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白檀未成熟胚的器官发生和植株再生(英文)



全 文 :北方园艺2016(05):119~123 ·生物技术·
第一作者简介:杨艳(1980-),女,博士,助理研究员,研究方向为林
木选育与栽培。E-mail:yangyanzupei@126.com.
责任作者:蒋丽娟(1965-),女,博士,教授,现主要从事能源植物培
育与利用等研究工作。E-mail:liljiang1965@sina.com.
基金项目:长沙市科技计划资助项目(kl307012-21)。
收稿日期:2015-10-08
DOI:10.11937/bfyy.201605032
Organogenesis and Regeneration of SymplocospaniculataFrom
Immature Zygotic Embryos in vitro
YANG Yan1,JIANG Lijuan2,TANG Yuxi 1,LI Changzhu1,TANG Jie1,LI Yongjin1
(1.Hunan Forestry Academy,Changsha,Hunan 410004;2.Colege of Life Science and Technology,Central South University of Forestry and
Technology,Changsha,Hunan 410004)
Abstract:Taking immature zygotic embryos of Symplocos paniculata as materials,using modified MS medium,the
organogenesis and regeneration were researched.The results showed that the mMS medium could induce calus greatly,
the highest induction rate 92.5% was obtained on the medium with 0.2mg/L 6-BA+0.1mg/L NAA.72.4%of
caluses initiated green bud-like structures when they were cultured on mMS plus 0.25mg/L 6-BA+0.15mg/L NAA.
The caluses with green bud-like structures were subcultured onto mMS medium without plant growth regulators for 15
days,76.6%of them developed into shoots.Shoots elongated to 1.5cm long on 1/2mMS medium plus 1.5%sucrose 20
days after culture.
Keywords:Symplocos paniculata;immature zygotic embryos;organogenesis;regeneration
中图分类号:S 792.29 文献标识码:A 文章编号:1001-0009(2016)05-0119-05
  Symplocos paniculata (Thunb.)Miq.belongs to
Symplocaceae.It is native in mixed forests at an elevation
of 800-2 500min the Himalayas,and eastern Asia,
including China,Japan,and Korea[1].Sapphire berry is a
deciduous shrub or smal tree with rough yelowish-brown
bark,creamy white fragrant flowers,and blueberry-like
fruits.This plant is known for edible and medicinal usages.
The bark is used for producing a kind of yelow dye[2],
and the juice of the bark is applied externaly to sprains
and muscular swelings[3].The plant has been used in
traditional medicines for the treatment of inflammation[4],
menorrhagia,bowel complaints,eye diseases,ulcers[5],and
typeⅡdiabetes[6].It is also used as a gargle for giving firmness
to spongy and bleeding gums[5].The fruit is edible and
can be used in jams,jelies,and sauce[7].In addition,the
seed has oil up to 36.6% with unsaturated fatty acids,
such as oleic acid and linoleic acid[8].This plant has
recently been investigated as an ideal raw material for
preparation of biodiesel[8-10].The domestication and cultivation
of this wild plant is necessary for protecting the natural
genetic resources from over-exploitation.
Propagation from seed germination requires
stratification[11].It is better to sow seeds in a cold frame
in late winter,and they need 12months to germinate[11].
Seed propagation of sapphire berry is limited due to long
period of stratification,and propagation of a specific cultivar
is impossible because of seedling variability.Semi-hard
wood cuttings,7-10cm long with a heel,can form roots
in about 4weeks and have a good percentage in July or
August[12-13].However,vegetative propagation of sapphire
berry by cuttings may be subject to seasonal variations.
There is an urgent need to develop a stable and eficient
protocol for the large-scale propagation of sapphire berry
in order to meet its ever-increasing demand and conserve
its germplasm and genetic diversity.
Tissue culture provides a fast clonal regeneration for
reforestation projects,nursery production and preservation
of natural genetic resources[14].Tissue culture of Symplocos
tetagona had been conducted[15].They found that the calu-
ses formed on 1/2MS medium supplemented with 2,4-D
(2,4-Dinitrophenylhydrazine)2mg/L when mature zy-
gotic embryos were cultured for 25days,and axilary
shoots developed on WPM medium plus 6-BA or kinetin
(N6 -furfuryladenine)at the concentration of 0.2-
1.5mg/L.However,litle information is available for plant
regeneration of Symplocospaniculata.The aim of this
study is to develop in vitro regeneration protocol for
Symplocos paniculatafrom immature embryos.
911
·生物技术· 北方园艺2016(05):119~123
1 MATERIALS AND METHODS
1.1 Test materials
Green immature fruits(4-5mm in diameter)of
sapphire berry were colected in mid-to-late August
(about 12weeks after polination)from an approximately
20-year-old superior tree grown in Dawei Mountain(Liuyang,
Hunan).
