全 文 ：广 西 植 物 Guihaia 23(3)：259—263 2003年 5月
仙人掌的微繁殖
程 磊，胡宋英
(上海中医药大学中药学院，上海 200032)
摘 要：成功建立了仙人掌离体快繁的实验体系，并且对影响微繁殖的一些因素，诸如激素组合、外植体的物
理状态、大量元素的含量等进行了研究。结果表明：BA对仙人掌芽增殖具明显作用，MS+BA 5．0 mg／L+
IBA 0．1 mg／L为最适增殖培养基；接种方式实验表明劈接优于整棵。钙、镁离子浓度对试管苗生长没有影
响，但影响生根数及根长；NAA抑制根的伸长，但一定浓度可促进生根。总体而言，最适的生根培养基为 1／2
MS。同时发现块接比单芽接具有优势。试管苗在形态上出现一些变异。实验结果对仙人掌科其它植物的快
速繁殖具有参考意义。
关键词：仙人掌；离体培养；增殖；茎段；纵劈；茎节
中图分类号：Q943．2 文献标识码：A 文章编号：1000—3142(2003)03—0259—05
M icropropagation of Opuntia
dillenii(Ker-Gaw1．)Haw．
CHENG Lei．HU Song—ying
(Shanghai University of Traditional Chinese Medicine，College of Traditional
Chinese Materla Medica，Shanghai 200032，China)
Abstract：A protocol is described for rapid in vitro propagation of the valuable chylocaula Opuntia dillenii
(Ker-Gaw1．)Haw．in vitro．The influence of various combinations of growth regulators，the physical stage of
explants and the concentration of the macroelements were also evaluated．It was found that bud variation was
dependent on BA supply，the synergistic combination of 5．0 mg／L and 0．1 mg／L IBA induced the optimum
frequency(5．3 buds／stem segment)．Stem segment with longitudinal split was prior to whole segment on bud
induction．The concentration of Ca +，Mg +affected root number and length，but had no efects on plantlet
height and growth．Though root length of plantlets declined with increased NAA concentration，0．2 mg／L
NAA promoted root number．Considering together，the best rooting medium was half—strength MS．It was ob—
served that sprout tuber was superior to single nodal plate on bud number as well as bud growth．Some mor—
phological variations exhibited in the transferred test—tube plants．This micropropagation procedure may pro—
vide the basis for improvement of this chylocaula and was beneficial to the tissue culture of other plants of Cac—
taceae．
Key words：Opuntia dillenii；in vitro；micropropagation；stem segment；longitudinal split；nodal plate
Opuntia dillenii(Ker—Gaw1．)Haw． is a pe—
rennial chylocaula of the Cactaceae，Opuntia genus
with planate green nodal plate，obovate to elliptic，
with clumped arbusculate．It is distributed in Flor一
收稿日期：2001—12—21 修订日期：2002—06—24
作者简介：程 磊(1976一)，男，江苏阜宁人，博士生，中药学专业。
ida of America，the W est Indies，Mexico and South—
America．And there are also some natural or half—
wild species grow in China，Australia and India．
Cactus(O．dillenii)can be propagated by seeds or
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26O 广 西 植 物 23卷
by stem cuttings． It usually used in grafring as
stock of Zygocacuts truncatus(Hqw．)K．Schum．
or Schlumbergera bridgesii(Yu et al，1993)．
The stems，flowers and fruits of cactus are
commonly used as forages，foods and medicines in
ancient China．It had already regarded as a tradi—
tional Chinese medicinal material listed in“Tradi-
tional Chinese Medicinal Dictionary”(Jiangsu New
Medicinal Academy，1986)．
Recent reports indicated the analgesic and an—
ti—inflammatory properties of O．dillenii(Park et
al，1998；Loro et al，1999)．Besides，the composi：
tion(Chen et al，1998；Elkossori et al，1998)and
chemical constituents(Qiu et aZ，2000)of Cactus
was investigated． Further，Cactus pear fruit may
become a new source for a natural food addictive
(Saenz et al，1998；Zhang et al，1992；Fu et al，
1993)．And exploitation perspective of Cactus has
been proposed(Wang et al，2001)．
W e only found very few previous reports a—
bout in vitro culture techniques of Cactaceae：Chen
et al(1999)only reported the grafting of 0．dille—
nii，but didn’t referred to it’s micropropagation；
Cai et al(2000)just published propagation of Echi—
nocactus grusonii very simply． Escobar et al
(1986)，Mohamed et al(1995)，Chen et al(2001)re-
spectively reported the tissue culture of O． amy—
claea，0．ficu~indica，0． milpa alta detailed，
which referred to the proper medium ，transplanted
conditions and position of explants．
In the resent paper，we report a rapid propaga—
tion through stem segments of wild plants of O．
dilleni followed by sHccessful establishment of re—
generated plants in pear and vermiculite．This sys—
tern will rapidly provide many plants for investiga—
tions of the efficacy and potentia1 commercia1 appli—
cation．
1 Materials and methods
1．1 Plant materials
New sprouted nodal plate．
1．2 Methods
1．2．1 Surface sterilization and inoculation
Juvenile nodal plates were excised from mater—
nal plants． The explants were washed thoroughly
for 30 min under running tap water，followed by
surface sterilized by dipping in 70％ ethanol for 30
