． 8． Cultivating
conditions were as follows:the temperature was 25 ℃，the in-
tensity of illumination was 2 000 － 3 000 lx，the illumination
time was 10 h．
Callus cultivation The C． rigens leaves were cultivated on
the MS medium that contained different kinds of hormone for
induction，the leaf induction situation was observed one month
later and the induction rate was calculated．
Induction rate = The number of explants that produced
callus /The number of inoculated explants × 100%
Induction of adventitious buds The viridis calluses of
close-up type were transplanted to the MS medium with differ-
ent concentrations of hormone，and the adventitious buds for-
mation rate was calculated．
Agricultural Biotechnology
Agricultural Science ＆ Technology，2010，11(4):64 －68
Copyright 2010，Information Institute of HAAS． All rights reserved．
DOI:10.16175/j.cnki.1009-4229.2010.04.036
Adventitious bud formation rate = The number of ex-
plants that produced adventitious buds /The number of inocu-
lated explants × 100%
Results and Analysis
Effects of TDZ，6-BA and NAA on the callus induction of
C． rigens leaves
The orthogonal experiment was used in this study，differ-
ent kinds of hormone including TDZ，6- BA and NAA were
added to MS medium for the induction of callus of C． rigens
leaves，and the result was observed 30 d later． It could be
concluded in Table 1 that the inductivity of induced callus on
MS + TDZ (0．80 － 1．00 mg/L)+ NAA (0．05 －0．10 mg/L)，
in addition，most of the induced calli were dark green and of
close-up type (Fig． 1 － C) ，while some parts of loosen-type
calli were light green，they were loosen and grown faster
(Fig． 1 －D)．
A:The calli of leafs in vitro after 2 days;B:Formed the calli after 15 days;C:Formed compact calli after 30 days;D:Formed loose calli
after 30 days;E:The adventitious buds in early differentiation stage;F:The adventitious buds in late differentiation stage．
Fig． 1 TDZ － induced callus
Table 1 Effects of TDZ，6-BA and NAA on the callus induction of C． rigens leaves
TDZ
mg/L
6-BA
mg/L
NAA
mg/L
Explants
number
The number
of explants
that produced
callus
Callus
rate∥% Situation of callus
0 2． 0 0． 10 40 0 0 The explants were of etiolation and browning，there was no callus produced．
1． 00
3． 0 0． 10
1． 00
4． 0 0． 10
1． 00
0． 1 0 0． 10 5 12． 5 There was a small amount of calli，which was of rarefaction．
0． 05 6 15． 0
0． 5 0 0． 10 35 87． 5 The yield of calli was moderately and parts of the calli were light green，whichwas compact．
0． 05 36 90． 0 The yield of calli was moderately and parts of the calli were light green，whichwas compact．
0． 8 0 0． 10 40 100 The growth velocity of calli was quite fast，the yield of calli was relatively high，and parts of calli could produce the buds．
0． 05
1． 0 0 0． 10 40 100 The growth velocity of calli was quite fast，the yield of calli was relatively high，and parts of calli could produce a small amount of multiple shoots．
It was generally thought that the compact calli had the po-
tential to differentiate adventitious buds［2］． The callus could be
differentiated by most plants in medium that contained 6-BA
and NAA，indicating that the callus induction of C． rigens
leaves needed the cytokinin which owned higher activity to
promote cell division;in the medium that contained 6-BA and
56LI Ye-feng et al． Discussion on the Regeneration Technology of Gazania rigens L． Leaves
NAA，callus was difficult to be induced by explants，and most
of the explants were of etiolation and browning (Fig． 2) ，and
the adventitious roots were produced (Fig． 3) ，which indica-
ted that 6-BA could not promote the induction of callus in C．
rigens leaves effectively．
