Life Science Journal 2013;10(2)

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Plant Micropropagation from in vitro cultured bulb scales of Lilium lancifolium

Lu Sun, Zhongze Zhou*, Kemeng Cheng
School of Resources and Environmental Engineering, An hui University, Hefei, Anhui 230601, China
sunluanhui@sina.com
Abstract: Lilium lancifolium Thunb. is one of the best lilies which are edible in China but the efficient shoot
regeneration system has not been developed. The purpose of the present study is to establish an efficient and
reproducible protocol for induction of shoots in vitro from L. lancifolium bulb scales Shoot regeneration from in
vitro culture scales of L. lancifolium was tested on a orthogonal test with 4 factors and 3 level, containing different
concentrations of BA6-benzylaminopurine (6-BA) and a-naphthaleneacetic acid (NAA) and sucrose. The best
adventitious shoot induction was showed when grown on media containing MS (Murashige and Skoog, 1962)
+1.5mg/ L 6-BA and 0.1 mg/L NAA+20g/L sucrose according to the result. The highest adventitious shoot
proliferation multiple (4.1) was observed when grown on media containing1.0 mg/ L 6-BA and 0.1 mg/L NAA.
Regenerated shoots were rooted after 20 days grown on media containing 1/2MS+ 0.4mg/L NAA.
[Lu Sun, Zhongze Zhou, Kemeng Cheng. Plant Micropropagation from in vitro cultured bulb scales of Lilium
lancifolium. Life Sci J 2013; 10(2):2689-2692]. (ISSN: 1097-8135). http://www.lifesciencesite.com 373
Keywords: Lilium Lancifolium, Bulb scales, Micropropagation, Orthogonal test
1. Introduction
The genus Lilium, with approximately 220
species, belongs to the large family of the Liliaceae. At
present, 80 species of Lilium are found in the temperate
and subtropical zones of the northern hemisphere
(Woodcock and Stearn 1950; Feldmaier and McRae,
1982) and 47 species 18 varieties of Lilium are mainly
for the edible and medicinal use. Conventional breeding
in the lily is hampered by its heterozygous state and self
-incompatibility among the species of the different
Lilium groups (Van Tuyl et al., 1990). The development
of efficient systems for transformation could accelerate
the breeding process, but these techniques require an
efficient and reproducible protocol for the induction of
adventitious shoots. In lily, plant regeneration has been
achieved from a vast array of explants ranging from
flower organs to bulb scales (Han et al., 1997; Mitsuguet
al., 2002; Kumar et al., 2005).
Tissue culture techniques have been successful
for rapid propagation of some members of the genus
Lilium, including L. Iongiflorum (Nhut, 1998), L.
rubellum (Nimii et al., 1997), L. lancifolium
(Marinangeli and Curvetto, 1997), L. auratum
(Takayama and Misawa, 1979, 1980, 1983), and L.
testaceum (Wozniewski et al., 1991). However, there
have been few reports about micropropagation of L.
lancifolium where bulb scales originating from
regenerants through in-vitro culture are used.
L. Lancifolium known as DaoChui lotus, tiger
lily, is a perennial herb, as the Liliaceae Lilium of
bulbous perennial flowers. Along with the development
of the economy, the lily cut flower production will
become the mainstreams and it needs a lot of ball. At
present, it is not able to meet the needs of edible
production. The lily bulb is mainly for vegetative

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2689

propagation, breeding bulb quantity easily degenerated

and their breeding cycle is longer. Tissue culture can
not only solve the degradation of the lilies but also can
solve the problem of detoxification and propagation.
The objective of the present study was to
establish cultural conditions where shoot organogenesis
from bulb scales could be improved. Furthermore, the
results of micropropagation that used bulb scales
segments are described.
2. Materials and methods
Plant material
Primary explants consisted of the bulb scales of
the L. lancifolium. Bulbus were washed thoroughly
under running tap water for 2 hours, and then submerged
in 70% ethanol for 20s. They were then rinsed three to
five times with distilled water and placed in a
0.1%(wt/vol) aqueous solution of HgCl2 for 13 min and
rinsed three to five times in sterile distilled water. Under
aseptic conditions, the bulb scales were excised from
the bulbs and the lower portion (0.5 cm 0.5 cm) of the
inner scales was used as the explants.
Culture conditions
Pseudo-bulblet induction culture
The explants were inoculated on MS
containing different concentrations of hormone and
sucrose. At first, with a L 9 (33) orthogonal designed
experiment we established the optimum association of
6-BA, NAA and sucrose with two bulb scales per glass
jar and 10 replications (glass jars) for each
treatment(Table 1). The total induced pseudo-bulblet
number of L. longiflorum was recorded.

