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蔷薇属植物无菌体系的建立(英文)



全 文 :Development of an in vitro culture system of Rosa
spp. Plants
GUO Yan-chao1,2,ZHAO Liang-jun2*
1. Coastal Agriculture Institute,Hebei Academy of Agriculture and Forestry Sciences,Tangshan 063200;2. China Agricultural University,
Beijing 100193
Abstract [Objective]This study was to develop an in vitro tissue culture system of Rosa spp.[Method]Using ten species of Rosa spp. plants
as experimental materials,different combinations of hormones were designed to establish their in vitro tissue culture system with the stem seg-
ments as explants. [Result]All ten tested varieties germinated when the nodal segment explants were cultured on the sprouting medium MS +
0. 5 mg/L BA +0. 01 mg/L NAA and grew vigorous shoots,and the sprouting rate was up to 70%. Of the ten tested rose varieties,each has a
respective optimal proliferation medium,and the multiplication rates for all the varieties reached 3. 0%. The axillary buds were vigorous and nor-
mal in leaf color. The optimal medium for rooting and acclimation was 1 /2MS medium containing 0. 1 mg /L or 0. 2 mg /L NAA,in which the roo-
ting frequency reached 90% -100% and the root system was developed. After acclimation and transplant,the survival rate was as high as 95%.
[Conclusion]An in vitro tissue culture system of Rosa spp. has been established in this study,which lays foundation for the molecular breeding
of Rosa spp.
Key words Rosa spp. plants;Tissue culture
Received:August 12,2011 Accepted:September 5,2011
Supported by National Natural Science Foundation of China
(30871733).
* Corresponding author. E-mail:zhaolj5073@sina. com
As one of the native producing regions,China has abun-
dant Rosa spp. resources,accounting for 41% of total world-
wide resources[1]. Due to the characteristics of multiform vari-
eties,pealike flower color,diversiform flower types,specific
flower aroma,Rosa spp. plants have become preferred mate-
rial for the greening of courtyard and garden. Presently,Rosa
spp. plants breeding is one of the most hotspots of flower
breeding,and the breeding targets are mainly involved in the
improvement in flower color,flower aroma,flower type,flow-
ering habit,fresh cut flower life,pole type and disease resist-
ance[2 -3]. By directly manipulating target gene,molecular
breeding technique possesses the advantage of solely impro-
ving a single trait while maintaining other traits unaltered.
Thus,molecular breeding technique has been more and more
used in improving ornamentals,displaying huge application
potential in the genetic improvement of Rosa spp. plants.
Establishment of tissue culture and genetic transformation
system of Rosa spp. plants is the precondition for breeding
using somatic cloning variation and gene engineering[4]. A
number of reports have focused on the in vitro tissue culture,
root induction and transplant of Rosa spp. plants[5 -8]. Howev-
er,different Rosa spp. varieties vary in tissue culture charac-
teristics because of the impact of genotype[5]. Therefore,in-
vestigation on the tissue culture conditions,especially optimal
hormone combinations in medium for different varieties is pro-
vided with important value.
Materials and Methods
Experimental materials
Two China rose varieties(Rosa White Meidilan and Rosa
carefully) and eight Rosa spp. varieties(Rosa. multiflora
‘inermis3’,Rosa. multiflora‘inermis4’,Rosa. multiflora‘in-
ermis11’, R. multiflora f. carnea, Rosa odorata, Rosa
wichuraiana,Rosa gallica and Rosa indica)as experimental
materials were collected,bred and planted in Ornamental Hor-
ticulture Parterre of China Agricultural University.
Disinfection of explants
Explant disinfection was referred to the method proposed
by CUI Guang-rong et al[9]. Middle-upper portion of lignifying
one year old tresses were cut from plants growing on bared
land,and rinsed in proper concentration of detergent solution
with a soft brush after removing the leaves and stipules;the
tresses were then cut into 4 -5 cm long stem segments and
placed into a one liter beaker which was then covered with
gauze;the beaker was flushed with running water for 20 min;
the stem segments were next cut into smaller segments each
containing a single bud,and placed in a sterilized plastic bot-
tle with suitable double distilled water for test. These explants
were surface-disinfected in laminar flow cabinet as following
conditions:disinfect in 70% ethanol for 30 s,rinse in double
distilled water for three times,immerse in 0. 1% HgCl(w/v)
for 8 min,rinse in double distilled water for six times,place in
sterilized Petri dishes for slightly drying for test.
Axillary bud sprouting and subculturing for proliferation
The stem segments disinfected were plated on the sprou-
ting medium MS +0. 5 mg/L BA + 0. 1 mg/L NAA to induce
the emergence of axillary buds. The sprouting medium formu-
la was referred to literatures related[10 -11]. Each treatment
was replicated for three times and each contained ten stem
segments. The sprouting rate was recorded 20 days later.
