全 文 ：热带亚热带植物学报 2006，14(3)：246—249
Journal ofTropical and SubtropicalBotany
两步外植体法高频再生麝香百合
刘菊华， 金志强， 韦带莲， 谭光兰， 徐碧玉
(中国热带农业科学院热带生物技术研究所，热带生物技术国家重点实验室，海 口571101)
摘要：采用两步外植体法，即以百合鳞片叶为初始外植体，以从初始外植体上长出的芽为次级外植体，成功建立了麝
香百合的高频离体再生系统。对不同的 BA浓度及次级外植体的不同部位对再生效果的影响，以及组培苗移栽前低温
处理的影响进行了研究。结果表明：不同部位的次级外植体中，以短缩苹切片出芽快、整齐、芽数多且粗壮；以MS附加
1．0 mg L·一BA和 0．1 mg L。-NAA的培养基最适于麝香百合的分化。 一个中等大小已脱春化的鳞茎通过两步外植体法
能扩繁出54 000株左右的新植株，从鳞片叶开始至开花仪需 8个月，而且4℃低温处理对开花期的影响不大。
关键词：麝香百合；组织培养；成芽；开花
中图分类号：Q943．1 文献标识码：A 文章编号：1005—3395(2006)03—0246—05
A Two—step M ethod for the Efi cient
’‘‘ ‘ ‘ ’Lilium longOelonMicropropaRation Ol tt tJ rum
LIU Ju—hua， JIN Zhi—qiang， WEI Dai一1 ian， TAN Guang—lan， XU Bi—yu
(State Key Laboratory ofTropical Crop Biotechnology,Institute ofTropical Bioscienee and Biotechnology,
Chinese Academy of Tropical Agricultural Sciences，Haikou 571 101，China)
Abstract： Bulb scale segments of Lilium longiflorum were used as original explants and shoot sections excised
from shoot clusters arosed from the first explants as the secondary explants．The efects of diferent concentrations
of BA(benzyladenine)and diferent parts of plant segm ents on shoot formation were studied．After roots form ed，
plantlets were treated at 4 0C for 0，7 and 30 days．The results indicated that M S medium supplemented with
1．0 mg L BA and 0．1 mg L一。NAA was optimum for shoot form ation and proliferation by using short basal stem
segm ent as the second explant．Moreover，cold treatment had liRle efect on flowering stage．About 54 000 new
plantlets could be obtained from one bulb．It took only 8 months from bulb scale to flowering．
Key words：Lilium longiflorum；Tissue culture；Shoot formation；Flowering
Lilium species (Liliaceae) are one of the three
major flower bulbs in the commercial market⋯ and
chosen as flesh cut flowers for their attractive colours．
Lilies are usually propagated by scaling，a technique
which produces 3-5 bulbs from each bulb scale，
depending on species，cultivar，and scale size．Scale
propagation produces limited numbers of bulbs[21．
Therefore， micropropagation in vitro is essential to
produce large num bers of bulblets in lily within a
short period of time．
In the micropropagation of Lilium species， there
were many studies on the regeneration of lily by using
bulb scale fragm ents【2-61，leaves ，anthers[81，flower
filaments 0】，stems and tepals⋯ to initiate cultures．
Received：2005——10——14 Accepted ：2006-03-13
Foundation item：The Scientifc Fund ofSouth China Tropical Agricultural University(No．Rky0529)
Corresponding author
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第 3期 刘菊华等：两步外植体法高频再生麝香百合 247
However，bulb scales in vitro were used as the best
explant source for the production of bulblets[3-5,12-15]．
Han et a1．嘲 documented a new method by the
formation of shoots with abnorm ally swollen basal
plates and the regeneration system had been greatly
improved． However， the exact number of new
plantlets obtained by micropropagation techniques
remains unknown． Moreover time and labor cost are
the main limiting factors in micropropagation．
In order to improve the micropropagation
eficiency of Liliurn longiflorum，we had developed a
two—step explant method， i．e． bulb scale segments
were used as original explants and shoot sections
excised from shoot clusters arosed from the first
explants were used as the secondary explants to
micropropagate new plantlets．
1 M aterials and methods
l-1 Treatment of the first explants
Medium—sized (50 g) healthy bulb scales of
Lilium longiflorum． stored in dark at 4"C for 69—9O
days to accomplish their vernalization，were used as
the first explants to induce shoots． Each bulb
contained 1 0—1 5 bulb scales．After washing in running
tap water for 1 2 hours， bulb scales were sterilized on
surface in 75％ ethanol for 30 seconds． then sterilized
