全 文 ：武汉植物学研究 2007，25(2)：185～191
Journa／of Wuhan Botanical Research
影响辣椒离体再生芽伸长因素研究
张余洋 ，¨ ，汤银珠 ，“，叶志彪 ，张婵娟 ，李汉霞 2”
(1．华中农业大学，国家蔬菜改良中心华中分中心，武汉 430070；2．华中农业大学，园艺植物生物学
教育部重点实验室，武汉 430070；3．华中农业大学，作物遗传改良国家重点实验室，武汉 430070；
4．中国科学院武汉植物园，武汉 430074)
摘 要：以辣椒子叶为外植体，比较不同浓度BA和I从 激素组合对辣椒再生芽诱导的差异，利用筛选出的高效芽
诱导培养基为基础，研究了赤霉素、芽诱导时间、培养基有机成分、不同激素组合和品种等因素对辣椒不定芽伸长
的影响。结果表明：不同基因型辣椒子叶再生能力不同，BA 3．0 mg·L +I从 0．5 mg·L 的激素配比对不定芽诱
导频率最高；不定芽的伸长百分率随着 GA，浓度的增加而增加，GA，的适合浓度为 1．0～2．0 mg·L～；不定芽诱导
时间对不定芽的伸长有一定的影响，诱导21 d的不定芽，其伸长频率砚显高于诱导14 d的不定芽；B5有机成分在
辣椒不定芽的伸长中效果优于MS有机成分；激素组合对不定芽伸长有一定的影响，Zeatin+GA 激素组合对伸长
效果最好，BA+I从 +GA3伸长效果较好，BA+PAA(苯乙酸，phenylacefic acid)+GA，伸长效果次之；不同品种辣
椒不定芽的伸长能力有一定差异，楚风和苏椒五号再生芽伸长能力最佳。与 I从 和 NAA相比，IBA对再生芽生根
效果较好。
关键词：辣椒；子叶；再生；芽伸长
中图分类号：S634；Q945．3 文献标识码：A 文章编号 ：1000．470x(2007)02．0185．07
Factors Afecting in vitro Shoot Elongation of Pepper
ZHANG Yu．Yang ， ’，TANG Yin．Zhu - ’，YE Zhi．Biao · ，ZHANG Chan．Juan ，LI Han．Xia - ’’
(1．National Centerfor Vegetable Improvement(Central China)，Huazhong Agricultural University，Wuhan 430070。China；2．Key
Laboratory ofHorticultural Plant Biology，M／n／stry ofEducation，Huazhong gr l Un／vers／ty，Wuhan 430070，China；
3．National Key Laboratory ofCrop GeneticImprovement，Huazhong Agricaltural University，Wuhan 430070，C~na；
4．WulmnBotanical ，I，TheChineseAcademy of&／ences，Wuhan 430074。China)
Abstract：A detailed study was carried out to investigate key factors affecting shoot elongation of pepper．
Using the cotyledons of pepper as explants，the optimal phytohormone combination of BA +IAA for bud
induction was screened out to investigate the efect of GA ，bud．inducting time，organic constituents in
media，diferent phytohorrnone combinations and cultivars on shoot elongation． e results were as
follows：Seven cuhivars showed ability diferences for bud induction．and BA 3．0 mg·LI1+I从 0．5 mg·L一
could give the highest response． e shoot elongation frequency increased with GA3 concentration，with
the optimal concentration of 1．0—2．0 mg·L～． e buds subjected to 21．day induction showed higher
dongation eficiency than that of 14-day induction．B5 vitamins were more eficient than MS vitamins for
shoot elongation． e phytohormone combination of Zeatin+GA3 exhibited higher elongation eficiency
than comb inations of BA+IAA +GA3 or BA+PAA+GA3．Elongation ability varied with pepper cuhivars，
with Chufeng and Sujiao No．5 having the best elongation ability．Compared with LAA and NAA．IBA
gave better rooting efect．
Key words：Capsicum annuum L．；Cotyledon；Plant regeneration；Shoot elongation
Pepper(Capsicum annuum L．)is an important
crop grown worldwide for its use as vegetable，spice an d
ornamental plant．Unfortunately，it is highly susceptible
to fungal or viral pathogens，and these cause considerable
damage to the crop[
． One ofthe solutions to this problem
is the development of pathogen-resistant pepper varieties
using genetic transformation techniques，which in turn
require eficient plant regeneration protocols[
． On the
收稿 日期12006-08-14，修回日期：2006-02-14。
基金项 目：国家 自然科学基金资助项目(30270914)。
作者简介：张余洋(1979一)，男，博士，讲师，从事蔬菜生物技术研究。汤银珠(1980一)，女，硕士，助理研究员，从事植物生物技术研究。
李汉霞(1958一)，女，教授，硕士生导师，从事植物生理与分子生物学研究。
· 同为第一作者。
··通讯作者(Author for corespondance．E．mail：hxli@mail．hzau．edu．on)o
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186 武 汉 植 物 学 研 究 第25卷
other hand，the abundan t novel genes emerging in post—
genomic era have entailed function deciphering via
transgenic approach．An essential step towards development
of tran$gt~ c plants t}lIough 4 er m-瑚edi砒ed trans—
formation is the development of an efficient regeneration