When the axillary buds grew to 1 cm in length,they were
cut and inoculated on proliferation medium for multiplication.
The hormones and concentrations tested listed in Table 1
were referred to literatures[9 -10,12 -13]. Each treatment
was replicated for three times and each contained three ex-
plants. Growth of materials on various proliferation media with
different hormone combinations was observed and classified.
Rooting,acclimation and transplant
Related researches usually chose 1 /2 MS as basic medi-
um,appended with low concentration of auxin NAA or cytokin-
in BA,to induce root growth. In this study,three concentra-
Agricultural Science & Technology,2011,12(10):1433 -1436,1448
Copyright 2011,Information Institute of HAAS. All rights reserved. Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2011.10.031
tions (0.1,0.2 and 0. 5 mg/L)of auxin NAA were designed
to optimize the rooting medium. Vigorous roots were trans-
ferred to rooting medium for recording the rooting rate 25 days
later. Each treatment was replicated for three times and each
contained three plantlets.
Table 1 Combinations and concentrations of plant growth regulators
tested in proliferation medium mg/L
Medium code
Combination and concentration
BA NAA GA3
1 0. 5 0. 010 0
2 0. 5 0. 050 0
3 0. 5 0. 100 0
4 0. 8 0. 010 0
5 0. 8 0. 050 0
6 0. 8 0. 100 0
7 1. 0 0. 010 0
8 1. 0 0. 050 0
9 1. 0 0. 100 0
10 0. 5 0. 005 0. 1
11 0. 5 0. 010 0. 1
12 0. 8 0. 005 0. 1
13 0. 8 0. 010 0. 1
14 0. 8 0. 050 0. 1
15 1. 0 0. 005 0. 1
16 1. 0 0. 010 0. 1
17 1. 0 0. 100 0. 1
The vigorous plantlets with two to five roots each longer
than 1 cm,were used for acclimation by opening the bottle.
Seven days later,the plantlets were taken from the medium
and the rudimental medium around the roots was removed by
washing with running water. The plantlets were then trans-
planted to nutritional pots with sterilized matrices sturfy soil
and vermiculitm (m∶m =1∶1)and cultured in HPG-400H illumi-
nator under following conditions:light intensity of 10 000 lx,pho-
toperiod of 14 h day /10 h night,temperature of 21 ℃ day/16 ℃
night,humidity of 60%. The survival rate was recorded two
weeks later.
Culture conditions
MS medium,appended with 3% sucrose,0. 6% agar
(pH 5. 96)was used as basic medium in this study. Except
GA3 was filter-sterilized,all other plant growth regulators were
added into medium and autoclaved along with medium at 121
℃ for 15 min. The growing conditions were temperature of
(25 ±2)℃,light intensity of 50 μmol /m2·s and photoperiod
of 16 h day /8 h night.
Results and Analysis
Results of axillary bud sprouting and induction
Most of the varieties began to grow axillary buds four to
seven days after being placed on sprouting medium,though
they differ in sprouting time. Ten days later,the axillary buds
grow up to be young seedlings. With the culture duration las-
ted,the young seedlings grew higher day by day and genera-
ted two to three bud clusters(Fig. 1).
A:R. multiflora‘inermis3’;B:R. multiflora‘inermis4’;C:R. multiflora‘inermis11’;D:R. odorata;E:R. wichuraiana;F:R. carnea.
Fig. 1 The in vitro sprouting of Rosa spp. plants
As shown in Table 2,the sprouting rates of all ten Rosa
spp. varieties tested were all as high as 70%. The highest
sprouting rate of axillary buds was found in inermis3 (97%)
and the lowest was observed in R. carefully(70%). The re-
sults indicate that the sprouting medium is suitable for all ten
varieties in the sprouting of axillary buds.
Sampling season has some impacts on the sprouting of ax-
illary buds. The samples collected during April - May showed
earlier sprouting of axillary buds,higher sprouting rate and lower
contamination rate,while those collected in autumn or winter
had lower sprouting rate and higher contamination rate be-
cause the buds got into natural dormancy. Therefore,spring
is the optimal time to collect sample.
Results of subculture for proliferation of axillary buds
At the beginning of plating on proliferation medium,the
young seedlings of some varieties showed lowered growth vig-
4341 Agricultural Science & Technology Vol. 12,No.10,2011
A:R. gallica;B:R. White Meidilan;C:R. odorata;D:R. indica.
Fig. 2 The rooting of Rosa spp. plants
A:R. gallica;B:R. odorata;C:R. wichuraiana;D:R. White Meidilan;E:R. indica;F:R. carnea.