Fig． 2 The leaves in 6-BA medium after 30 days
Fig． 3 The transplanted calli in 6-BA medium induced by TDZ
after 30 days
Effect of TDZ and NAA combination on adventitious buds
of C． rigens leaves
Effects of TDZ and NAA combination on the number of
adventitious buds of C． rigens leaves The induction of
adventitious buds of C． rigens leaves explants was carried out
by MS medium that contained different concentrations of TDZ
and NAA combinations，60 d later，the data was recorded．
Table 2 showed that the increasing of TDZ concentration could
significantly increased the number of adventitious buds;when
the TDZ concentration was the same and the NAA concentra-
tion increased，the number of adventitious buds was also im-
proved;when the TDZ concentration was up to above 1． 00
mg/L，the increasing of the number of adventitious buds was
not obvious，which was the close to that of 2． 00 mg/L of
TDZ;when the TDZ concentration was of 2． 00 mg/L，vitre-
ous shoots were produced． These results demonstrate that in
the induction process of adventitious buds by the use of TDZ，
the higher TDZ concentrations were not needed，but certain
concentrations of NAA was needed．
Effects of TDZ and NAA combination on the formation
rate of adventitious buds of C． rigens leaves The in-
crease of TDZ concentrations could significantly improve the
regeneration frequency of leaves;when the TDZ concentra-
tion was the same，the regeneration frequency of adventitious
buds would increase with the increasing of NAA concentra-
tions． When the TDZ and NAA concentration was up to 0． 50
mg/L，the regeneration frequency of adventitious buds could
achieve 100%． It could be concluded from Table 3 that the
TDZ concentration range that needed for the regeneration of
adventitious buds of C． rigens leaves was wide(0．50 － 2．00
mg/L) ，which would reach higher regeneration frequency
combined with 0． 05 － 1． 00 mg/L of NAA． Contrary，when
TDZ concentration reached above 1． 00 mg/L，vitrification
phenomenon was appeared，the stems and leaves were slen-
der，and browning brittle leaves were produced，which went
against the continuing growth of adventitious buds． Therefore，
the optimum concentrations of TDZ for the effective differenti-
ation of green callus were of 0． 50 －1．00 mg/L (Fig． 4)．
Table 2 Effects of different concentrations of TDZ and NAA on the
number of adventitious buds of the C． rigens leaves
TDZ∥mg/L NAA∥mg/L Number ofexplants
Number of
adventitious buds
0． 1 0． 05 15 4
0． 10 4
0． 50 6
1． 00 8
0． 5 0． 05 15 15
0． 10 17
0． 50 23
1． 00 26
0． 8 0． 05 15 25
0． 10 28
0． 50 34
1． 00 36
1． 0 0． 05 15 30
0． 10 35
0． 50 36
1． 00 47
2． 0 0． 05 15 29
0． 10 37
0． 50 38
1． 00 45
Table 3 Effects of different concentrations of TDZ and NAA on for-
mation rate of adventitious buds of C． rigens leaves
TDZ
mg/L
NAA
mg/L
Number of
explants
Explants number
from adventitious
buds treated
by hormone
Formation rate
of adventitious
buds∥%
0．1 0． 05 15 2 13． 3
0． 10 2 13． 3
0． 50 4 26． 7
1． 00 5 33． 3
0． 5 0． 05 10 66． 7
0． 10 13 86． 7
0． 50 15 100． 0
1． 00
0． 8 0． 05
0． 10
0． 50
1． 00
1． 0 0． 05
0． 10
0． 50
1． 00
2． 0 0． 05
0． 10
0． 50
1． 00
66 Agricultural Science ＆ Technology Vol． 11，No．4，2010
Rooting and transplanting
When the adventitious buds were grown to 2 －3 cm long
and 2 －3 pieces of leaves，they were cut and transplanted to
the rooting medium，basic medium was 1 /2 MS，and different
concentrations of NAA were added，30 d later，the rooting sit-
uation was observed． It could be concluded from Table 4 that
the adventitious roots were difficult to be produced by adventi-
tious buds when there was no NAA contained in medium;
when NAA concentration was of 0． 1 mg/L，the rooting rate
