全 文 ：热带亚热带植物学报 2004，12(6)：528—532
Journa／of Tropical and Subtropical Botany
利用辣椒疫霉培养滤液体外筛选
胡椒抗瘟病无性系研究
刘进平，郑成木
(华南热带农业大学农学院，海南 儋州 571737)
摘要：在胡椒 (PZper nigrum Linn．)茎尖丛生增殖技术的基础上，以印尼大叶种 “Lampong Type”无菌实生苗作外植体
源，利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择。辣椒疫霉培养滤液
的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著，过滤灭菌方式可以保持辣椒疫霉培养滤液的毒性，而高温高压
灭菌方式则不能。随着辣椒疫霉培养滤液浓度的增加，茎尖和丛生芽的存活率和增殖率都在下降。在存活的茎尖或丛
生芽培养中，一部分可正常增殖，其余的形成愈伤组织，或者保持生长停滞的休眠状态。在选择性培养基上继代培养2
次后进行生根和移栽，利用离体叶片针刺接种法对温室条件下生长的移栽植株进行抗瘟病测定。以3次抗病检测均无
明显症状的植株作为抗病株。随着辣椒疫霉培养滤液浓度的增加，得到的再生植株数量降低，但其中抗病株的比例提
高。利用过滤灭菌方式加入选择性培养基的处理中，25％、50％和 75％的辣椒疫霉培养滤液分别获得 1株、4株和3株
抗病株，分别占各处理再生植株总数的 1．54％、20．00％和42．86％，共获得8株，占该组处理再生植株总数的8．70％。
关键词：胡椒；胡椒瘟病 (根腐病)；辣椒疫霉；培养滤液；体外选择；体细胞无性系变异；抗病性
中图分类号：$332．2 文献标识码：A 文章编号：1005—3395(2004)06—0528—05
／n vitro Selection of Black Pepper(Piper nigrum Linn．)Somaclones
Resistant to Foot Rot Using Culture Filtrate of Phytophthora capsici
LIU J in—Ping， ZHENG Cheng—mu
(Agronomy Colege，South China Univers of Tropical Agriculture，Danzhou 571737，China)
Abstract：Based on shoot—tip multiplication technique，the application of in vitro selection of black pepper
somaclones resistant to foot rot disease caused by fungus Phytophthora capsici was carried out using a large—leaf
variety Daye(Lampong Type)which is widely cultivated in Hainan but highly susceptible to P capsici as explant
resources．The results demonstrated that sterilization methods significantly influenced on the toxicity of the culture
filtrate of P capsici．The fungal filtrate toxicity could be maintained using filtrate sterilization by addition of the
fungal filtrate into selective medium instead of autoclave sterilization． Th e survival rate of shoot tips and multiple
shoots decreased with the increasing concentrations of the fungal culture filtrate． Some cultures blackened and
died finally,and some formed calli or remained dorm ant．After 2 subcultures onto the same selective medium
，
microshoots were rooted in vitro and transferred to the greenhouse conditions for screening the resistance to
fungu s P capsici．The plants exhibited no external symptoms of the disease during three successive times of
resistance assay once two weeks were considered to be resistant
． As the concentrations of fungal culture filtrate
increased，the total number of regenerated plants obtained declined but the frequencies of plants resistant to
capsici increased．1(1．54％)，4(20．OO％)and 3(42．86％)disease resistant somaclonal variants were obtained at
concentration of 25％，50％ and 75％ fungal culture filtrate as selective agents
， respectively，with the total number
Of 8plants resistantto capsici．
Recieved：2004—-05—14 Accepted：2004-09——16
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第6期 刘进平等：利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究 529
Key words：Black pepper；Piper nigrum；Foot rot；
Somaclonal variation；Disease resistance
— Black pepper(Piper n 驴／zm Linn．)is an impor—
tant tropical spice crop．Whether in cultivation area,
yield,or economic value，black pepper has been the
leader among spices， and known as“king of the
spices’or“black gold”．Major countries in production
and supply of black pepper include India, Vietnam,
Indonesia， Malaysia， Thailand， Brazil， Sri Lanka
and ChinatlJ．
Foot rot disease(Phytophthora foot rot，quick
wilt，or sudden—death)is the most serious and dreaded
disease afecting black pepper which is caused by the
soil borne fungus Phytophthora caps ici(formerly
kn own as Phytophthora palmivora var． piper~， or
Phytophthora palmivora(But1．)Butler MF4)．This
disease is prevalent in all pepper growing tracts of the
world．This“black pepper killer”is one of the major
constraints in black pepper production t2j．Therefore，
improvement of crop resistant to foot rot disease has
been becoming a major objective of black pepper
breeding．
Two essential prerequisites for plant breeding are
the presence of suficient genetic variation and the
availability of eficient selection procedurest ．There is
no real germ plasm which is resistant to foot rot foun d
in P nigrum．Although P colubrinum is reported to
be resistant to fungal pathogen P capsici and can
serve as potential donor of various resistance traits in
black pepper improvement programme[4]，but conven—
tional hybridization and grafting Of P colubrinum and
P nigrum have not been successful as yet due to graft
as well as sexual incompatibility ． Asymmetric
hybridization and genetic transform ation for the
transfer of quick wilt resistance from colubrinum
tO P nigrum is not practical due to lack of the eficient
in vitro regeneration systems and disease—resistance