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Table1. Orthogonal test with3 factors and 3 levels L

9(33) for induction of shoots from L. lancifolium bulb
scales
6-BA (mg/L )
NAA (mg/L )
sucrose (g/L )
1.0
0.1
10
1.5
0.2
20
2.0
0.3
30
Adventitious bud proliferation culture
The induced pseudo-bulblets were excised when
they reached a size of 23cm and transferred onto MS
medium supplemented with different hormone
concentrations for adventitious bud proliferation. The
adventitious bud proliferation experiment was also set
up in a completely randomized block design with two
pseudo-bulblets per glass jar and 10 replications (glass
jars) for each treatment.The induced adventitious buds
were placed with medium onto the following media:
1 MS+0.5 mg /L 6- BA+ 0.1mg /L NAA
2 MS+1.0 mg /L6- BA+ 0.1mg /L NAA
3 MS+1.0 mg /L 6- BA+ 0.2mg /L NAA
4 MS+1.5 mg /L 6- BA+ 0.1mg /L NAA
Rooting culture
For the rooting, the shoots (about 3cm in length)
were excised, and placed vertically into half-strength
MS basal medium containing four NAA concentrations
(0.2, 0.4, 0.6 and 0.8 mg/L) and 15 g /L sucrose,
solidified with 7 g /L agar. The rooting experiment was
also set up in a completely randomized block design
with two shoots per glass jar and 10 replications (glass

jars) for each treatment.

Medium was adjusted to pH 5.8 before autoclaving
at 121 , 1 atm for 30 min. Cultures were incubated at
251C with a photoperiod of 12 h per day at a light
density of 40umol m2 s1 of (in light condition) and
7080% relative humidity.
3. Results and discussion
pseudo-bulblet induction culture
L. lancifolium bulb scales was cultured on the
nine kinds of media with different concentrations of
hormone and sucrose, after 7 days of incubation, most
bulb scale explants of L. lancifolium showed elongation
and enlargement. Regenerated shoots appeared within
7-11 days of culture initiation. While they resembled
bulblets morphologically, they did not exhibit
dormancy or an arrested phase in their development.
Bulb scales gradually turned into green and bulb scales
have obviously thicken at the same time, pale green
embryoid was the first appeared in turned green bulb
scales, then the adventitious bud was directly induced
by the embryoid .When the callus differentiation
particles and adventitious bud were not induced, the
bulb scale explants gradually turned into browning and
eventually died. The detailed orthogonal experiments
and related results are listed in Tables 2 respectively. By
variance analysis, the order of influence for induction
adventitious buds is BA6-benzylaminopurine (6-BA),
a-naphthaleneacetic acid (NAA) and sucrose.The
optimum experiment conditions is: 1.5 mg/L6-BA,
0.2mg/L NAA and 20g sucrose(Figure 1).

Table 2 Effect of various combinations of BA6-benzylaminopurine (6-BA), a-naphthaleneacetic acid (NAA) and
sucrose on pseudo-bulblet induction from bulb scales of L. lancifolium
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
T9
F

6-BA (mg/L)
1.0
1.0
1.0
1.5
1.5
1.5
2.0
2.0
2.0
109.8

NAA (mg/L)
0.1
0.2
0.3
0.1
0.2
0.3
0.1
0.2
0.3
11.48

sucrose(g/L)
10
20
30
30
10
20
20
30
10
8.1

Total number of adventitious buds

82
80
77
79
86
82
66
72
64

The time of turning green

7
8
10
9
8
7
9
9
12

Adventitious bud proliferation culture

Within 40 days, each pseudo-bulblet developed into a rosette of shoots. The number of shoots on each rosette
varied with the amount of 6-BA and NAA. The best proliferation medium was MS +6-BA 1.0mg/L+NAA 0.1mg/L,
in which the average number of shoots can reach 4.1 and length 1.8 cm (Figure 3 .Table3).
Table 3 The effect of 6-BA and NAA on the number of adventitious bud proliferation from pseudo-bulblet
Treatment
P1
P2
P3
P4

6-BA
(mg/L)
0.5
1.0
1.0
1.5

NAA
(mg/L)
0.1
0.1
0.2
0.1

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The total number of adventitious buds proliferation from

10 bottle of pseudo-bulblets
63
123
111
93

2690

Mean number of shoots per

pseudo-bulblet
2.10.1
4.10.1
3.70.1
3.10.1

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Rooting culture
When transferred to half MS medium
supplemented with different concentration NAA, all of
treatments used in the present study induced rooting.
However, the improvement of rooting was found in
half-strength MS medium supplemented with 0.20.8
mg /L NAA (Figure 4 .Table4).
Table 4 Effect of NAA on rooting of L. Lancifolium
regenerated shoots after 20 days of culture
Treatments
NAA(mg/L)
Number of roots per
shoot
R1
0.2
3.780.78
R2
0.4
5.942.34
R3
0.6
3.800.39
R4
0.8
2.780.99

Figure 4 elongated shoots rooted after20 days o f

culturing

Figure 1 Pseudo-bulblet of L. lancifolium derived

vitro-grown sprout of bulb

Figure 2 callus of L. lancifolium derived from in from

in vitro-grown sprout of bulb.

