全 文 ：广 西 植 物 Guihaia 25(5)：459—463 2005年 9月
大叶种胡椒实生苗茎尖培养和合子胚培养研究
刘进平，郑成木
(华南热带农业大学农学院，海南儋州 571737)
摘 要 ：利用各种表面消毒方法对采 自海南岛三个地区的胡椒大田植株的外植体进行消毒试验，由于内源性
污染，除胡椒成熟种子外，其它各种大田外植体的表面消毒均未能成功。以胡椒成熟种子无菌萌发的实生苗
茎尖作外植体 ，在 1／2MS(MS或 B5)+1．5 mg／L BA+0～0．2 mg／L IAA(或 NAA)上可实现丛生芽增殖。
茎尖水平或竖直接种方法显著影响茎尖的增殖；水平接种茎尖的生长和增殖效果优于竖直茎尖接种方式。茎
尖增殖率随 BA浓度的增加而提高，但 BA浓度大于 2．0 mg／L时会使苗芽的质量降低，愈伤组织产生严重，
苗芽细小，抽出不明显 ，颜色发黄甚至变自。附加或不附加 100 mg／L AdSO4对丛生芽增殖没有明显影响。
生根培养基以 1／2MS+1．0 mg／L IBA+0．5～1．0 mg／L IAA为最优，生根率可达 100％；在细沙 ：土 ：椰糠
(1；1：1)的基质中常规炼苗 ，成活率可达 98 以上。液体纸桥法对胡椒种胚进行培养 ，在不附加任何生长调
节物质的培养基(MS、Bs或SH)上只产生单苗，而在附加不同种类和不同浓度的生长调节物质的培养基上则
诱导形成愈伤组织，但未能实现分化；以胡椒无菌萌发的实生苗胚轴和叶片切段作外植体进行培养，较易诱导
产生愈伤组织 ，但难 以实现分化。
关键词 ：胡椒 ；茎尖培养 ；胚培养
CLC number：Q943．1 Document code：A Article ID： 1000—3142(2005)05—0459—05
I vitro culture 0f shoot tips from seedlings
ancl zyRotic em bryos 0t Pt P er 1 ru7 1 ■ ’ n 一■ ●
LIU Jin—ping，ZHENG Cheng—mu
(Agronomy College．South China University of Tropical Agriculture，Danzhou 571737，China)
Abstract：Experiments on surface-sterilization methods were carried Out with various explants collected from
field—grown black pepper(Piper nigrum Linn．)c．v．Daye(Lampong Type)，extensively cultivated in China
．
Due tO endogenous contaminants，contamination of all types of explants except for seeds was not yet effectively
controlled．In vitro clonal propagation of black pepper with shoot tips excised from aseptic seedlings through
multiple-shoot multiplication methods is successfuly achieved．The best establishment and proliferation Of
shoot tips was obtained on 1／2MS(MS or Bs)basal medium supplemented with 1．5 mg／L BA and O～O．2 mg／
L IAA(or NAA)．Excised microshoots were rooted in vitro on 1／2MS in the presence of 1．0 mg／L IBA and
0．5～1．0 mg／L IAA with the optimum rooting results．Plantlets had been successfuly acclimatized and trans—
ferred to the greenhouse conditions．Complete plants were grown from mature and immature zygotic embryos
of black pepper incubated On filter paper bridges in test tubes containing liquid SH (M S or B5)basal medium
with not any growth regulators，and cali were induced with different combinations 0f auxins and cytokinins
．
Subculture of those cali onto the multiplication medium and differentiation medium led tO browning and death
finally，and no plant regeneration occurred．The morphogenetic potential of other explants such as leaf pieces
and hypocotyl segments from aseptic seedlings was also investigated in vitro
．
Callus induction was relatively
Received date：2004一10—08 Accepted date：2005-04—2O
Biography：LIU Jin-ping(1970一)，Male，Born in Qin County．Shanxi Province．Doctor，Associate professor，working in plant
genetics and breeding and plant tissue culture．
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46O 广 西 植 物 25卷
easy on MS(1／2MS，B5 or SH)basa1 medium fortified with a wide range of auxin—cytokinin combina i。“。’b
most attempts to regenerate plants from the calli were unsuccessful due to serious browning occurred during
the subculture onto the multiplication medium and differentiation medium．
Key words：Piper nigrum ；shoot—tip culture；embryo culture
Abbreviations：AC，Activated charcoal；AdSO4，
Adenine hemisulphate；B5，Gamborg medium(Gam—
borg t a1．，1968)；BA，N 一Benzylaminopurine；
CH，Casein hydrolysate~CM ，Coconut milk；2，4-D，
2，4-Dichlorophenoxyacetic acid；IAA，Indole acetic
acid；IBA，Indole-3一butyric acid；Kt，Kinetim MS，Mu—
rashige and Sk00g(1962)basal medium ~NAA，a—Naph—
thaleneacetic acid PVP，Polyvinylpyrrolidone SH，
Schenk and Hildebrandt(1972)basal mediurm
Black pepper(Piper nigrurn Linn．)is an ever—
green climbing shrub and one of the most impor—
tant tropical spice crops． In addition to flavoring
food and beverages，the fruit of black pepper is ex—
tensively used for culinary purposes such as in the
treatment of asthma，pains，disease of throat，etc
(Joseph et a1．，2001；Nybe et a1．，2001)．Whether
in volume，value or cultivation area，black pepper
has been the leader，and known as“king of the
spices”or“black gold”(Nambiar et a1．，2000)．
Black pepper is believed to have originated in the
forests of Western Ghats of Peninsular India，and
at present mainly cultivated in India，Vietnam，In—
donesia，M alaysia，Thailand，Brazil，Sri Lanka，Chi—
na and some other tropical countries． In China，