genes clonedt~．Somaclonal variation and vitro
selection techniques have akeady been used for
obtaining potentially disease—resistant plants of various
crops， and some cultivars have， been released that
derived from somaclonal v撕 ation[3．7-9． Therefore．
application of in vitro selection of black pepper
Phytophthora capsici；Culture filtrate；In vitro selection；
somaclones resistant to the foot rot may be an attrac—
tive alternative．
In the present paper we describe the in vitro
selection of black pepper somaclones resistant to the
foot rot based on the in vitro shoot—tip multiplication
techn ique rep orted by Liu& Zheng[Ⅷ．To our kn ow—
ledge，there have been no reports of use of this system
to produce disease—resistant black pepper plants．
1 Materials and Methods
l-1 Plant materials an d culture eonditiOilS
Shoot tips and multiple shoot buds．initiated from
shoot tips excised from 50·day·old aseptic seedlings of
a black pepper variety，Daye(Lampong Type)，exten—
sively cultivated in China but highly susceptible to P
capsici，were used as plant materials for selection．Initia-
tion medium (IM)and multiplication medium (MM)
wereMSmedium +1．0mgL- BA+0．1 mgL- IAA+3％
sucrose+0．75％ agar．A11 cultures were incubated at 26±
2oC under continuous illum ination of 56 la mol m。2s
cool—white fluorescent ligIlts．
1．2 Preparation of culture filtrate media and
seleetion
The isolate ofP．capsici，kindly supplied by Plant
Protection Institute． Chinese Academy of Tropical
Agricultural Science， was cultured on carrot agar
medium．To obtain filtrate．cultures were grown in flasks
containing 200 ml of liquid carrot medium agitated at
l00 rpm for 2 weeks at 25oC． The suspensions were
filtered to remove the conidia and mycelia and
sequentialy filtered through filter paper r中60~045)．
The undiluted culture filtrate was used in preparing
toxic media．Toxic media were prepared by substi—
tuting fungal culture filtrate for 0％ (0 ml L )，25％
(250 ml L )，50％ (500 ml L )，and 75％ (750 ml L )
of the distilled water in the IM or MM described
above．The toxic media were autoclaved for 20 min
at l2loC before or after addition of the fungal
culture filtrate through 0．2 la m aseptic N札 GENE
milipore membrane． Shoot tips excised from aseptic
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530 热 带 亚 热带 植 物 学 报 第 l2卷
seedlings were grown on the selective media for 4
weeks，and surviving shoots and multiple bud clusters
from each botle were pooled and transfered every 4
weeks into the same fresh selective media for 8 weeks．
After 1 2 weeks of culture， the number of shoot tips
survived，forming calus，being dorm ant and form ing
new shoots were measured After selection,shoots were
rooted in 1／2 MS+1．0 mg L mA +0．5 mg L。 IAA+
3％ sucrose+0．75％ agar．After 4 weeks of culture,the
plantlets were established in pots containing a mix of
coir dust，sand and soil(in the proportion of 1：1：1)and
were later transferred to the greenhouse．
13 Evaluation of regenerated plants for resistance
to P．capsici
Screening of regenerated plants for resistance to
P capsici was conducted using a modified method of
Kueh& Khew【 】and Lin & pan t ．Two weeks after
vitro inoculation of zoospore suspension into wounds，
created by pun cturing， in three full expan ded leaves，
the reaction of 3 leaves of individual regenerants to P
caps ici was observed and the percentage of infected
leaftissue was converted to a graded series：0(no lesion
onleaf),1(appearanceoflesionlessthan25％leafareas),
2(25％一50％)，3(50％一75％)，and 4(75％一1 00％)．An
average disease severity index(ADSI)was calculated
from these scores according to the form ula：ADSI
『∑(score x num ber of leaves corresponding to each
score)】／(3 x4)x 1 00％． The disease response of the
individual regenerants was therefore calculated and
classifedinto4groups：resistant 0A，andthismeans
that the plants exhibited no external symptoms of the
disease during three successive times of resistance
assay once two weeks were considered to be resistant)，
moderately resistant <30％)moderately suscep—
tible s，30／o-60／o),and susceptible(s60A-100％)．
2 Results
The results demonstrated that sterilization
methods had a significantly influence on the toxicity
of the culture filtrate of P capsic fTable l ． The
percentages of shoot tips survived after selection
ranged from 93．33％ to 81．42％ with the autoclave
sterilization of tle culture filtrate．and from 70．1 5％ to
3O．56％ with the filter sterilization． It showed that the
fungal filtrate toxicity could be maintained using
filtrate sterilization instead of autoclave sterilization．
In addition，The percentage of survived shoot tips and
