全 文 ：第 45 卷 第 2 期
2 0 0 9年 2 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol.45 , No.2
Feb., 2 0 0 9
爬行卫矛下胚轴高频离体再生体系的建立＊
尚爱芹1 , 2　孙振元3　赵梁军1
(1.中国农业大学农业与生物技术学院　北京 100094;　2.河北农业大学园艺学院　保定 071001;
3.中国林业科学研究院林业研究所　国家林业局林木培育重点实验室　北京 100091)
关键词:　爬行卫矛;直接器官再生;组织学观察;离体培养;木本观赏植物
中图分类号:S718.46;Q943.1　　　文献标识码:A　　　文章编号:1001-7488(2009)02-0136-06
Received on February 2, 2008.
Foundat ion project:This research was supported by Beijing Municipal Science and Technology Commission(H020620110130).
＊ Author for correspondence:Zhao Liangjun.
High Efficient Regeneration in Vitro from Hypocotyl of Euonymus fortunei var.radicans
Shang Aiqin1 , 2　Sun Zhenyuan3　Zhao Liangjun1
(1.College of Agriculture and Biotechnology , China Agricultural University　 Beijing 100094;　2.College of Horticulture ,
Hebei Agricultural University　 Baoding 071001;　 3.Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration　
Research Institute of Forestry , Chinese Academy of Forestry　 Beijing 100091)
Abstract:　In this paper , a protocol for efficient shoot regeneration was successfully developed from hypocotyl explants of
Euonymus fortunei var.radicans.Some factors that influenced shoot regeneration such as different combinations of plant growth
regulators, types of medium and inoculation ways were studied in order to establish an efficient plant regeneration for
transformation.The results showed that hypocotyl explants were horizontally cultured on a basic medium composed of MS medium
supplemented with 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA for induction and development of adventitious shoots.Ninety-four
percent of regeneration frequency and 5.1 shoots per explants were obtained after 30 days of culture.Regenerated shoots
proliferated efficiently on a shoot multiplication medium consisting of MS medium containing 1.0 mg·L-1 BAP and 0.1 mg·L-1
NAA.Microshoots were rooted on a rooting medium made up of MS medium enriched with 0.5 mg·L-1 IBA and 0.5 mg·L-1
IAA.After hardening , 90% of plants were successfully established under greenhouse conditions.Histological observation
revealed that shoot primordium originated from subepidermal cells of hypocotyl explants and directly developed into adventitious
shoots without callus formation.
Key words:　Euonymus fortunei var.radicans;direct organogenesis regeneration;histological observation;in vitro culture;
woody ornamental plant
　　Euonymus fortunei var.radicans , an indigenous
species of China , is a climbing , everygreen-woody plant.
Having several desirable traits such as tolerance to sulfur
oxids and dioxide , and wide adaptability to diverse
ecological conditions(Pan et al., 2004), this species is
widely grown as an ornamental plant in temperate and
subtropical areas of China.Euonymus plants are often
seriously destructed by aphis (Hodgson et al., 1999).
E .fortunei var.radicans is often attacked by aphids
(Myzus persicae)and coccid(Ceroplastes japonicus)too.
Chemical treatment not only costs much but also leads to
environmental pollution.Novel cultivars bred for insect
resistance would greatly improve the usefulness of E.
fortunei var.radicans.
Genetic improvement of woody plants by conventional
methods is constrained by their long juvenile period ,
complex reproductive biology and high degree of
heterozygosity (Vigouroux , 1992). Plant genetic
transformation may help to solve these problems by
introducing desirable traits into these species.To perform
such a transformation , establishment of an efficient in
vitro plant regeneration system must be developed.
Explant was one of the most important factors that
influenced the efficient in vitro plant regeneration system.
The types of explant , sampling time , explant age and
polarity were all limiting factors to shoot regeneration.In
Euonymus fortunei efficiency of adventitious shoots
induction from stem was higher than that from leaf disc
(Yin et al., 2004), but the regeneration frequency of
adventitious shoots from hypocotyls was higher than that
from stem(Wang et al., 2004).In pre-experiments , we
found the shoots regeneration frequency from leaf disc was
lower than that from hypocotyl(data not shown).Against
these results , we used hypocotyl as explant to establish
high efficient regeneration system of E .fortunei var.
radicans.