protoco1．
To date． researchers worldwide have Hlade great
progress towards pepper regeneration 一l3]．However．tisue
culture techniques in pepper Lag behind other solaJls~ous
species ，mainly due to its recalcitrance to regeneration
especially at the shoot elongation stage[14]
． The production
of distorted leafy structures which resists elongation
or I| 把 鹧 of distorted leaves without normal shoots has
been a maj0r constraint in the plant regeneration protocols
reported for pepper[
． 3his problem was partialy resolved
by plant regeneration from decapitated．rooted hypoco~
explants or half-seed explants usmg hormone—free Murashige
and Skog(MS)medium or medium supplemented
with 6一Benzyl—aminopurine(BA)and indole．3。acetic
acid(I从 )[9,16,17]．On the other hand，organogenesis
in vitro from diferent pepper cels an d tissues
， as
protoplasts 引
， hypocotyls，cotyledons，an d young
leaves，has been repo rted by culturing the explants in
MS basal medium supplemented with exogenous
regulators[ ’引
． Recendy Pozueta—Romero et
growth
[卸】
have overcome the difculty of shoot elongation by
designing an explant which can provide nutrients f0r
calus diferentiation and regeneration in tomato an d
bell pepper．However，al these researches repo rted low
regeneration eficiencies and tl1e protocols described in
the literatures ltre genotype specific and in some cases
vary from laboratory to laboratory[引
．
The lack of a reliable system for regeneration of
pepper coupled with strong genotype dependency an d
low tran sformation frequ encies have hampered tIle
development of transgenic pepper breeding[21] an d
made it necessary to optimize regeneration protocols f0r
specific cuhivars[
． In this paper．we investigated the
key factors afecting the shot elongation in several
cultivated peppe r species．
1 M aterials and M ethods
1．1 Plant materials and experimental conditions
Seeds of commercialy important pepper(Capsicum
口，l，lnn，1)including 5 chili pepper cultivars(Bianhong
No．1，Bianjiao No．1，Sujiao No．5，Chufeng and
Xiangjiao No．21)and 2 sweet pepper cultivars(Qien瑚
and Teda)were used．The seeds were surface sterilized
in 70％(v／v)ethanol for 30 sees folowed by 3％(
)sodium hypochlorite for 15 mins，and rinsed with
three times in sterile distiled water．The sterilized seeds
were sown on half-strength MS medium[~]c0ntaining
1．5％ sucrose and allowed to germinate in darkness for
3 days and山en transfefred in light conditions at 25℃ ±
2~C．Cotyledons were excised aseptically at 10 d seed—
lings age at the petioles．Incubation was in a culture
room at 25oC 4-2℃ witIl a regime of 8 h dark／ 16 h
light wi山 山e intensity of 1 800 Ix．All media were so—
lidifed by 0．74％ agar and the pH was adjusted to 5．8
prior to autoc laving at 121 oC for 15 mins．Observation
was recorded after every．two．week subculture for one
mon山．AU treatments in山e experiments were repeated
three times．The data were subjected to Duncan’S new
multiple ran ge test to check the sign ificance at 95％
confidence interva1．
1．2 Adventitious bud induction
The bud induction mediUll consisted of MS me出啪
containing 3％ sucrose．supplemented with regulator
combinations of BA (0，3．0，4．0 or 5．0 mg·L )
and IAA(0，0．2，0．5 or 1．0 mg·L )．About fifteen
explants were inoculated per plate(9 am diameter)in
山e medium．
1．3 Adventitious shoot elongation
The diferentiated shots were excised from
explants an d transfefred to elongation medium ． The
elongation medium utilized 山e MS containing 3％
sucrose as a basal medium supplemented wi山 BA
3．0 mg·L‘ and IAA 0．5 mg·L一 to investigate the