The induction medium consisted of Murashige and
Skoog[16]basal medium,modified MS(mMS)or woody plant
medium[17]supplemented with 30g/L sucrose,0.65% (w/v)
agar,and plant growth regulators.Modified MS(mMS)was
prepared by adding 50mg/L Na2SO4in inorganic salt and
reducing Zn2SO4·7H2O to 8mg/L in trace elements.
1.2 Test methods
1.2.1 Test materials treatment Fruits were thoroughly
washed for 4hours in running tap water.They were sur-
face disinfected for 5minutes with 10%(v/v)commercial
bleach(5.25%sodium hypochlorite)and then rinsed in
distiled water 5-6times.Seeds excised from fruits were
soaked in 75%(v/v)ethanol for 5minutes,and subse-
quently in 50% (v/v)commercial bleach containing 0.1%
Tween-20for 15minutes,and rinsed 5-6times with
sterile distiled water.The pericarp and testa of al seeds
were removed.
1.2.2 Explants inoculation The white immature zy-
gotic embryos(1.5-2.0mm in length)were transferred
to Pyrex glass flasks containing 25mL induction medi-
um.6-BA (6 -benzylaminopurine)at 0.15 mg/L,
0.20mg/L,0.25mg/L or 0.30mg/L plus 0.10mg/L
NAA(α?naphthaleneacetic acid)was tested.Al media
were autoclaved at 121℃for 20minutes after adjusting pH
5.8using NaOH or HCl.Al cultures were maintained
under dark condition in a culture room at(25±2)℃.A
total of 20explants(immature zygotic embryos)were used
for each treatment,and each treatment was repeated 3
times.20days after cultured,the quality and the presence
or absence of caluses were recorded.
1.2.3 Organogenesis and plant regenerationm MS
basic media supplemented with 6 -BA at 0.20 or
0.25mg/L and NAA at 0.10 mg/L,0.15 mg/L or
0.20mg/L were used for calus diferentiation and plant
regeneration.mMS without any plant growth regulators
was prepared as the control.Al medias were added with
30g/L sucrose and 0.65% (w/v)agar,and pH was ad-
justed to 5.8.A total of 20flasks with 5caluses(5mm
in diameter)per flask were used for each treatment,and
each treatment was repeated 3times.30days after cul-
tured under dark condition,the days for caluses to difer-
entiate,and the presence or absence of caluses with green
bud-like structures were recorded.Diferentiated caluses
were cultured on mMS basic medium without phytohor-
mones under the culture room for 15days to elongate the
shoots.Regenerated shoots were transferred to the mMS
basic medium at 1/2mMS plus 15g/L sucrose and kept in
culture under light condition with a 12hours photoperiod
and 50μmol·m
-2·s-1 from cool white fluorescent
tubes.
1.3 Item determination
The percentage of explants with caluses and caluses
with bud-like structures were calculated.The calus induction
rate was calculated using the equation,induction rate(%)=
(explants with caluses/total explants cultured)×100;the
calus diferentiation rate was calculated using the equation,
diferentiation rate(%)= (diferentiated caluses/total
caluses cultured)×100.
1.4 Data analysis
Analysis of variance(ANOVA)was performed using
JMP?9.0(SAS Institute,Inc.,Cary,NC).Tukey’s Honestly
Significantly Diferent(HSD)test at P<0.05was applied for
means separations.
2 RESULTS ADN ANALYSIS
2.1 Calus induction
The immature zygotic embryos began to expand
5days after culture(Fig.1A).Caluses with globular struc-
tures formed on the hypocotyl and radicle of zygotic embry-
os 10days after culture(Fig.1B,C),and the entire embryo
diferentiated to caluses with horn-shaped organogenesis
tissue 15days after culture(Fig.1D,E,F).20days after
culture,the number of immature embryos with caluses
was counted while the color and texture of the caluses
were also recorded(Table 1).
Both basic medium and plant growth regulator
significantly afected calus induction;however,no interactive
efects occurred(Table 1).The majority of immature
embryos cultured on MS (average 64.6%)and mMS
(average 85.1%)media formed caluses,while 32.4%
(average)of immature embryos cultured on WPM media
had caluses.Most of the caluses formed on immature
embryos that cultured on MS and WPM media were watery
or soft in texture.This kind of calus was dificult to further
diferentiate and eventualy died during subculture.
However,the caluses formed on immature embryos that
021
北方园艺2016(05):119~123 ·生物技术·
cultured on mMS medium were usualy loose,milky white
with strong diferentiation capacity.As the concentration
of 6-BA increased,there was a quadratic trend for the
percentage of caluses formed on immature embryos.
When immature embryos cultured on mMS supplemented
with 0.20mg/L 6-BA and 0.10mg/L NAA,the percent
of embryos with caluses was the maximum,92.5%.