Hypocotyl has been proven to be one of the most
often employed explants for shoot regeneration in many
woody species , such as Liquidambar styraciflua (Kim et
al., 1997), Eucalyptus nitens and E. globulus
(Bandyopadhyay et al., 1999), Psidium guajava(Singh
et al., 2002), Annona squamosa (Nagori et al.,
2004), Euonymus japonicus `Cuzhi (Shang et al.,
2005)and Feronia limonia (Vyas et al., 2005).To the
best of our knowledge , shoot regeneration from hypocotyl
of E.fortunei var.radicans has never been reported.
　　Direct shoot regeneration system has been used to
genetic transformation.Cui et al.(2004)reported that
the direct shoot regeneration system was successfully used
to achieve the transformation of Antirrhinum majus in a
short time period.So the goal of the study reported here
was to establish an efficient plant regeneration system from
hypocotyls segments of E.fortunei var.radicans , which
could be employed for its transformation manipulation.
1　Materials and methods
1.1 　Seed germination 　Seeds of E.fortunei var.
radicans collected during October , 2003 from
Experimental Station of Chinese Academy of Forestry ,
Beijing , China were surface sterilized with 70% ethanol
for 1 to 2 min and then treated with HgCl2 for 20 min ,
following by 4 or 5 times of washing in sterile distilled
water.Sterilized seeds were inoculated on a basic medium
(BM)composed of Murashige and Skoog (1962)(MS)
medium containing 3% sucrose and 0.6% agar (w v).
The medium pH was adjusted to 5.8 prior to autoclaving
at 121 ℃ for 15 min.
1.2 　 Shoot regeneration 　Four weeks after seed
germination , hypocotyl explants (each 5 to 6 mm in
length)were excised and used for shoot regeneration.
Otherwise stated elsewhere , the explants were cultured
horizontally on a shoot regeneration medium (SRM)
composed of BM containing 0.5 mg·L-1 BAP and 0.01
mg·L-1 NAA for 4 weeks under the culture conditions as
described for seed germination.After 30 days of culture ,
shoot regeneration was evaluated by regeneration
frequency and mean number of shoots per explant.
Regeneration frequency was defined as percentage of
explants producing shoots longer than 5 mm.In this
stage , 3 experiments were designed to optimize parameters
for shoot regeneration.In the first experiment , the
explants were cultured horizontally on BM supplemented
with various concentrations and combinations of 6-
benzylaminopurine (BAP), kinetin (KT), thidiazuron
(TDZ)and naphthaleneacetic acid(NAA)(Tab.1).In
the second experiment , the explants were cultured
horizontally on 5 different media:BM , half-strength BM ,
B5(Gamborg et al., 1968), salts of MS plus B5 s
vitamines(MB)and woody plant medium(WPM)(Lloyd
and McCown , 1980).All tested media contained 0.5
mg·L-1 BAP and 0.01 mg·L-1 NAA.In the third
experiment , effect of orientation of explants on SRM on
shoot regeneration was investigated.Explants were placed
horizontally , vertically and vertically inverted ,
respectively , on SRM.
1.3 　 Histological observation 　Hypocotyl explants
cultured on SRM for various durations(4 , 7 and 11days)
were collected , fixed in formalin∶acetic acid∶alcohol ,
1∶1∶18 , for 24 h , dehydrated through an incremental
ethanol series(50%, 70%, 85% and 95%)and stored
in 100% ethanol.After embedding in paraffin , 10-μm-
thick sections were cut by a microtome and stained
(Jensen , 1962)with 1% safranine and 1% fast green.
The sections were observed with an Olympus optical
microscope.
1.4　Multiplication of shoots　Regenerated shoots (2
to 3 cm long) were separated from explants and
subcultured onto a shoot proliferationmedium composed of
BM containing 1.0 mg·L-1 BAP and 0.1 mg·L-1 NAA
for shoot multiplication.Culture conditions were the same
as for seed germination.Subculture was performed every 4
weeks.
1.5　 Rooting and acclimatization 　Otherwise stated
elsewhere , proliferating shoots about 2 to 3 cm in length
were excised and transferred to half-strength MS
supplemented with 100 mg·L-1 activated charcoal(AC),
0.5mg·L-1 IBA and 0.5 mg·L-1 IAA , to induce root
formation.One experiment was carried out to test effect of
137　第 2期 尚爱芹等:爬行卫矛下胚轴高频离体再生体系的建立
concentrations and combinations of auxin on root formation
(Tab.4).Percentage of rooting and root number per
shoot were recorded after 30 days of culture.Culture
conditions were the same as for seed germination.