Fig. 3 Rosa spp. plants transplanted to pots
or,which was mainly shown in yellowed leaf,dwarfed and
compacted bud cluster and diminished leaf size. This phe-
nomenon could be attributed to be an adaptation process of
young seedlings to different hormones and combinations add-
ed into the subculture medium. Eight weeks after subcultu-
ring,the vigorous young seedlings of all ten varieties on sev-
enteen proliferation media showed enhanced growth vigor and
differentiation capability to some extent (with cluster buds,
vigorous growth,peak green leaf and extended leaf blade) ,
but they differed in optimal proliferation medium,growth status
of axillary buds and proliferation folds(Table 3) ,which were
probably related with the genotype of varieties[14].
In addition,plants of some varieties (including R. odora-
ta)became dwarf and compact on the medium containing
GA3,which could not be used in subsequent experiment due
to the diminished leaf size;in the medium without GA3,plants
produced extended leaf and suitable leaf size. Some other va-
rieties (including R. carnea and R. wichuraiana)showed vig-
orous growth and extended leaves only on the medium contai-
ning GA3 . This suggests that the varieties vary in the re-
sponse to GA3 among different genotypes.
Table 2 The sprouting of axillay buds
Species Explantnumber
Explants producing
axillay buds
Sprouting
rate∥%
R. White Meidilan 30 23 77
R. carefree 30 21 70
R.‘inermis3’ 30 29 97
R.‘inermis4’ 30 28 93
R.‘inermis11’ 30 27 90
R. carnea 30 26 87
R. odorata 30 23 77
R. wichuraiana 30 28 93
R. gallica 30 23 77
R. indica 30 26 87
The data in the table was got 25 days after culture.
Result of rooting and acclimation of plantlets
As shown in Table 4,the plantlets of all tested varieties
generated adventitious roots on rooting medium 1 /2 rooting
medium appended with 0. 1,0. 2 or 0. 5 mg/L of NAA. The
roots emerged gradually about 10 days after been transferred
to rooting medium. The roots appeared to be an actinomor-
phic shape,with a mean root number of 3 - 10 and rooting
rate of 70% -100%. The medium appended with 0. 1 or 0. 2
5341GUO Yan-chao et al. Development of an in vitro culture system of Rosa spp. Plants
mg/L NAA showed optimal rooting effect,displaying vigorous
and stuggy growth. After acclimation and transplant,the sur-
vival rate was reached as high as 95%.
Discussion
The achievement of rapid propagation of Rosa spp.
plants was firstly made in the bud proliferation and rooting of
Rosa multiflora young seedlings in 1970[15]. Since then the in
vitro culture of Rosa spp. plants has been studied and repor-
ted extensively.
Referring to the related literature in the past 20 years,cy-
tokinin,auxin and gibberellin have been broadly applied in the
tissue culture of Rosa spp. plants. Cytokinins could enhance
the number of clustered buds,of which 6-BA is proved most
effective[15 -17]. In the tissue culture of Rosa spp. plants,
6-BA has the working concentration of 0. 5 -3. 0 mg/L usual-
ly,and is combined with other auxins for application. Low
concentration of 6-BA could promote growth while high con-
centration would cause vitrification[13]. Auxins can induce the
elongation of cells,and the most broadly used auxin is NAA.
Beside this,additional low concentration of GA3 in proliferation
could promote the proliferation of axillary buds and enhance
proliferation rate[18], facilitate the elongation of axillary
buds[19 -21].
Table 3 The optimal proliferation media of different cultivars and the
growth of axillary buds
Species
Optimal
medium code
Proliferation
rate∥%
R. White Meidilan 16 4.5
R. carefree 5 3.1
R.‘inermis3’ 5 3.2
R.‘inermis4’ 5 3.3
R.‘inermis11’ 5 3.5
R. carnea 14 3.6
R. odorata 5 3.8
R. wichuraiana 11 3.3
R. gallica 2 3.5
R. indica 3 3. 5
Table 4 Influence of NAA concentrations on the root induction
NAA concentration
mg/L
Rooting rate∥%
R. White Meidilan R. carefree R.‘inermis3’ R.‘inermis4’ R.‘inermis11’
0. 1 100 100 100 100 100
0. 2 80 90 90 90 90
0. 5 70 80 90 90 90
NAA concentration
mg/L
Rooting rate∥%
R. carnea R. odorata R. wichuraiana R. gallica R. indica
0. 1 100 100 100 100 100
0. 2 90 100 90 90 90
0. 5 90 90 90 90 80
On axillary bud induction medium MS +0. 5 mg/L BA +
0.1 mg/L NAA,all ten varieties sprouted out axillary buds
successfully,and the buds were vigorous and leaves were ex-
tended. When been transferred subculture medium for prolif-
eration,the varieties differ in response,which demonstrated
that axillary buds were largely influenced by plant hormones
during subculture process. Of the varieties,0.1 mg/L of GA3
was required for the well proliferation of R. wichuraiana and
R. carnea;while it was an obstacle for other eight tested
materials,where the plantlets showed compact plant type and
narrowed leaves on the medium with 0. 1 mg/L of GA3,grew
cluster buds and proper leaf size after the GA3 was removed
from the proliferation medium. The plantlets of all varieties
tested generated adventitious roots on rooting medium 1/2 roo-
ting medium appended with 0.1,0. 2 or 0. 5 mg/L of NAA,but
those apended with 0.1 or 0. 2 mg/L NAA showed optimal roo-
ting effect,displaying vigorous and stuggy growth.