multiple shoots and newly initiated shoots decreased
with the increasing concentrations of the fungal
culture filtrate using the same sterilization method．Of
all shoot tips survived， some form ed new shoots，
others profusely form ed calus or remained dorm ant．
Th e num ber of shoots produced from treated shoot
tips was generally low compared to the controls·
After 2 subcultures onto the same selective
medium ．microshoots were rooted n vitro and trans—
ferred to the greenh ouse conditions and screened for
resistance to P capsici．The plants which exhibited no
external sym ptoms of the disease during three
successive times of resistance assay once two weeks
were considered to be resistant．No resistant plant was
produced with autoclave—sterilized fungal culture
Table 1 Efects ofconcentrations ofculture filtrate ofP capsici and sterilization methods on growth ofshoot tips ofblack pepper
I I } I } } } 娶 莨 蔹
誊 I ，
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第 6期 刘进平等：利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究 53l
filtrate．As the concentrations of fungal culture filtrate
increased，the total number of regenerated plants
obtained declined but the frequencies of plants
r esistant to capsici increased．1(1．54％)，4(20．O0％)
and 3(42．86％1 disease resistant somaclonal variants
were obtained at concentration of 25％．50％ and 75％
filter—sterilized fungal culture filtrate as selective
agen~，respectively,with the total num ber of 8 plants
resistant to P capsici(Table 2 ．
Eight somaclones wim the resistance to capsici
were obtained through n vitro selection with the
fungal culture filtrate of P capsici．In contrast to
those，there was no one plant having disease resistance
found in the un selected control plants and source
plants．Morphological variations such as leaf color
change(turned to yelow—green)， leaf size change
Coecame smal1)，aberrant leaf laminae，dwarfism and
bushiness in apices were also observed ex vitro．
3 Discussion
Two prerequisites for a successful n vitro
selection system with pathogen culture filtrate
containing toxins are that toxins which play an
important role in pathogenesis or disease development
,
and that the resistance is expressed at cellular level as
well as the whole plant levelt3,7, 堋．The work ofLee[141
demonstrated that toxin inoculation of Phytophthora
palmivora(i．e．Phytophthora capsici)was as efective
as fungal inoculation on black pepper cultivars
， Piper
colubrinum and P sarmentosum for the screening of
black pepper for foot rot resistance．Our results
showed that in vitro selection with the filter—sterilized
fungal culture filtrate of 尸． capsici for foot rot
resistance is efective and inhibition of shoot tip
growth and multiplication with the selective medium
containing 5O％ fungal culture filtrate is most suitable．
Although the inh ibition of the shoot tip growt h and
proliferation increased with the concentrations of
the fungal culture filtrate，but the number and the
percentage of shoot tips and multiple shoots survived
and of newly initiated shoots dramatically decreased
．
Only 7 shoots were produced from 22 survived shoot
tips and 3 somaclones were screened to be resistant to
脚 tophthora capsici when treated with 75％ fungal
culture filtrate．
Toxic pathogen culture filtrates and purified
toxins have been used for in vitro selection and
regeneration of disease—resistant plants[15-24
． In
consideration of sensitivity of plant cultures to
phytotoxins，protoplasts，cell suspension，and callus
were used as the selection material un its in most
experiments． But， one prerequisite of a successful in
vitro selection system is the establishment of a plant
regeneration system． Since there were only several
reports on the successful regeneration of black
pepper f q 。 and black pepper lacked an eficient and
reliable system of plant regeneration from the
undiferentiated cultures， the use of an organized
culture system such as shoot tip and multiple shoot for
in vitro selection may be an attractive way，because it
may reduce or eliminate some of the problems
Table 2 Reaction ofsomaclones obtained with diferent treatments ofselection to P capsici
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532 热 带 亚 热 带 植 物 学 报 第 12卷
associated with undiferentiated culture systems such
as the loss of morphogenetic potential[ j瑚．This study
showed that in vitro selection of black pepper
somaclones resistant to the foot rot based on the
shoot—tip proliferation may be feasible，and further
studies of the somaclones are needed to understand the
stability and inheritance of the disease resistance．
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