Plantlets with well-developed roots were removed from
culture bottles and washed free of agar.After then , they
were grown into black plastic pots containing a mixture of
greenhouse compost at 24 ℃under dim light conditions
and covered with a plastic bag to maintain high humidity
and prevent plantlets from wilting.After 2 weeks , the
plastic bag was gradually opened to reduce humidity.
After 4 weeks of hardening , the plants were moved to
greenhouse conditions.Survival of plants was recorded
after 4 weeks of transfer to greenhouse.
1.6　 Data analysis　In all experiments , at least 100
explants were included in each of treatment of three
replicates , and each experiments were carried out twice.
Results were described by percentages of hypocotyl
segments that regenerated shoots(regeneration frequency)
(means±SE)and the number of shoots per regenerating
explant (means ±SE).Results were analyzed with
ANOVA and Duncan s multiple range test(P =0.05)
using the Statistical Analysis System package (SAS
Institute v.9.1).
2　 Results
2.1　 Influence of plant growth regulators on shoot
regeneration　Of the three cytokinins tested , BAP was
most effective in induction of adventitious shoots , while
TDZ completely failed to induce shoot regeneration ,
regardless of its concentration and combination with NAA
(Tab.1).TDZ at 0.5 ～ 2.0mg·L-1 alone or combining
with NAA at 0.01 mg · L-1 promoted only callus
formation.Although either BAP or KT alone was able to
induce formation of adventitious shoots , regeneration
frequency was low:15%～ 40%.Treatments combining
BAP with NAA significantly improved shoot regeneration
with the best results(92% of regeneration frequency and
4.2 of shoots per explant)obtained using 0.5 mg·L-1
BAP and 0.01 mg·L-1 NAA (Tab.1).Compared with
the other treatments by SSR , the results were significant.
Therefore , shoot regeneration medium containing 0.5 mg·
L
-1
BAP and 0.01 mg·L-1 NAA was always used in all
the following experiments.
2.2　 Influence of the media on shoot regeneration　
Tab.1　Shoot regeneration from hypocotyl explants of
E.fortunei var.radicans cultured on BM medium
supplemented with various concentrations and combinations
of BAP , TDZ , KT and NAA①
Treatment
(mg·L-1)
Cytokinins Auxin
Number
of
explants
Number
of
dif feren-
tiation
Regeneration
frequency %
Mean number
of shoots
per explant
BAP NAA
0.5 0 106 42 40±3 c 1.8±0.2 d
0 0 112 38 34±5 d 1.2±0.1 e
0.5 0.01 116 107 92± 2 a 4.2±0.2 a
1.0 0.01 110 80 73±6 b 3.1±0.1 b
2.0 0.01 100 71 71±5 b 2.3±0.2 c
TDZ NAA
0.5 0 102 0 0 h 0 f
1.0 0 106 0 0 h 0 f
0.5 0.01 110 0 0 h 0 f
1.0 0.01 102 0 0 h 0 f
2.0
KT
0.5
1.0
0.5
1.0
2.0
0.01
NAA
0
0
0.01
0.01
0.01
100
107
110
104
108
102
0
16
22
22
31
29
0 h
15±3 g
20±5 f g
21± 6 f
29± 0 d e
28± 5 e
0 f
1.1± 0.1 e
1.2± 0.1 e
1.1± 0.1 e
1.2± 0.1 e
1.1± 0.1 e
　　① Data were recorded after 4 weeks of culture and presented as means
± SE.Data followed by the same letters in the same column were not
significantly different at P = 0.05 by Duncan s multiple range test.The
same below.
Although hypocotyl explants inoculated on all the media
tested were able to regenerate adventitious shoots , optimal
regeneration frequency(92%)and highest number(4.6)
of shoots per explants were obtained when MS medium
was used (Tab.2).This result was not significant
difference compared to that of B5 and WPM media , but
there was a significant difference compared to that of
1 2MS and MB media.Based on these results , MS was
consistently adopted in the following experiments.
