全 文 ：武汉植物学研究 2007，25(1)：79～83
lollm曲of Wuhan Botanical Research
猕猴桃高频直接再生体 系的建立
田娜，徐子勤’，何近刚
(陕西省生物技术重点实验室，西北大学生命科学学院，西安 710069)
摘 要：为了建立猕猴桃高频再生体系，以Ms为基本培养基，猕猴桃(Actinidia deliciosa Qinmei)茎及叶片为外植
体，研究了2，4-D、6．BA和NAA在美味猕猴桃愈伤组织形成及分化过程中的作用。方差分析结果表明，6-BA能够
显著促进愈伤组织形成，6-BA和NAA可以显著促进愈伤组织形成和分化，而2，4-D抑制愈伤组织形成。附加
2．0 ms／L 6-BA、1．0 ms／LNAA和6OO ms／L水解酪蛋白的Ms培养基是茎段培养的最佳培养基，在该培养基上，以
再生的无菌苗为起始材料，一个月时叶圆盘的直接再生频率达到 100％，平均每个叶圆盘产生 9．33个芽，其中
23．21％芽高度超过0．5 am。
关键词：高频；直接再生；美味猕猴桃
中图分类号：Q943．1 文献标识码：A 文章编号：1000—470X(2007)01—0079-05
Establishment of High Frequency and Direct Regeneration
System of Kiwifruit(Actinidia deliciosa Qinmei)
rI1AN№ ，xU zi—Qin’，HE Jin—Gang
(Provinda／Key Laboratory ofBiotechnology ofShaanx／，Northwest ，Xi’an 710069，China)
Abstract：In order to establish a high frequency regeneration system of kiwih-uit，e cts of 2，4一D，6一BA
and NAA on caUus induction and diferentiation of stem and leaf explants of Actinidia deliciosa Qinmei
were studied wi山 MS as basic medium．The results revealed山at calus could be stimulated significan tly
by 6一BA。and its diferentiation could be improved by 6一BA in combination wi山 N从 ．In contrast。2。4一D
had an negative efect on callus induction．MS medium supplemented wi山 2．0 ms／L 6一BA an d 1．0 ms／L
NAA was optimal for shoot regeneration of stem cuttings an d leaf discs．On山e optimal medium，山e leaf
discs from the regenerated sterile plan ts could produce secondary shoot directly an d shot regeneration
rate reached 10o％ ．Each leaf disc produced 9．33 shots on average．an d 23．21％ 0f山em exceed
0．5 cm in height．
Key words：Hi gh frequency；Direct regeneration；Actinidia deliciosa Oinmei
Kiwifmit(Actinidia deliciosa Qinmei)is a valuable
economic woody plant native to China．Among 26 kinds
of main h-uits，kiwih-uit is one of the most nutritious
species rich in Vc，Mg，K and microelements．In addi—
tion，kiwifruit is the only fruit containing chlorophyll
after maturation． Kiwifruit csxl restrain skin can cer，
improve blood circulations，prevent thrombus forma-
tion，an d also be extensively used in food processing．
Approxima tely 62 species of kiwih-uit were distri—
buted in diferent aI~as of China．occupying 97．8％ of
al the species in the world[¨
． In recent years．kiwifruit
varieties have been widely domesticated by peasan ts，
but there are a lot of questions in cultivation through
traditional method have been pro~ sed． Kiwfruit is a
type of dioecious plants wi山 a complicate genetic back—
ground，an d ~lorescences of the male an d the female
are hard to meet，so crossbreeding between species is
dificult[引
． In addition，traditional seedling propagation
of kiwifruit，due to time consum ing，low survival rate
an d unstable propemes，could not meet the requ ire—
ments[3】．Besides，traditional cutage and engrafting
were limited by lack of adequ ate materials．Th e propa-
gation of this species in a large scale becomes the bet—
tleneck．Nevertheless．山ese restrictions can be over-
come by combination of tissue culture with genetic
transformation or other biotechnologies theoretically．
收稿13期：20064)74)3，修回13期：2006-11-28。
基金项 目：陕西省自然科学基金重点项 目(2OOlSm 4)资助；陕西省省级重点实验室科研计划项 目(04JS07)资助；陕西省教育厅科学研
究计划项 目(05JK304)资助。
作者简介：田娜(1981一)，女，在读硕士研究生，主要从事基因工程与转基因植物方面的研究。
· 通讯作者(E-mail：ziqinxu@nwu．edu．cn)。
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武 汉 植 物 学 研 究 第 25卷
In comparison with other kiwifruit species，A．deli-
ciosa has the highest yield of fruit in China．So it is
important to establish an eficient and feasible regener-
ation system for its propagation．Early in 1975，stems
an d roots of kiwifruit had been successfuly applied in
tissue culture by Harada．In the last years of 20th
century，protoplast，anther，endosperm，an d embryo
culture of kiwifruit were also repo rted in succession．
Progresses of kiwifruit tissue culture have been achieved
in several countries，including New Zealand，Italy，Chile，
and Australia，an d regeneration systems of leaf an d
immature emb ryo have been establishedt4·51． Previous
work showed that antibiotics，casein hydrolysate and