production of black pepper is dominated by Hain—
an，Yunnan Province and other regions． Of those，
Hainan is the largest producer of black pepper，
with a production staggering around 80 of the
total production in China，and its production value
ranks second just after that of rubber(Cai，1999)．
In vitro culture technique is the basis of in
vitro rapid clonal propagation of black pepper and
the application of biotechnology such as in vitro se—
lection，somaclonal variations，asymmetric somatic
hybridization and gene engineering in black pepper
cuhivars improvement． There are few earlier re—
ports on in vitro propagation of black pepper with
shoot—tips of seedlings of c．v．Panniyur 1(Mathews
P￡口Z．，1984)and mature vines of c．v．Panniyur 1，
Karimunda and Arivalli(Philip et a1．，1992)． In
addition，plantlets have been obtained from embry—
ogenic calli derived from zygotic embryos of black
pepper c．v．Panniyur 1 and Karimunda(Joseph et
nZ．，1996)．Daye(Lampong Type)，a black pepper
variety extensively cultivated in China，is high—
yielding but susceptible to foot rot．W ith the ob—
jective of employing in vitro culture techniques for
rapid clonal propagation and somaclonal improve—
ment particularly for resistance to foot rot，we first
examined plant regeneration potential of various
explants from the black pepper c．V．Daye．
1 Materials and methods
Stem segments，shoot tips，leaf，inflorescences，
seeds and zygotic embryos of field-grown plants of a
local black pepper variety，Daye(Lampong Type)，were
used as explants． From these nodal segm ents，leaf
blades were carefully removed，leaving behind the basal
stumps of the petioles covering the dormant axillary
buds．Then these nodal segments were washed under
running tap water for about 30~ 60‘rain．These nodal
segm ents were surface_disinfested in a solution of
0．11 mercuric chloride for 8～ 15 min with frequent
shaking after several seconds dip in 75 9／6 alcohol and
followed by three rinses with sterilized distilled water．
These nodal segm ents were trimmed into single node
explants around 2 cm long with the petiole stumps and
both ends of the nodal segm ents removed in a laminar
air flow cabinet． The shoot tips of approximately 2
mm lengths were excised from the treated nodal seg—
ments． Immature leaves and inflorescences were sur—
face-disinfested in a solution of 0．11 9／5 mercuric chlo—
ride for 8～ 10 min after several seconds dip in 75 al—
cohol and folowed by three rinses with sterilized dis—
tiled water．Then these explants were aseptically cut
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5期 刘进平等：大叶种胡椒实生苗茎尖培养和合子胚培养研究 461
into 0．25～ 0．36 nln％2 and 5～ 6 mm respectively．In
order to obtain seed explants，completely ripe fruits
were collected and rubbed in detergent solution with
the outer fleshy mesocarp removed． The seeds were
washed under running tap water for 10 min and surface
sterilized with 0．1 mercuric chloride for 15~20 min
followed by three rinses with sterile distilled water．
The zygotic embryos were dissected aseptically from
surface sterilized seeds．
Due to endogenous contaminants，contamina—
tion of all types of explants except for seeds and
embryos were not yet effectively controlled．Shoot
tips，leaf pieces and hypocotyl segments from asep—
tic seedlings were actually used as explants to in—
vestigate their morphogenetic potentia1．These ex—
plants were aseptically cut into pieces of appropri—
ate sizes，i．e．shoot tips(approximately 2 mm in
length)。leaf pieces(O．25～ O．36 mm。)and hypocot—
yls segments(5~ 6 mm in length)respectiVely．
1／2MS，MS，SH，B5 were used as the basal
media and they were supplemented with various
growth adjuvants such as 2，4-D(0~3 mg／L)，IAA
(1 mg／L)，NAA(0～ 5 mg／L)，IBA(2 mg／L)，BA
(O～ 4 mg／L)，Kt(0～ 1 mg／L)，CM (10 9／6 or
3O )，CH(400 mg／L)，AdSO4(100 mg／L)in dif—
ferent combinations and concentrations． In order to
control browning and necrosis of the callus，0．3
AC or 10 9／5 PVP was added．The PH of the media
were adj usted to 5．8 prior to autoclaving and al
the media were supplemented with 3 sucrose，
gelled with 0．8 agar except specially noted．The
culture bottles each containing 25 mL culture
media were autoclaved under a pressure of 1．1 kg／
cm。for twenty rain． Solidified media and liquid