in 0．1％ HgCl，for l5 minutes and then washed in
deionized water for three times． Bulb scales were cut
into 1 cm ×1 cm sections． each bulb scale could
be divided into 3—5 sections．These sections were
cultured on shoot induction media．
1．2 Treatment of the second explants
Leaves． sinail bulbs and short basal stems which
came from the shoots of first explants were cut into
segm ents in size of2 1TIn (width)xl0 ilil(1ength)
(1eaves)，4-5 il／1(width)x 7-8 inil(1ength)(smal
bulbs) and 8—10 mll(diameter) x 2 inll(height)
(short basal stems)．Each shoot could be divided into
1 5—1 8 segm ents． All these segm ents were used as the
secondary explants and were cultured on shoot
induction medium ，too．
1．3 Culture condition
The shoot induction media was MS【 6】supple—
mented with BA． 0．1 mg L NAA (a-Naphtha—
leneacetic acid)，30 g L sugar and 7 g L。agar．The
concntration of BA was chosen from 0．5，1．O，1．2，1．5，
1．8．and 2．0 mg L。。．
The root induction medium was 1／2 M S
containing 0．25 mg L‘ IBA(Indole一3-butyric acid)．
All experiments were perform ed in petri dishes
and glass bottles． Each vessel contained 1 5 ml
medium． Media were adjusted to pH 5．8 before
autoclaving at 121℃ for 15 rain． Cultures for shoot
induction and proliferation were performed under the
light with l 6 h photoperiod per day at quantum flux
density of 50 m0l m s。。from fluorescent lamps． All
cultures were incubated at 24±2℃ ．
1．4 Low temperature treatm ent
After washing in running tap water， form ed
plantlets were treated at 4℃ for 0．7 and 30 days．and
then transferred to greenhouse at temperature Of 28—
30~C (day)／20-25~C (night)and at ilum ination
intensity of about 1 000 ktmol m。 s1．
1．5 Calculation of the number of plantlets
The total num ber of plantlets from one bulb
could be calculated according to this form ula：T=s xax
bxcxd
total plantlet num ber／bulb；s：the number of
bulb scale／bulb；0：the segm ent number／bulb scale；6：
shoot number／segm ent： c：segm ent num ber／shoot；d：
shoot cluster number／segment．
2 Results
2．1 Shoot induction and proliferation
The original explant expanded quickly and
form ed shoots when cultured on the medium
with different concentration of BA after 50 days．
Considering the num ber and fresh weight of shoots
induced， the MS medium containing 1．0 mg L。1 BA
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248 热带亚热带植物学报 第 l4卷
was optimum for the formation and proliferation of
shoots(Plate I：A；Table 1)．The segments of leaves，
small bulbs， and basal stems excised from the shoots
arosed from the first explants were cultured in the
same medium (MS medium containing 1．0 mg L
BA1．Fourteen days 1ater，the first batch of shoots from
secondary explants form ed(Plate h B1．Shoot clusters
form ed after 26 days． The size， number and flesh
weight of shoots induced from the leaf segm ents were
less than those of the shoots cluster generated from
short basal stems． Considering the number and flesh
weight of shoots． the sections of short basal stems
were the best secondary explants for quick form ation
of shoots clusters．and followed by small bulbs(Plate
I：C，Table 2)．
0．25 mg L一 IBA to induce roots(Plate I：D)．Thirty
days later，strong roots form ed from shoots．After low
temperature treatment as mentioned above， all the
plants within 4 months stepped into flowering stage at
the same time(Plate I：E1．This result indicated that
there was little efect of cold treatm ent time on
flowering time．
2．3 Number of plantlets
In this study． number of plantlets could be
calculated according to this form ula：T=s xaxb xc xd，in
which s=lO，a=3，b=30，c=15，d=4．Considering the
survival rate of 1 00％ (data not shown)， each bulb
could produce about 54 000 new plantlets． It is
suggested that the micropropagation efi ciency of
Lilium longiflorum had been greatly improved．