efect of GA3， bud—inducting time， phytohorm one
comb ination and cultivar on shot elongation． Both MS
an d B5[243 vitamins were individuany applied in bud
elongation medium supplemented with BA 3．0 mg·L一
and IAA 0．5 mg·L一 to investigate efect of organ ic
constituents of medium on shot elongation．
1．4 Ro ting of regenerated sho ts
After a l—month incubation on the elongation
medium，the shots of 1．5-2．0 cm in length induced at
the base of cotyledons were excised from the cotyledons
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第2期 张余洋等：影响辣椒离体再生芽伸长因素研究(英) 187
and transferred to half-strength MS medium containing
1．5％ sucrose supplemented with alpha—naphthaleneacetic
acid(NAA)， A or indole一3一butyric acid(IBA)at
diferent concentrations(O．1，0．2，0．5，1．0 mg·L )．
The efect of phytohormones on rooting was investigated
after 14 days．
2 Results and Discussion
2．1 Efect of genotypes on shoot induction from
cotyledons
has significant influence Ol shoot induction
eficiency as could be seen from Table 1．AU 5 pepper
cultivars displayed significan t diferences in regenera—
tion ability，varying from 0 to 100％ ． Similar results
were obsen，ed as Ochoa-Alejo and Ireta—Moreno[ 】．
Th erefore，genotype is one of the most important factors
on reliab le and efficient regeneration in pepper．Mean—
while，in this research，the diference among genotypes
becam e indistinctive when cultured with optimal phyto—
hormone combinations e．g． BA 3．0 mg·L +IAA
0．5 mg。L～ ，on which al genotypes reached 100％
respo nse． In addition， Chufeng exhibited 100％
respo nse with all combinations of BA and IAA．thus
presented the most applicab le donor of explants for
genetic tran sformation．
Th e present experiment showed a varied sensitivity
of the cuhivars tested to h 0l ⅡⅨme．Similarly elongation
rate of buds an d shots development depended on the
peppe r genotypes that confirm the cro88一cultivars difer-
ences was also obsenred in other experiments[2引 ． A
varied cultivar response to cytokinins may depend
on the activity of enzymes in the metabolism of these
phytohorm ones and exogenous cytokinins added can
inhibit the synthesis of endogenous cytokinins and
disturb the regeneration process[
． Apart from the
genotype dependency of the protocol，variations between
laboratories for the r~eneratiou protocols of the same
genotype can be expe cted because of variation in the
growing conditions of the mother plants producing
seeds[圳 ．
2．2 Efect GA3 on sho t elongation
Th e main obstacles of pepper regeneration lie in
the adventitious sho t elongation． Although diferent
phytohorm ones have been tried in adventitious bud
elongation of pepper，the eficiency remained dissatis—
fying．Many studies have included GA3 in the medium
for shot elongation[ 。，圳
． In this research．it could be
concluded from Fig．1 that the addition of GA3 acceler-
ated the elongation of adventitous buds in Chufeng．
Maximum elongation eficiency of 5 1％ was obtained
using combination of 3．0 mg·L BA +0．5 mg·L一
IAA +1．5 mg·L～ GA3．Addition of GA3 promoted the
adventitious bud elongation as was consistent with
previous research[驯
． Th is might be due to the action
of GA3 on cel division an d growth[如]
． Th e elongation
eficiency increased with GA3 concentration to certain
extent，but concentration of up to 3．0 mg·L一 would
inhibit the elongation of adventitious bud．Th e optimal
Table 1 Frequencies of bud induction from different genotypes of pepper on medium supplemented、 th BA and IAA
· The induction frequency of adventitious bud wa8 presented by the percentage of explants with b1Jd8 after inductior．for 1 month