Tab.2　The effect of different media on shoot regeneration
from hypocotyl explants of E.fortunei var.radicans ①
Medium
Number
of
explants
Number
of
diff eren-
tiat ion
Regenera-
tion
frequency %
Mean number
of shoots
per explant
MS 100 92 92±2 a 4.2±0.2 a
1 2MS 100 60 60±8 c 1.6±0.2 bc
B5 100 86 86±4 ab 1.5±0.1 c
MB 100 73 73±6 bc 1.9±0.2 b
WPM 100 80 80±7 abc 1.4±0.3 c
　　① All media tested contained 0.5 mg·L-1 BAP and 0.01 mg·L-1
NAA.
2.3 　 Influence of explant orientations on shoot
regeneration 　 All three orientation treatments of
138 林 业 科 学 45 卷　
hypocotyl explants on the medium were able to produce
adventitious shoot formation. Both horizontal and
vertically upright orientation on the medium produced
comparable and significantly high shoot regeneration ,
compared with vertically inverted orientation (Tab.3).
When the explants were vertically inverted on the
medium , callus formation was found at both proximal and
distal parts , while adventitious shoots were regenerated
only at distal part of the explants (Plate Ⅰ-1).The
explants that had been vertically cultured with upright
position on the medium produced both callus and
adventitious shoots only at distal part(Plate Ⅰ-2).The
explants , when placed horizontally on the medium ,
developed adventitious shoots at all parts of explants
(Plate Ⅰ-3).
Tab.3　Influence of explant orientation on shoot regeneration
of hypocotyl explants of E.fortunei var.radicans ①
Orientation of
explants
Regeneration
frequency %
Mean number of
shoots per explant
Horizontally 92±2 a 4.2±0.2 a
Vertically upright 94±5 a 5.1±0.2 a
Vertically inverted 52±2 b 2.3±0.3 b
　　①Medium used contained 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA.
2.4 　 Histological observation 　When hypocotyl
explants were cultured on SRM for 4 days , small and
green protuberances were observed.These protuberances
eventually developed into adventitious shoots in about 11
days without callus formation.Histological studies
revealed that adventitious shoots directly generated from
subepidermal cells of hypocotyl explants without a phase
of callus formation.Meristematic activities occurred in
subepidermal cells of the hypocotyl explants after 4 days
of culture (Plate Ⅰ-4).Continuous division of these
subepidermal cells formed meristematic nodules after 7
days of culture (Plate Ⅰ-5).Further differentiation of
these meristematic cells eventually led to formation of
well-developed bud primordium after 11 days of culture
(Plate Ⅰ-6).Leaves developed directly from both sides
of the shoot bud after 15 days of culture (Plate Ⅰ-7).
2.5　Multiplication of shoots and establishment of
rooted cuttings in the greenhouse　When adventitious
shoots produced on the hypocotyl explants reached 2 ～ 3
cm in length , they were excised from the parent culture
and transfered to BM containing 1.0 mg·L-1 BAP and
0.1 mg·L-1 NAA for multiplication.After 30 days of
culture , a 3 ～ 5 multiplication rate was obtained (Plate
Ⅰ-8).A further increase in BAP concentration reduced
multiplication rate , while an increased NAA promoted
callus formation (data not shown).Treatments of either
IBA(0.5 ～ 2 mg·L-1)or IAA (0.5 ～ 2.0 mg·L-1)
were able to induce root formation of microshoots(Tab.
4).There was no difference of concentrations in root
formation.The best results of root formation (91% of
rooting percentage , 3.6 of mean root number and 11 mm
of root length)were obtained using a treatment combining
0.5 mg·L-1 IBA and 0.5 mg·L-1 IAA (Plate Ⅰ-9).
Ninety percent of plants survived hardening and were
successfully established under greenhouse conditions.