s，then immersed in 0．1％ HgC12 for 9 min，fol—
lowed by 5 rinses with sterile distilled water．Ex—
posed ends of each explant were given a fresh cut
before they were cultured in MS medium．
1．2．2 Culture medium and conditions
The culture medium used for the present work
was basal medium with 3 9／5(w／v)sucrose and 0．
8 (w／v)agar．The media were further augmented
with different concentrations of hormones． The
pH of the media was adjusted to 5．8．The follow—
ing media were used：
MS0：MS(Contro1)；MS1：MS with 2．0 mg／L
BA，0．1 mg／L IBA；MS2；MS with 5．0 mg／L BA，
0．1 mg／L IBA；MS3：MS with 8．0 mg／L BA，0．1
mg／L IBA；MS4：MS with 10．0 mg／L BA，0．1 rag／
L IBA．
CMo：half-strength MS(1／2 MS)with 59．86
mg／L Ca抖，18．01 mg／L Mg。 ；CM1：1／2 MS with
strengthened(1×)Ca (119．71 mg／L)and Mg
(36．03 mg／L)；CM2：1／2 MS with strengthened(2
×)Ca (179．57 mg／L)and Mg抖(54．04 mg／L)．
Ro：1／2 MS(Contro1)；R1；1／2 MS with 0．1
mg／L NAA；R2：1／2 MS with 0．2 mg／L NAA；R3：
1／2 MS with 0．5 mg／L NAA．
The cultures were maintained at 2 5±2℃ with
12一h day light at an intensity of 60#mol·m一 ·s‘
(National MZD 30 w)．
1．2．3 Multiplication of nodal plate cultures
Nodal plates(1．3cm height)from MS medium
were cultured on propagational media(Mo—M4)to
evaluate the effect of combination of hormones．
Subsequent subcultures were at 30一day intervals．
Nodal plates sprouted in M2 medium were used in
the following experiments．
1．2．4 Comparison of inoculation manner
Nodal plates were subcultured in two man—
ners：one inoculated with the whole nodal plate；
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3期 程 磊等：仙人掌的微繁殖 261
the other nodal plates split longitudinally， then
placed the longitudinal section exposed to the me—
dium．
1．2．5 Effect of Ca + and Mg2+
Nodal plates were subcultured on media(CMo
— CM2)to evaluate the effect of Ca and Mg on
growth of shoots．
1．2．6 Induction of rooting
Nodal plates were subcultured in rooting
media(Ro—R3)to evaluate the influence of NAA
concentrations．
Rooted plants were washed in tap—water and
then transplanted into peat+ vermiculite(1 ：1)
pots，covered for 10 days with storage bags to avoid
dehydration．Then acclimatized plants were trans—
ferred to ambient conditions．
Observations were recorded on the number of
buds per explant，number of roots per shoot，root
length，shoot height and plantlet fresh weight after
30 days；rooting data was recorded after 10 days．
2 Results
2．1 Shoot regeneration
Shoot regeneration from O． dillenii nodal
plate explants cultured on M S basal and MS medi—
um supplemented with various concentrations of
BA in combination with IBA is summarized in Ta～
ble】．
Table I Efleets of combination of hormones on
propagation of O．dilleni
Note：buds(> 3 mm)are assessed by counting)“一”means no
bud；“+”means the quantity of buds(<3 mm)．
The results presented in Table 1．show that
the number of buds produced increased as the BA
concentration increased from 0 to 5．0 mg／L．How～
ever，explants on 8．0 mg／L and 10．0 mg／L BA
produced few，small and glomerate buds，indicating
an upper limitation BA concentration for the micro—
propagation of O．dillenii(Plate I：1)．However，
it must be pointed out that shoots which sprouted
first were obtained on M，medium 18 days after in—
duction，the first sprouting was delayed on M 1 me—
dium on the 22nd d，and took place after 30 days on
M 3，M4 media．In additional，the higher concentra—
tion of BA was found to promote callus formation．
2．2 Inoculation manner
This experiment(Table 2)showed that buds
produced from split explants were about twice as
much as that of whole explants(Plate I：2)．
Table 2 Effects of inoculation fashion on
propagation of O．dillenii
Note：M eans data followed are significantly different using test of
significance(t> to． 05)．
2．3 Growth of shoots
The effects of Ca and M g on the growth of
O．dillenii are shown in Table 3．
Table 3 Eff~ts of concentration of Ca2+ and
M g2+ on growth of O．dilleni
The shoots cultivated on CM1 produced more
roots than those cultivated on other media． The
mean length of the roots produced on CM1 and
CM3 media was significantly higher than that ob—
tained on CM2．But there’s not an evident effect of
treatments on shoot height and plantlet fresh
weight．The above results showed that Ca and
Mg抖 affect the induction and elongation of roots
．
The present study reveals that the relatively low
concentration of Ca and Mg of M S is beneficial
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262 广 西 植 物 23卷