． Mean index±SD are
shown； ·· Meansinthe 8813ne columnfollowed by diferentlettersindicate significant diferences byDuncan’8multiple rangetest atP． 05．
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第2期 张余洋等：影响辣椒离体再生芽伸长因素研究(英) 189
constituents and phytohormones especially GA3．
2．5 Efect of phytohormone combination on shoot
elongation
When the optimal combination of BA +IAA was
used for buds induction．an d three kinds of phytohor-
mone combination were used for shoot elongation，
Zeatin+GA3 had the best elongation efect，BA+LAA
+GA3 took the second place，and BA +PAA + GA3
had the least efect(Table 2)．Comb ination of
2．0 mg·L一 Zeafin+1．5 mg·L— GA3 gave the highest
elongation efficiency of 56．25％ ．Although Zeatin was
less eficient in bud induction，it has high eficiency in
sho t elongation in combination of GA3．
To our knowledge，very few reports were available
on utilizing Zeatin for shot elongation of pepper so far．
About 35％ of the induced shots elongated on MS
medium plus 2 mg·L一 Zeatin or comb ination of BA
(2 mg·L )and GA3(with several concentration
grads from l to 3 mg·L一 )[7]-Meanwhile，after shot
induction，no stem elongation was induced with Zeatin
or GA alone at an y of the concentrations tested[ ¨ ．
The highest response eficiency of 85％ was obtained
with combination of Zeatin(9—18 Ixmol／L)with GA3
(2．89 p~mol／L)，but maximum elongation percentage
of only 25％ ——30％ bud clumps elongated on a medi·-
um containing BA in comb ination with GA [28]
．
In a previous repo rt．Cao et al[32]found that BA
was beter than Zeatin for bud induction，but prevented
shot elongation．Th ese data on pe ppe r shots elongation
show that Zeatin，when comb ined with GA3，has a positve
efect on pepper in vitro regeneration．To clarify the role
of Zeatin an d GA3 more comprehensively，additional
experiments should be carried out to determine the
more optimal concentrations for Zeatin．Wh ether Zeatin
an d GA3 have some interactions is not repo rted yet，
although Tieu et al reported that Zeatin and gibberel—
lic acid was ab le to break dormancy．However，this was
the first detailed research on the efect 0f Zeatin an d
Table 2 Efect of phytohormone combinations on pepper(Chufeng)shoot elongation‘
· The explants were incubated for bud induction with phytohormone combination of BA+L ．and then phytohormone comb ination of BA +
I从 +GAs，Zeatin+GA3 or BA +P A^ +GA3 was used for shoot elongation ； ·· Means in the 8anle column followed by diferent leters
indicate sig~aificant diferences byDuncan’8multiple rangetest atPwith elongated shoots．mean index±SD al shown．
GA3 on shot elongation of pepper in vitro regeneration．
2．6 Efl6ect of cultivars on sho t elongation
As could be drawn from Fig．4，7 cultivars all
exhibited eficient shot elongation on the same medi．
um(MS+2．0 mg·L Zeatin+1．5 mg·L GA3)
with ability diference to some extent．Chufeng reached
the highest elongation frequency of 5 l％．folowed by
Sujiao No．5 with 38％ elongation frequency． The
lowest elongation frequ ency occurred on Teda．
2．7 Ro ting of cultilr~
Norm ally elongated shoots were excised from the
cotyledous explants and transferred to rooting medium
supplemented with NAA． IAA or IBA at diferent
Values followed by diferent letters indicate sig~aificant diferences
(PFig．4 Efect of cultivars on shoot elongation
from cotyledon explants of pe ppe r
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第2期 张余洋等 ：影响辣椒离体再生芽伸长因素研究(英) 191
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