Tab.4　Effect of auxin treatment on rooting of microshoots of
E.fortunei var.radicans
Treatment (mg·L -1)
IBA IAA
Root ing
percentage %
Mean number
of roots
Mean length of
roots mm
0.5 0 64±5 cd 2.6±0.2 b 8± 1.0 b
1.0 0 72±3 bc 2.5±0.3 bc 7± 1.0 bc
2.0 0 64±5 cd 2.7±0.1 b 7± 1.0 bc
0.5 0.5 91±2 a 3.6±0.2 a 10±1.0 a
1.0 0.5 79±6 b 2.1±0.2 cd 8±0.5 b
0 0.5 71±8 bc 2.5±0.4 bc 6±1.0 cd
0 1.0 65±5 cd 2.0±0.1 d 5±0.5 de
0 2.0 60±2 d 2.0±0.2 d 4±0.2 e
3　Discussion
In previous studies , the role of BAP in adventitious
shoot bud differentiation has been demonstrated in a
number of cases using different explants.With Annona
squamosa , Nagori et al.(2004)compared effects of 3
cytokinins on adventitious shoot formation of hypocotyl
explants , and found that the BAP was much more
effective than TDZ and KT in shoot regeneration.Similar
results of effects of BA on shoot regeneration from
hypocotyl explants were also observed in Feronia limonia
(Vyas et al., 2005)and Sesbania rostrata (Jha et al.,
2002).Although BA alone was able to induce shoot
regeneration , a combination of BA and auxin frequently
evoked a significant response of treated explants to
adventitious shoot formation.Vyas et al.(2005)
reported in Feronia limonia , the response of medium
containing 4.4 μmol·L-1 BA and 0.05 μmol·L-1 NAA
was definitely better than that obtained with individual
BA.It was also reported that the effects of BA and NAA
on direct adventitious shoot bud formation were highly
significant in Phragmites communis(Guo et al., 2004).
A recent study by Shang et al.(2005)on regeneration
139　第 2期 尚爱芹等:爬行卫矛下胚轴高频离体再生体系的建立
from hypocotyl of Euonymus japonicus belonging to the
same genus of E.fortunei var. radicans , also
demonstrated a combination of BA at 1.5 mg·L-1 and
NAA at 0.05 mg·L-1 resulted in the highest frequency of
shoot regeneration.Results of the present study showed
that addition of BAP to the shoot regeneration medium was
able to induce adventitious shoot formation , and it was
more effective than other cytokinins.We also observed
that a combination of BAP (0.5 mg·L-1)with NAA
(0.01 mg·L-1)significantly increased shoot regeneration
of hypocotyl explants.These results were consistent with
those of Nagori et al.(2004), Jha et al.(2002), Vyas
et al.(2005), Guo et al.(2004), and Shang et al.
(2005).
TDZ has been reported to promote adventitious shoot
formation from hypocotyl explants of several plant species
such as Liquidambar styraciflua (Kim et al., 1997),
Linum (Mundhara et al., 2002)and Psidium guajava
(Singh et al., 2002).In the present study , treatments
using TDZ alone or combining with NAA completely failed
to develop adventitious shoots , but only induce callus
formation.Similar results were reported by Nagori et al.
(2004), who used hypocotyl segments of Annona
squamosa.These data again reflected that effect of TDZ
on organogenesis depended on its concentration and plant
species.
Orientation of explants on the medium was reported
to influence adventitious shoot formation.Changing the
orientation of explants from vertically upright to horizontal
drastically decreased the frequency of shoot induction as
well as the length of the shoot apical end (Saini et al.,
2002).In a study of Vigna mungo (Saini et al.,
2002), epicotyl explants inserted vertically in the medium
or placed horizontally on the medium formed adventitious
shoots , while all explants inserted in an inverted position
did not regenerate shoots , but developed callus.Saini et
al.(2002) suggested that the orientation might affect
shoot regeneration by interacting with polarity of explants.
Results from the present study also demonstrated that
orientation of explants on the medium influenced
adventitious shoot formation.
Type of medium had an effect on the adventitious
shoot bud regeneration.In sweet and sour cherry(Prunus
avium), Tang et al.(2002) reported that shoot
regeneration was more efficient when using WPM medium
as their basal medium than MS and other media.
Whereas , with Annona squamosa , Nagori et al.(2004)
found the highest frequency of shoot regeneration was
obtained on B5 medium , the maximum number of shoot
buds per explant was obtained on MS medium.MS was
more suitable for shoot regeneration from hypocotyl
explants of Euonymus japonicus (Shang et al., 2005).
Results from the present study showed that MS was more
efficient than any other media.
In conclusion , a highly efficient protocol for shoot
regeneration of hypocotyl explants of E.fortunei var.
radicans was developed in the present study.With the
optimized parameters , 94%of regeneration frequency and
5.1 of shoots per explant were achieved.The regenerated
shoots were micropropagated , rooted and finally
established under greenhouse conditions.
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141　第 2期 尚爱芹等:爬行卫矛下胚轴高频离体再生体系的建立