media with filter paper bridges were used to cul—
ture the zygotic embryos．All the cultures were in—
cubated at 26± 2℃ under continuous illumination．
2 Resuhs and discussion
2．1 Surface-sterilization of various explants from
field-grown plants
Surface—sterilization of stem segments，shoot
tips，leaf，inflorescences of field—grown plants lead
to 100 percent of contamination and fungi were ma—
jor contaminants responsible for the results．White
hyphae still grew on the dead explants especially
on cuts even surface-sterilization with exceeding 15
min led to lethality of the explants．Re—sterilization
with 0．1 1 9／5 mercuric chloride had no positive
effect on the contamination problems of the ex—
Dlants． M oreover，contamination was relatively
higher with the seeds surface sterilized with 0．1
mercuric chloride for 15～ 2O min(the percentage of
contamination varied between 30 9／6～6O 9／6，some—
times to 100 )．These results indicated that inter—
hal contamination was responsible for the high con—
tamination percentage． Exogenous micro—organ—
isms can be effectively eliminated by surface steri—
lants(Bonga，1 982)，but endogenous contaminants
are very difficult to eliminate and is a serious prob—
lem with explants of field grown trees(Arnold et
口Z．，1986；Duhem et a1．，1988)．As for black pep—
per，the similar contamination problems were en—
countered by other researchers(Philip et a1．，
1 992)．This indicated that it is important in vitro
culturing with material free of endogenous contam—
inants for successful establishment of culture．The
internal contamination problem was overcome by
using the explants from in vitro grown seedlings in
this study．
2．2 M ultiplication of black pepper through in vitro
culture of shoot tips from aseptic seedlings
Effects of different concentrations of BA and
orientation of explants on shoot multiplication of in
vitro culture of shoot tips from seedlings of black
pepper were shown in Table 1． Average multipli—
cation rate of explants was greater with shoot tips
placed horizontally compared with those placed
vertically．Some explants placed vertically initiated
sporadic multiple shoots but most of them did pro—
duce single shoot and serious callus at all concen—
tration levels of BA． In terms of multiplication
rate，shoot growth and callus formation on the cuts
of explants，horizontal orientation of shoot tips was
found to be more suitable for shoot proliferation，
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462 广 西 植 物 25卷
compared with those shoot tips placed vertically on
the media in the establishment of first culture．The
explants placed vertically on 1／2MS without BA e—
ventually died． In the case of horizontal orienta—
tion，as the concentration of BA increased，the mul—
tiplication rate increased．Combination of 1．5 mg／
L BA and 0～O．2 mg／L IAA(or NAA)made the
newly initiating shoots more strong and healthy
(Plate I：A)．While the quality of newly forming
shoots was affected with the internodes of these
multiple shoots highly condensed and giving a ro—
sette-like appearance as the concentration of BA a—
bove 2 mg／L(Plate I：B)． Although maximum
number of shoots per explant was observed on this
medium，but the leaves of some of these multiple
shoots were thick，condensed，yellow or even white
with callusing tendency． Addition of adenine sul—
phate had no notably influence on proliferation of
multiple shoots． The best establishment and pro—
liferation of shoot tips was obtained on 1／2M S bas—
al medium supplemented with 1
． 5 mg／L BA and 0
～ O．2 mg／L IAA(or NAA)．Therefore。In vitro
clonal propagation of black pepper with shoot tips
excised from asept ic seedlings used as explants
through multiple—shoot multiplication methods
could be successfully achieved．
Al1 shoots of size up to 3 cm were separated
from the shoot clusters and transferred into rooting
medium．Four auxin treatments with 1／2MS used
as basal medium were tested． The optimum roo—
ring results was obtained l／2MS in the presence of
1 mg／L IBA and 0．5～ 1．0 mg／L IAA(Table 2，
Plate I：C)．Rooted plantlets had been successfully
acclimatized and transferred to the greenhouse con—
ditions(Plate I：D)．
The orientation of the explants on the medium