was up to 100%，and the roots were stout (Fig． 5) ;when
NAA concentration was more than 0． 1 mg/L，the rooting rate
decreased． Therefore，the optimum NAA content in rooting
medium was 0．1 mg/L．
When the root length was of 3 － 4 cm，after hardening-
seedling for 7 d，the seedlings were taken out and the medium
on the roots was washed，and then transplanted into the
mixed matrix(Vermiculite∶Perlite =1∶2) ，14 d later，they were
transplanted into pot soil，and the plants grew well (Fig． 6)．
Fig． 4 The adventitious buds induced by TDZ
Fig． 5 The root induction of adventitious buds
Fig． 6 The growth situation of regenerated plantlet in pot
Conclusions and Discussions
Many researches about tissue culture of ornamental
plants have been reported at home and abroad［3 －7］，however，
the researches of establishment of clone and rapid propaga-
tion of C． rigens have not been reported yet． In this study，
the C． rigens leaves were used as explants，after cultivated in
medium that contained different hormones，the optimum medi-
um for callus differentiation，adventitious buds production and
rooting of C． rigens leaves was screened，and the propose of
plantlet regeneration and establishment of clone were
reached．
Plant growth regulator TDZ owns strong cytokinins activi-
ty，therefore，it shows the promote effect of differentiation of
adventitious buds［8 －10］． In a certain range，the higher the con-
centrations of TDZ the higher formation rate of adventitious
buds． The results of this study showed that the optimum TDZ
concentrations that suitable for the growth of adventitious buds
and callus of C． rigens leaves were of 0． 5 － 1． 0 mg/L;TDZ
had obvious proliferation effect on plants，but when TDZ con-
centration increased to 1． 0 mg/L，the formed tube seedlings
began to be smaller，with the continued increase of concen-
tration of TDZ，parts of tube seedlings showed evident tufted
effect，the stems and leaves were slender，and browning brit-
tle leaves were produced． Therefore，in the same time of im-
proving the proliferation rate of tube seedlings，the tube seed-
lings quality should also be considered to improve its trans-
planting seedling rate．
Hormone combination and concentration ratio are very
important to the differentiation of explants，the production of
adventitious buds mainly dependsed on types and concentra-
tions of cytokinins and auxin［11］． Results in this study showed
that TDZ and NAA had significant effect on the induction of
callus and differentiation of adventitious buds of C． rigens
leaves，but whether high or low concentrations of 6-BA could
barely affect the induction of callus of C． rigens leaves，indi-
cating that high efficiency cytokinins were needed for the in-
76LI Ye-feng et al． Discussion on the Regeneration Technology of Gazania rigens L． Leaves
duction of callus of C． rigens to promote the cell division．
Table 4 Effect of NAA concentrations on rooting
Medium
NAA
mg/L
Number of adven-
titious buds
Rooting number
of adventitious buds
Rooting
rate∥%
Growth situation of adventitious root
1 /2MS 0 0 0 There was no root grew
0． 1 20 100 The roots was long and thick，with larger amount of roots
0． 5 20 18 90 The roots was long and thin，with larger amount of roots
1． 0 16 80 The roots was long and thin，with less amount of roots
2． 0 15 75 The roots was long and thin，with less amount of roots
References
［1］YANG JJ(杨俊杰)，FU HM(付红梅)． The main cultivation tech-
niques of Cazania rigens(勋章菊的栽培技术要点)［J］． Agriculture
Engineering Technology:Greenhouse ＆ Horticulture(农业工程技
术:温室园艺)，2008(6):61 －62．
［2］AN LJ(安利佳)，JIANG CY(姜长阳)． Introduction to plant tissue
culture(植物组织培养导论)［M］． Dalian:Liaoning Normal Universi-