  Note:A,Caluses induced from immature zygotic embryo 5days after culture;B and C,Globular caluses formed and proliferated 10days after culture;D,E
and F,Leaf primordia initiated 15days after culture;G,Light green leaf primordia developed on the mMS basal medium containing 0.25mg/L 6-BA and 0.15
mg/L NAA;H,Green shoots regenerated on mMS mediums without plant growth regulators for 15d;I,Shoots elongated on 1/2mMS medium plus 1.5%su-
crose 20days after culture.
Fig.1 Regeneration of Symplocs paniculatafrom immature zygotic embryo via organogenesis
  Table 1 Calus induction of Symplocos paniculata on different media supplemented with 6?BA and NAA
Basal medium Concentration of 6-BA/(mg·L-1)Concentration of NAA/(mg·L-1)Induction rate of explants/% Calus morphology
0.15  0.10  50.1±0.3gd  Yelowish white,watery texture
MS  0.20  0.10  73.2±1.3c Green,firm texture
0.25  0.10  69.8±2.4c Green with white tissue scattered on the surface,soft texture
0.30  0.10  65.3±0.9c White,watery texture
0.15  0.10  81.2±1.0bc  Light yelow,firm texture
 
mMS
0.20  0.10  92.5±0.9a Milky white,loose texture with granular tissue
0.25  0.10  86.3±0.8ab  Milky white,soft texture
0.30  0.10  80.3±0.4bc  Yelowish green,soft texture
0.15  0.10  21.2±0.6f Milky white,watery texture
 
WPM
0.20  0.10  37.9±0.5e White with slightly green,soft texture
0.25  0.10  37.0±0.3e White,soft texture
0.30  0.10  33.5±0.9e Brown,watery texture
  Note:Diferent lowercase letters in the same column show significant diference at 0.05level.The same below.
121
·生物技术· 北方园艺2016(05):119~123
2.2 Organogenesis and plant regeneration
Milky white caluses in loose texture were subcultured
(Table 2).It took 18days for the caluses that cultured on
the medium containing 0.25mg/L 6-BA and 0.15or
0.20mg/L NAA to diferentiate into green bud-like
structures(Fig.1G),whereas 22days for those on the me-
dium plus 0.20mg/L 6-BA and 0.10mg/L,0.15mg/L
or 0.20mg/L NAA.However,there was no sign of dif-
ferentiation of caluses observed on the medium without
any hormones.This data was excluded for analysis.The
percent of caluses that diferentiated into bud-like struc-
tureswas significant among 6-BA levels,NAA levels,and
the interactive efects were also significant(Table 2).The
percent of caluses with bud-like structures responded
quadraticaly to the increased 6-BA.Regardless of the
NAA concentration,the percent of caluses with bud-like
structures was 2-3times higher,compared to those cul-
tured on the medium with 0.2mg/L 6-BA.The maximum
value of diferentiation rate was 72.4% when the calu-
ses were cultured on 0.25mg/L 6-BA and 0.15mg/L
NAA. 
The caluses with green bud-like structures were
subcultured on to mMS medium without plant growth
regulators for 15days.A total of 76.6%of caluses with
bud-like structures developed into shoots(Fig.1H).They
were then transferred on to 1/2mMS medium plus 1.5%
sucrose.Shoots elongated,and the height of shoots was
1.5cm after 20days of culture(Fig.1I).
Table 2 Symplocos paniculatacaluses differentiated on modified
MS supplemented with 6?BA and NAA
Concentration of 6-BA
/(mg·L-1)
Concentration of NAA
/(mg·L-1)
Time of diferentiation
/d
Rate of caluses
with shoot/%
0.20  0.10  22  23.3±1.5e
0.20  0.15  22  25.7±1.8d
0.20  0.20  22  25.6±0.8de
0.25  0.10  22  66.0±1.5b
0.25  0.15  18  72.4±1.0a
0.25  0.20  18  57.9±0.6c
3 CONCLUSION
In conclusion,caluses were induced from immature
embryos cultured on mMS supplemented with 0.2mg/L
6-BA and 0.1mg/L NAA with calus induction percent-
age of 92.5%.The induced caluses were cultured on
mMS plus 0.25mg/L 6-BA and 0.15mg/L NAA,and
72.4% of the caluses differentiated into green
bud-like structures.These tissues were subcultured on
to mMS medium without plant growth regulators for
15days,76.6% of which developed into shoots.Shoots
elongated to 1.5cm long on 1/2mMS medium plus
1.5%sucrose 20days after culture.Shoots were stil un-
der test to develop roots.This protocol might provide a
new approach for the rapid propagation of symplocos
paniculata. 