might have a significant effect on growth and pro—
liferation of explants from other crops(Gunatilleke
et(fZ．，l988)，but the effect had not been reported
in in vitro cult ure of black pepper by early re—
searchers(M athews et a1．，l984；Philip et(fZ． ，
l992)．HorizontaI orientation of the explants mav
have more contact with the medium and hence be
helpful for absorption of nutrients through a bigger
surface area． On the other hand，this orientation
may have a positive effect on releasing axillary
shoots from apical dominance．
Table 1 Effects of different concentrations of BA and
orientation of explants on shoot multiplication of in
vitro culture of shoot tips from seedlings of black
pepper(Piper nigrum)(cultured on 1／2MS)
Table 2 Effect of 4 auxin treatments on in vitr0
rooting of black pepper(Piper nigrum)on 1／2MS
2．3 In vitro culture of the zygotic embryos
At the end of 8 weeks of incubation of the zy-
gotic embryos on the solidified medium with no
growth regulator，the zygotic embryos j ust slightly
swelled with different degree of browning
． W hen
transferred onto the same medium ，the growth of
the zygotic embryos were arrested and eventually
died of browning．In vitro response of zygotic am—
bryos culture on filter paper bridges in culture tube
containing liquid SH supplemented with no growth
regulator and different combinations of auxins and
cytokinins was summarized in Table 3
． W ith no
growth regulator，the cult ured embryos germinated
into complete plantlets with 63．1 6 of germlna—
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5期 刘进平等 ：大叶种胡椒实生苗茎尖培养和合子胚培养研究 463
tion percentage(Plate I：E)．With different combi—
nations of auxins and cytokinins，calli were initiated
and proliferated with different frequencies of callus
induction(PlateⅡ：F)．The calli could be classified
into two types：one was watery，white or pale white
due to browning produced with one or two auxins；
the other was compact，white produced with the
combinations of auxins and cytokinins． W hen
transferred onto the same medium or 1／2 SH with
no growth regulator，the callus proliferation was
fast but all calli became browned and eventually
died(Plate n：G)．The results showed that all calli
produced in the study were with no regeneration a—
bility．Joseph et a1．(1 996)reported that plants re—
generated from embryogenic callus derived from
zygotic embryos wit h liquid SH without growth
regulators，but the zygotic embryos germinated and
gave rise into single complete seedlings with same
medium in our study．The reason for this might be
genotype—specific in vitro response．
Table 3 In vitro response of zygotic embryos of black
pepper c．V．Daye cultured on filter paper bridges in
culture tubes containing liquid SH supplemented
with no growth regulator and combinations
of auxins and cytokinins
Growth regulators
(mg／I )
Percentage of
embryos showing Annotation
cal】us induction
2．4 In vitro culture of leaf pieces and hypocotyl seg—
ments from aseptic seedlings
Callus induction and proliferation with the leaf
pieces and hypocotyl segments from aseptic seed—
lings of black pepper c
． v．Daye cult ured on MS(1／
2M S，Bs or SH)basal medium added with different
combinations of BA(0～ 15 mg／L)，NAA(0～ 5
mg／L)，Kt(0～ 1 mg／L)，2，4-D(0～3 mg／L)，CM
(10 or 3O )and／or CH(400 rag／L)were easily
achieved(PlateⅡ：H，I)．The calli were white wa—
tery，white spongy or compact slime—like． Neither
direct somatic embryogenesis nor adventious shoot
formation was observed，but root development was
occurred on the explants directly or callus initiated
from the explants when the relative concentration
of auxins was higher． Following transfer to the
same media or 1／2 basal medium with no any
growth regulator，callus soon turned brown and
necrotic and addition of 0．3 AC or 10％ PVP had
no effect． This phenomenon was not experienced
by other researchers．W ith the exception of shoot
tips，zygotic embryos and nodal ring tissue，plant
regeneration from the explants has not been repor—
ted．The results of this study and other research—
ers’work showed some explants of black pepper
were recalcitrant to regenerate in comparison with
pipli(P．1ongum)and betel vine(P．bete1)(Bhat et
“Z．，1995；Geetha et a1．，199O)．
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