diferent carbon sources had strong efects on v／tro
regeneration of kiwifruit[ ·
． In recent years．most
researches concentrated on physiological Is,9]
，biochem．
icalt10,11】an d nutritional aspects[ · 】of kiwifruit tis．
sues and organ s． Consequently，a direct and eficient
regeneration system of kiwifruit is extremely needed．In
order to explore a highly frequent，feasible and time
saving regeneration system for kiwifruit，we compared
the efects of many factors on tissue culture of kiwifruit。
including 2，4-D(2，4-Dichlorophenoxy acetic acid)，
6-BA(6-Benzyl aminopurine)and NAA(Naphthyl acetic
acid)，and an eficient system of plant regeneration
was successfuly established．
1-1 Plant materials an d culture conditions
Stems of Actinidia deliciosa Qinmei were colected
from Hu County of Shaanxi Province． Youn g stems
were used as explants an d cultured in Erlenmeyer
flasks(100 mL)，under a 16 h photoperiod of2000 lx
light intensity at(25±2)℃．1}le basic medium was
MS containing 30 g／L sucrose，6OO mg／L casein hy-
drolysate，and 0．8％ (W／V)agar，and plant growth
regulators were added to the medium and the pH was
adjusted to5．8 with NaOH or HCI solution before auto．
claved un der 121 oC for 25 min．
1-2 CaⅡls induction an d shoot regeneration
The stems were washed un der running water for
6 h，folowed by surface．sterilization with 75％ ethanol
for 3 min and then 0．1％ HgCl2 for 8 min．After wash．
Lug 3 times with sterile distiled water，the stems were
cut into about 0．5 cm long，an d segments were inocu-
lated on MS medium containing diferent comb inations
of plan t growth reguhtors． One treatment had two
replicates，each containing 10 —15 explan ts． Callus
induction rate an d shoot numb er were analyzed statisti-
cally．For subculture，calli and shots were tran sferred
onto the same fresh medium at 20 d intervals．
1-3 Direct shoot regeneration from sterile rege-
neran ts
e sterile plantlets originated from regenerated
sho ts of cali．Young healthy leaves expanded fuly
were excised into discs about 0．3 cm ×0．3 cm in size ．
Th e leaf discs were cultured on MS medium containing
2．0 mg／L 6一BA and 1．0 mg／L NAA．The numb er of
sho ts regenerated directly from leaf discs was counted
after one month．Shots of 3 cm in height were im．
mersed in 100 mg／L IBA(Indolebutyric acid)f0r
10 min．an d then transferred onto l／2 MS medium
without growth regulators for root induction．
2 Results
2．1 Callus an d sho t induction
Seventeen media were designed to test the efects
ofdiferent growth regulators on callus and shot induc．
tion． raJ)le l showed that 2．4．D inhibited calus for-
mation obviously．6-BA could stimulate the formation
of yelow green calli，but could not induce callus
diferentiation subsequ ently．Comb inations of 6．BA an d
Table 1 E嘲 0f2．4-D and 6-BA on calls and shoot
induction from stem cutings
Notes：Type I calus，hard yeHow green calus with rough 8Ul"face．
s=standani dev／ation．C811u8 induction rate = callus number／
explant number× 100％． 一
， mean8 no valid record． ol 6)
8．746． +· represents extremely significant diference
， a=O．Ol，
the t~llle below．
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第 【刹 口娜等：赫懿佻高领直接再生体系的吐 (蝇
TaMe 2 Efect 6-BA In c0mbIlm“0n wiIh NAA 0n髓 U
and sh【川t I．duction 0r 1 m euuin~s
。嚣 Type ÷“ 一 “”⋯ ⋯ ‘
¨I l⋯ I |】i， lJ _』【’ 6Ij^ I¨ 6 H^⋯ I|】 I’l H ¨ |J1 2．4 D -⋯ hm j】 ，⋯ _1】l 【J vt-‘】¨ ⋯ l_¨ n ¨
b } Ⅱt- “， Ⅲ hv 6·nA in mm bi l”th N A^ 1¨ B ⋯ l J k nlh 1¨I
t J r¨ ，d Sh 1 B n I 【t l ln1Ihe d c~lli ( i⋯ L_ ⋯ t‘ IJlm ¨¨ H ． l¨， I H1 tl⋯ 11 nI ”l’ ．w1lh
l g b⋯ I Lu nl Ⅲ1 r s H I‘ H|_lf- jf J|l1 Th I t b⋯Wl 一 k of co1]i ，h lk ⋯ lI J儿Y¨ I：lf~ ⋯ b⋯ l
⋯ I r mi~ opy．1_兀H dl眦 d y ”l J ⋯ k ⋯ l Pla nltle nfIf 3 m In h f
H 1 Plant regeneration of Idwlfndt(AetO*idfi~ * )
薰菱
囊 蒸 鋈 一椰一～ _至
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武 汉 植 物 学 研 究 第 25卷
During diferentiation，the dark green calli with rough
tubercles would change into bright green calli with
smo0th brown tubercles at first，and t}len shoots were
produced from the tubercles(Fig．1：e，f)．
The results of variance analysis were listed in