ty Press(大连:辽宁师范大学出版社)，1997:55 －60．
［3］CHANG C，CHANG WC． Effect of thidiazuron on bud development
of Cymbidium sinense Willd in vitro［J］． Plant Growth Regulation，
2000，30(2):171 －175．
［4］CONFALONIERI M，BALESTRAZZI A，BISOFFI S． Invit roculture
and genetic engineering of Populus spp．:synergy for forest tree
improvement［J］． Plant Cell Tissue and Organ Culture，2003，72:
109 －138．
［5］CHEN BX(陈宝鑫)，WANG XX(王晓旭)，ZHANG QY(张倩怡)，et
al． Tissue culture and plantlet regeneration of Tadescantia flumi-
nensis (白花紫露草的组织培养与植株再生体系的建立)［J］． North-
ern Horticulture(北方园艺)，2009，9(6):84 －86．
［6］WANG JH(王静华)，HOU JS(侯建生)，LIU GL(刘桂林)，et al．
Tissue culture and plant regeneration from leaf of Siberian elm(白
榆的组织培养与叶片再生研究) ［J］Journal of Northwest Forestry
University(西北林学院学报)，2009，24(5):74 －77．
［7］ZHAO PP(赵培培)，CHE DD(车代弟)，WANG JG(王金刚)，et
al． Establishment of rose (Rosa chinensis var． floribunda)regen-
eration system(丰花月季再生体系的建立)［J］Journal of Northeast
Agricultural University(东北农业大学学报)，2008，39(6):30 －32．
［8］ LENG CL(冷春玲)． Research on virus-free and tissue-culture
techniques in strawberry(草莓脱毒及组培快繁研究)［J］． Northern
Horticulture(北方园艺)，2007(5):210 －211．
［9］LU RE(卢永恩)，LI HX(李汉霞)，YE ZB(叶志彪)． Shoot regener-
ation from hypocotyl explants of Chinese cabbage enhanced by
TDZ(激素 TDZ 对促进白菜下胚轴不定芽再生的效应分析) ［J］．
Journal of Huazhong Agricultural University(华中农业大学学报)，
2003(6) :591 －594．
［10］QIN L(秦玲)，LI M(李明)，HAN LX(韩礼星)． Effects of different
cytokinins on leaves in vitro regeneration of Fuji 2001 apple variety
(不同细胞分裂素对2001富士苹果离体叶片再生的影响)［J］． Jour-
nal of Fruit Science(果树学报)，2002，19(4):215 －218．
［11］LIN RS(林荣双)，WANG QH(王庆华)，LIANG LK(梁丽琨)． His-
tological study on adventitious bud formation and somatic embryo-
genesis of peanut (Arachis hypogaea L．)leaflets (TDZ诱导花生
幼叶的不定芽和体细胞胚发生的组织学观察)［J］． Bulletin of Botan-
ical Research(植物研究)，2003(2):169 －172．
［12］MIAO CB，WAN ZG，SUN BY． Effects of 2，4-D and 6-BA on cal-
lus induction and plantlet regeneration from mature embryos of
hsien rice［J］． Agricultural Science ＆ Technology，2009，10(4):
22 －26．
［13］YUE CJ(岳才军)，HE YP(何彦平)，RUAN HS(阮洪生)，et al．
Extraction and content determination of squalene in Amaranthus
tricolor Calli(苋菜培养细胞中角鲨烯的提取与含量测定) ［J］． Jour-
nal of Anhui Agricultural Sciences(安徽农业科学)，2010，38(10):
146 －147．
［14］WANG KC，LENG C，HUANG WG，et al． Transmission electron
microscopy observation of different callus structure in Heiya no．14
［J］． Agricultural Science ＆ Technology，2009，10(4):125 －127．
［15］CEHG HX(程焕欣)，LIANG YL(梁玉玲)，YAO HB(姚红宝)．
Effect of drought stress on total flavonoids content in Glycyrrhiza
inflata Callus(干旱胁迫对甘草愈伤组织总黄酮含量的影响) ［J］．
Journal of Anhui Agricultural Sciences(安徽农业科学)，2010，38
(11):123 －125．
［16］ZENG SJ，BIAN JC，FANG Z，et al． Callus induction and plant
regeneration from mature seeds of perennial ryegrass［J］． Agri-
cultural Science ＆ Technology，2009，10(6):33 －36．
Responsible editor:LI Ting-ting 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
勋章菊叶片再生技术的探讨(摘要)
李叶峰，盛亚丹，王彩晨，王 宁，陆小平* (苏州大学金螳螂建筑与城市环境学院，江苏苏州 215123)
［目的］研究勋章菊叶片再生技术，筛选适合勋章菊叶片再生的最佳培养配方。
［方法］以日本引进的勋章菊叶片为材料，置床于添加不同种类和浓度激素的 MS培养基中进行愈伤组织和不定芽诱导的正交试验，筛选勋
章菊叶片再生的最佳培养基配方。
［结果］在 MS + TDZ(0． 8 ～1． 0 mg /L)+ NAA(0． 05 ～1． 0 mg /L)培养基上可形成紧密型绿色的愈伤组织;叶片接种于 MS + TDZ(0． 5 ～ 1． 0
mg /L)+ NAA(0． 05 ～1． 0 mg /L)培养基上可形成许多不定芽，诱导效率达 100%;健壮的不定芽长至 2． 0 ～ 3． 0 cm 长时移至 1 /2 MS + NAA
(0． 1 mg /L)培养基中，其发根状况良好，生根率达 100%。
［结论］为勋章菊的高频快繁提供了新途径，并为勋章菊的遗传转化及培育新品种奠定基础。
关键词 勋章菊;愈伤组织;植株再生;苯基噻二唑基脲
基金项目 苏州大学第十二批大学生课外学术科研基金(KY2010114A);苏州市科技支撑(农业)项目(SNG0908)。
作者简介 李叶峰(1986 －)，女，安徽定远人，在读硕士，研究方向:园林植物生理生化方向。* 通讯作者，博士，教授，E-mail:szlxp@ yahoo． com． cn。
收稿日期 2010-03-11 修回日期 2010-05-25
86 Agricultural Science ＆ Technology Vol． 11，No．4，2010