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Responsible editor:YIN Jian-li Responsible proofreader:
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WU Xiao-yan
控蘖剂对超级早稻金优 458 生长发育及产量形成的影响(摘要)
钱银飞1,2,邱才飞1,邵彩虹1,陈先茂1,谢 江1,邓国强1,彭春瑞1* ,任天志2* (1.江西省农业科学院土壤肥料与资
源环境研究所,江西南昌 330200;2. 中国农科院农业资源与农业区划研究所,北京 100081)
[目的]研究控蘖剂对超级早稻品种金优 458生长发育及产量形成的影响。
[方法]试验设喷施控蘖剂和喷施清水两种处理,研究了控蘖剂对产量及其构成因素,稻株形态,孕穗期田间小气候及群体质量特征的影响。
[结果]喷施控蘖剂处理能显著减少无效和低效分蘖的发生,增加高峰苗期分蘖构成中高效分蘖(单茎 4叶及以上分蘖)所占比重。喷施控蘖
剂处理的穗长、穗粒数、穗着粒密度、二次枝粳数及着生其上的二次颖花数及结实率均显著高于未喷施控蘖剂的对照。同时喷施控蘖剂能增
加株高,中后期群体生物量和叶面积指数,提高孕穗期上三叶叶温和群体的透光性,增加叶片 SPAD值,从而增强光合作用能力,但也表现出
稻株节间长度增加,茎秆粗度减小等特点。
[结论]喷控蘖剂措施处理最终表现为茎蘖成穗率、有效穗数、穗粒数、结实率和千粒重协调上升而增加产量。
关键词 控蘖剂,超级杂交早稻,金优 458,生长发育,产量
基金项目 国家粮食丰产科技工程(2006BAD02A04) ;国家农业科技支撑计划(2007BAD87B08) ;江西省农科院博士启动资金(2009博-1) ;江西
省学科带头人计划;中国农科院博士后启动资金。
作者简介 钱银飞(1980 - ) ,男,江苏如东人,助理研究员,研究方向:从事植物栽培研究。博士。* 通讯作者。
收稿日期 2011-07-08 修回日期
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2011-09-19
(From page 1436)
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Responsible editor:DUAN Yong-bo Responsible proofreader:
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WU Xiao-yan
蔷薇属植物无菌体系的建立(摘要)
郭艳超1,赵梁军2* (1.河北省农林科学院滨海农业研究所,河北唐山 063200;2.中国农业大学,北京 100193)
[目的]建立蔷薇属植物的无菌快繁体系。
[方法]以茎段为外植体,以 10种蔷薇属植物为试验材料,在培养基中添加不同的同激素组合来建立其组织培养体系。
[结果]10种蔷薇属植物在萌发培养基MS +0. 5 mg /L BA +0. 01mg /L NAA上均可萌发,芽体健壮,萌发率可达到70%以上;10种蔷薇属植物
各自适宜的增殖培养基略有不同,在增殖培养过程中,增殖率可以达到 3. 0%以上,丛生苗健壮,叶色正常;健壮小苗在添加 0. 1 ~0. 2 mg /LNAA的
1 /2MS培养基上诱导生根,根系生长良好,生根率可以达到 90% ~100%;将生根后的小苗驯化后移栽,成活率在 95%左右。
[结论]建立了蔷薇属植物的无菌快繁体系,为蔷薇属植物的分子育种奠定了基础。
关键词 蔷薇属植物;组织培养
基金项目 国家自然科学基金资助项目(30871733)。
作者简介 郭艳超(1974 - ),女,河北定兴人,助理研究员,硕士,研究方向:园林植物抗逆生理及分子遗传育种研究,E-mail:guoyanchao2008@ sina.
com。* 通讯作者,教授博士,博士生导师,研究方向:观赏植物抗逆分子育种,E-mail:zhaolj5073@ sina. com。
收稿日期 2011-08-12 修回日期 2011-09-05
8441 Agricultural Science & Technology Vol. 12,No.10,2011