Table l Efiects ofBA concentration on jnduction ofshoots from bulb
scales ofLiliMm fo， g 。rMm cultlred f0r 50 days 3 Discussion
Diferent letters in same column represent significant diferences at
P=0．Ol by Ducan’s multiple range test．
Table 2 Effects ofdifierent shoot sections on the formation of
shoot clusters cultured for 40 days on MS medium
containing 1．0 mg L- BA and O．1 mg L。 NAA
Diferent letters in same column represent significant diferences at
P=-0．01 by Ducan’s multiple range test．
2．2 Rooting and flowering
Shoots were excised仔om shoot clusters and were
cultured on 1／2 MS medium supplemented with
Han et a1．【 】reported the cycle method from shoot
section to shoot cluster could efectively promote
shoot proliferation． Similar results were also reported
by Stanilova et a1．【 7】．Maesato et a1．【 8】and Nhut【 91．W e
have confirm ed that the two—step explant method
worked eficiently， and the segm ents of short basal
stems had the best regeneration capacity in Lilium
longiflorum． The total number of plantlets from one
bulb was about 54 000．
Difrerent kinds of cytokinins have diferent efect
on the shoot form ation．Han et a1．[21 reported that BA
with IAA was more favorable on shoot form ation．Our
result had diferent from it．According to our research．
when the BA concentration was 1．0 mg L～．the shoots
were very strong and the number of shoots increased
greatly(Table l，Plate I：A，C)．Therefore，we only
used the same kind of medium Of MS containing
1．0 mg L—BA and 0．1 mg L‘’NAA which was most
suitable to initiate shoot and proliferate shoot(Table
1)．Thus the whole process could be greatly simplified．
Lilium is one of the three bulb crops in the
commercial market【1】．To ensure flowering，it requires
vernalization． Han et a1．【 reported that the form ed
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第 3期 刘菊华等：两步外植体法高频再生麝香百合 249
bulblets generated flower stems after cold treatment at
5℃ for2—3 months．
Ranwala et a1．【加】 reported that mature plants
were stored in darkness at 3℃ for 2 weeks before
placing them in a postharvest evaluation room (22℃)
to accomplish vernalization．Therefore from bulb scale
to flowering it required too long time， which greatly
limited the contribution of flower to the market．
According to our research， if the bulbs had been
storaged at 4"C to accomplish their vernalization， the
new formed plantlets need not cold treatm ent at 4"C
before planting into greenhouse even in tropical area．
Thus the growthperiodcouldbe greatly shortenedand
onlyneed 8months． Itmightbedueto allthe cells of
bulb which could diferentiate into new plan tlets had
completed their vernalization during storage at 4~C
before culture． M ore interesting． even if the new
form edplan tlets hadbeentreated at4℃ for0． 7． 30
days before culture， their flowering dates were the
salleinthefirstyear(PlateI：E)．
Acknowledgements We wish to thank Professor W ang
Zhu-lian for his kindly providing experiment materials and Mr
．
LaiKangforhis carefulyplantingplantlets
． Althe authors are
grateful to W u Xin-rong for carefuly reading this manuscript
．
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Explanation ofplate
PlateI：
A：Theform ationofsho ts from bulbscalesonMSmedium +1
．0mgL
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l{^ d L NAA c‘ I rc after {day~；
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1¨ |_h¨1h 。l1⋯ 1d“‘ I’ l "_l _¨ u d‘⋯ ～ ⋯ ⋯I1 w c_1
lJ)T L BA nd m l AA {nr14山 ．
⋯Tl lI rn1．1I J⋯ { huuI 1 h r nuI11[hc cc 10】_’LI■㈣n ba~LI 【⋯
1u Ⅵs rnvdi with ¨ n IIA ：lld(1l1 L N^^ r
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【¨ 】Ⅲ } v
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⋯ 1h(，lJT t 0lf I r ”He1 h f，Ⅲt【 lⅢ nl uf 4[_inr 7 d~ys：c：cold
c¨¨ 1¨1cnl l 4 c n n d±
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