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[6] NA M K,YANG S,HE L,et al.Inhibition of protein tyrosine phosphatase
1Bby ursane-type triterpenes isolated fromSymplocos paniculata[J].Planta
Med,2006(72):261-263.
[7] FACCIOLA S.Cornucopia II:a source book of edible plants[M].Vista:
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[8] LIU Q,YANG Y,YIN X,et al.Fruit morphological development of oil
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[9] China Oil Plant Editorial Commitee.Chinese oil plant[M].Beijing:Science
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[11]BEAN W J.Trees and shrubs hardy in great britain[M].London:Cam-
bridge University Press,1919.
[12]HUXLEY A,GRIFFITHS M,LEVY M.The new royal horticultural
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221
北方园艺2016(05):123~127 ·生物技术·
作者简介:鲁京慧(1967-),女,河北承德人,本科,实验师,现主要从事
园林工程及园林史的教学与科研工作。E-mail:lsz520999@126.com.
收稿日期:2015-06-01
DOI:10.11937/bfyy.201605033
云南栽培金盏花遗传多样性RAPD分析
鲁 京 慧
(河北旅游职业学院 园艺系,河北 承德067000)
  摘 要:用采自云南昆明、曲靖和玉溪等地的8份金盏花种质资源为试材,利用改良CTAB
法提取DNA,筛选引物,优化PCR反应体系,对其进行遗传多样性RAPD(random amplified
polymorphic DNA)和NTSYS2聚类分析,以建立金盏花亲缘关系分析方法,明确分布在云南省各
地的金盏花种质资源之间的亲缘关系和遗传多样性。结果表明:优化的PCR反应体系能达到良
好的扩增效果,30条RAPD引物在8份金盏花种质资源中共产生165个位点,其中45个位点具
有遗传多态性,约占27.28%。CK与7号的相似性最小,用CK和7号作为亲本的后代会有较大
的分离。试验表明金盏花种植资源遗传多样性丰富,遗传关系与其地理关系密切相关,但并非严
格的限聚在一起,需结合表型更深入地分析。
关键词:金盏花;RAPD;遗传多样性
中图分类号:S 681.703.6 文献标识码:A 文章编号:1001-0009(2016)05-0123-05
  金盏花(Calendula officinalis L.)属菊科金盏菊属
一年生草本植物,又名金盏菊、山金菊等,适合于我国大
多数地区栽培,具有较高的经济价值。首先作为园林植
物中最为重要的观赏花卉之一,其在城市园林造景,局
部区域美化中具有独特作用,其次以金盏花的植株和花
为原料可提取叶黄素和精油,不但是化妆品中一种天然
的、具有良好抗氧化功能的原料,还可用于治疗多种皮
肤疾病,如外伤、皮疹、皮肤破裂等,具有抗感染和杀菌
消炎等功效[1]。近些年来,国内外学者主要开展金盏花
    
授粉结实和环境条件的关系[2]、水肥措施对经济性状的
影响[3]、盐胁迫对金盏花生长和逆境的生理响应机制[4]、
雄性不育两用系选育及植物学特性研究[5],而关于金盏
花种质资源的研究尚鲜见报道。
分子标记技术是分析种质亲缘关系的有力工具,其
中RAPD分子标记是建立在PCR基础之上。用筛选的
有10个碱基的单链随机引物,对基因组DNA进行PCR
扩增以检测其多态性位点,具有操作简单、无需专门设
计引物、对模板DNA质量要求不高、不受材料生长情况
限制,能反应材料的遗传信息差异等优点;虽然RAPD
分子标记有重复性不佳的缺点,但可以通过改良PCR反
应体系加以改善[6]。已有学者为了在金盏花雄性不育
的育种过程中使用RAPD分子标记技术,通过条件优化
  
白檀未成熟胚的器官发生和植株再生
杨   艳1,蒋 丽 娟2,汤 玉 喜1,李 昌 珠1,唐   洁1,李 永 进1
(1.湖南省林业科学院,湖南 长沙410004;2.中南林业科技大学 生命科学院,湖南 长沙410004)
  摘 要:以白檀未成熟胚为试材,以改良 MS为培养基,研究了白檀未成熟胚的器官发生和植株再生。结果表
明:改良 MS(mMS)能够很好的诱导愈伤组织,在添加了0.2mg/L 6-BA+0.1mg/L NAA的培养基中,诱导率
高达92.5%;胚性愈伤组织分化最佳培养基为mMS+0.25mg/L 6-BA+0.15mg/L NAA,分化率为72.4%;分化
出的胚性愈伤组织在空白mMS培养基上继代培养15d,76.6%的组织分化出芽,再转入1/2mMS+1.5%蔗糖培养基
上培养20d,芽长至1.5cm。
关键词:白檀;未成熟胚;器官发生;植株再生
321