for root induction and growth of O．dillenii(Plate
I：3)．
2．4 Rooting
From 85 shoots used to evaluate the rooting，
a1l the plantlets were obtained giving an average
rooting of 1 00 after 1 0 days’cultivation．
W ith regards to the rooting media，length of
roots declined with increasing NAA concentration．
The highest observed number of roots was ob—
tained using 0．2 mg／L NAA，indicating a little
Dromotion of NAA on root ind uction(Fig．1)．Con—
sidering together，the number of roots per shoot
and root length， the appropriate and economical
rooting medium is 1／2 MS(Plate I：4)．
80
20
0
0 0．1 0．2 0．5
Concentration of NAA／(mg／L)
2
～
0
1．5 e-
曲
芒
1
0．5
《
0
Fig．1 Effects of concentration of NAA
on rooting of O．dillenii
~ Roots／Shoot) ⋯●⋯Average length of root
3 Discussion
From these results it appears that it is possible
to micropropagate cactus with satisfactory results．
Among the growth regulators utilized to induce
shoot proliferation，MS with 5．0 mg／L BA，0．1
mg／L IBA，seems to give the best results．This re—
suit was different from the papers of other re—
searchers(Escober et al，1986；Mohamed et aZ，
1995；Chen et al，2001)，which may caused by dif—
ferent species of Cactaceae．
The required level of cytokinin in the medium
seems to be higher than other plants．But the vari—
ation tendency of bud sprouting is as same as previ—
OUS reports(Zhou et al，2000；Zhang et al，1996)．
Explants on high concentration of BA commonly
produced glomerate and malformed shoots(Zhou et
aZ，2000；Zhang et al，1996；Fracaro＆ Echeverriga—
ray,200 1)．All the nodal plates induced in the
Dresent experiment are cylindrical which lost their
natural flattened form． Newly developed nodal
Dlates from the acclimatized plantlets gradually dis—
played the flattened tendency(Plate I：5)．This re—
sult can be attributed to the high level of BA，
which is considered as an important factor on cell
division during plant tissue culture． Because the
effect of exophytohormone，cells of nodal divided
in all directions so as to form a cylindrical shape．
The influence of BA in vitro decreased as the pla—
ntlets transferred to ambient conditions． So the
newly sprouted nodal plates returned to norma1．
Similar variation was not reported in other papers
(Chen et aZ，1999；Cai et aZ，2000；Escobar et aZ，
1986：Mohamed et al，1995；Chen et al，2001)．
In the current study，the physical condition of
the tissue through explanting significantly affected
the micropropagation of O．dillenii． Split explants
induced more buds can be attributed to the large
contact surfaces to the medium，which can absorb
more nutrients and hormones．It was also reported
in other plants(Zhou et al，1999a，b)．
An interesting phenomenon was also observed
in the present study when sprout tuber and single
nodal plate were cultured in the M 2 medium with
the same conditions． The buds induced from
sprout tuber grew vigorously and rapidly， but
those induced ftom single nodal plate grew shortly
and slowly(Plate I：6)．This finding can be attrib—
uted to the build—up effect(Yan，1991)．Each ex—
plant diffused metabolites into the surrounding me—
dium during cultural periods． The levels of such
substance would affect the growth and prolifera—
tion of cultures，which are reached in high density
of explants populations， but not in low density
populations where the metabolites diffuse out into
a relatively large volume of culture medium．This
phenomenon was found in single cell cultivation
置 u、 00
石0us，sl00-1 0 Ja0暑T1Z
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3期 程 磊等：仙人掌的微繁殖 263
advantageous to the rooting of shoots． But the
present study reveals the inhibited effect of NAA
on rooting．
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程 磊，等：仙人掌的微繁殖
CHENG Lei，et a1．：M cropr。pagation of Opuntia dillenii(Ker Gaw1．)Haw
囱版 I
Plate I
1．Effects of combination of hormones on propagation。f0．dillenii(from leftto right：M。，MI，M2，Ma，M{)；
2 Effects of inoculation manners on propagation of 0 dillenii(1eft：whole；right：split)；3．Efects of concen—
tratkm of Ca¨+and Mg2+Oil growth of dillenii(from lefttO right：CM0，CM1，CM2)；4
． Effects of COncen—
trat[on of NAA on rooting of0．dillenii(from left tO right：R。，RI，R2，R3)；5．Newly developed nodal plate
from the acclimatized plantlet；6．Effects of sprout tuber and single nodal plate on propagation of O．dillenii
(Ieft：sprout tuber；right：single nodal plate)．
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