Table 3 and 4．which indicated t}lat 6一BA was very efi—
cient in calus induction． For diferentiation，6一BA，
NAA and t}leir interaction were very important．NAA
was the most impo rtant factor influencing plan t regene—
ration (F=108．810，showing extremely significant
diference)，6一BA was t}le second factor(F=59．029，
also showing extremely signifcant diference)and the
interaction between them was the third factor(F =
4．287，having signifcant diference)．
Table3 Variance analysis of efects of6-BA and NAA
on calus induction rate of stem cuttings
Notes：Fo
．
ol(2．9) 8．022，Fo
．
05(2．9) 4·256， Fo
． 05(4．9)
3．633．
Table 4 Variance analysis Of efects Of 6-BA an d NAA
Ol shoot number per calus
Notes：Fo
．
01(2．9) 8．02 2，F0
．
∞(2．9) 4．256，
．
∞(4．9)=3．633．
· repre8ent8 signifcant dⅢerence． =0．05，the s啪 e below．
2．2 Direct regeneration of shoot dusters from
leaf discs of regenerated plants
After cultured on MS medium contMning 2．0 mg／L
6一BA and 1．0 ms／L NAA for two weeks．the leaf discs
coming from sterile regenerants cuded upward，an d
folowed by shooting directly on the margin three weeks
later(Fig．1：g，h)．Some discs，especialy those with
veins and petioles，formed a few lumpish calli． One
month later，shoot number was recorded an d the shoot
induction rates were analyzed(Table 5)．Each leaf
disc produced 9．33 shots on average，2．17 of them
above 0．5 am high． During subculture process， the
shots an d calli grew continuously，and a few calli also
could produce sho ts． The shoots grew to 3 am in
height in two months(Fig．1：i)．
Table5 Shoot induction ra tes ofleafdiscs cultured
on MS medium supplemented th 2．0 mg／L 6-BA and
1．0 mg／L NAA for30 days
Notes：Shoot induction rate=number of leaf discs with sho~number
oftotal leaf discs×100％．Shot induction rate(>0．5 am)：
shoot number(>O．5 cm)／total shot nunlberx100％
3 Discussion
In the present work，two type of calli，term ed as
type I and I1 were induced from stem segment，and
both had the abilities to diferentiate． After being
transferred onto MS medium added 2 mg／L 6-BA and
1 mg／L NAA，the type I calli would turn to type I
cali and a lot of shots could be regenerated．
In addition．an eficient an d direct regeneration
system was established through stem and leaf culture．
All leaf discs from sterile regn eran ts could be success—
fully induced to produce many sho ts directly and a few
cali．Th is is pe rhaps attributed to that the leaves col-
lected from sterile regenerated plan ts were young an d
had strong ab ilities of diferentiation．Leaves of woody
plan t were frail an d sensitive to sterilant an d need more
favorab le culture conditions．In our study，the sterile
plan tlets were unnecessary to undergo SUl~ace·-steriliza·-
tion which maybe reduce the regeneration frequency．
Further more ，the explan ts for direct sho ts induction
originated from regenerated sho ts in vitro，so they did
not undergo a domestic process． According to our
observation，casein hydrolysate supplemented in the
medium could promote shot development．
Th e process of regeneration from sterile leaves was
time saving，highly frequent and practica1．In the pre—
sent work ，shots were induced directly from leaf discs
without undergoing the callus stage．Since the in vitro
plant regeneration via the pathway of direct clustered
bud induction can reduce tl1e rate of somatic clonal
mutation，it would be useful in genetic transform ation
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第 1期 田娜等：猕猴桃高频直接再生体系的建立(英) 83
第二届植物分子育种国际学术研讨会
2007年 3月23—27日在海南省三亚市召开
(htp：／／www．icpmb．org)
现代农业已经发展到了“分子农业”时代，基因工程、转基因技术等分子生物学手段广泛应用于植物的遗传育种，基因组
学的研究成为植物基因资源发掘的基本科学平台，分子育种成为植物育种的最主要手段之一。为加强与国际植物分子育种
领域的合作与交流，推动应用基因组学与植物分子育种科学的发展，111e Generation Chalenge Programme(全球挑战计划)、国
际水稻研究所 、海南省科学技术协会、中国农业科学院、海南省热带农业资源开发利用研究所等单位发起 ，定于 2007年 3月
23~27日在海南省三亚市举办“第二届植物分子育种国际学术研讨会”。
大会主题
本次大会主题为“应用基因组学与植物分子育种”。
主要议题
(1)应用植物基因组学：从基因组走向大田；(2)分子标记与植物育种：一种整合；(3)植物育种中新型的分子工具和技
术；(4)转基因新技术、产品及其市场；(5)设计育种：基因发现和性状改良的连接；(6)转基因植物中知识产权的问题。
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