Figure 3 Multiple shoots developing from one

pseudo-bulblet cultured in MS medium

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4. Discussion
In this experiment, applications of middle
levels of BA6-benzylaminopurine (6-BA) can enhance
meristem formation and shoot proliferation and may
manifest results similar to those caused by cytokinins in
many species (Reynolds, 1987). Likewise, Yun et al.
(2000),
studying
the
influence
of
6-BA,
a-naphthaleneacetic acid (NAA) and sucrose on the
development of L. lancifolium, found that 6-BA was the
most effective in promoting regeneration. We examined
their effects on shoot induction from bulb scale
explants, and found that organogenesis depended on the
specific type of plant growth regulator used in the MS
media. Several studies have demonstrated that BA is a
very effective promoter of shoot induction and
multiplication from Liliaceae (Godo et al., 1998; Nhut,
1998; Ulrich et al., 1999). Although plants treated with
higher levels of BA produced more shoots, they were
very short and compact, and some had abnormal leaves.
In contrast, lower concentrations may have little effects
on shoot growth (Ulrich etal., 1999; Han et al., 2001).
The pseudo-bulblets occurred from the bulb
scales accompanying with a few calluses in some
medium (Figure 2). The best pseudo-bulblet induction
medium designing the orthogonal test and the optimum
experiment conditions is MS +1.5mg/L 6-BA +0.2mg/L
NAA+20g/L sucrose. A rosette of shoot arised from the
pseudo-bulblet. The best adventitious bud proliferation
medium is MS +1.0mg/L 6-BA +0.1mg/L NAA and the
highest proliferation multiple is 4.1. Regenerated shoots
have been rooted and the best rooting medium is 1/2MS
+0.4mg/LNAA.
This research demonstrates the excellent
regeneration capacity for L. lancifolium. Our success
with in-vitro establishment clearly indicates that
micropropagation is an effective and useful technique
for the reproduction of this species. Mass production

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via tissue culture may also become a desirable

alternative to seed propagation, since the latter method
has inherently low germination rates and a slow growth
process.The orthogonal test is a scientific and systematic
design method by which concise test set with fewer test
cases is created. Another advantage of the orthogonal
test, compared to all factorial design, is reduction of
testing treatment amounts and determination of
scientific results (Zheng et al., 2010). In this orthogonal
test, only 9 treatments were performed compared to 71
treatments in all factorial experimental design, which
consumes more costs.
Acknowledgements:
Foundation item: the College Students
Innovation and Entrepreneurship Training Program
(Grant No.cxcy2012055).
Corresponding Author:
Zhongze Zhou,
School of Resources and Environmental Engineering,
Anhui University,
Hefei, Anhui 230601, China.
E-mail: zhzz@ahu.edu.cn
References
[1] Feldmaier C, McRae J. Lilien. Ulmer,
Stuttgart .1982.
[2] Godo T, Kobayashi K, Takami T, Matsui K, Kida T.
In-vitro propagation utilizing suspension cultures
of meristematic nodular cell clumps and
chromosome stability of Liliumformolongi hort.
Sci Hortic. 1998; 72: 193-202.
[3] Han DS, Niimi Y, Nakano M Regeneration of
haploid plants from anther cultures of the Asiatic
hybrid lily Connecticut King. Plant Cell Tissue
Organ Cult.1997; 47: 153158.
[4] Han BH, Yae BW, Yu H J, Sun JS.In-vitro
propagation of Spathiphyllum floribundum cv.
Cupid. Kor J Plant Tiss Cult.2001; 28: 209-213.
[5] Li Z, Qiu HP, Cui XY, Duan CQ. Optimization on
Anthocyanins Extraction from Wine Grape Skins
Using Orthogonal Test Design. Food Sci
Biotechnol .2010; 19: 1047-1053.
[6] Kumar, S., Kashyap, M., Sharma, D.R. In vitro
regeneration and bulblet growth from lily bulb scale
explants as affected by retardants, sucrose and
irradiance. Biol. Plant. 2005; 49, 629632.
[7] Mitsugu, H, Hitomi, M, Fuminori, K. Regeneration
of flowering plants from difficile lily protoplasts
by means of a nurse culture. Plantarum .2002; 215,

880884.
[8] Murashige T, Skoog F. A revised medium for rapid
growth and bioassay with tobacco tissue cultures.
Physiol Plant .1962; 15:473-497.
[9] Marinangeli P, Curvetto N. Bulb quality and
traumatic acid influence bulblet formation from
scaling in Lilium species and hybrids. Hort
Science .1997; 32:739-741.
[10] Nhut DT. Micropropagation of lily (Lilium
Iongiflorum).Plant Cell Rep. 1998; 17:913-916.
[11] Nimii Y, Nakano M, Saito S .Production of
commercial rubellum Baker bulbs: Effects of
volume and renewal of liquid medium on in-vitro
growth, bulb rot infection during cold treatment,
and post-in-vitro growth of bulblets. J Jpn Soc
Hortic Sci. 1997; 66:113-119.
[12] Reynolds JF .Chemical regulation in tissue
culture:An overview. Hort Science .1987;
22:1192-1194.
[13] Takayama S, Misawa M. Differentiation in Lilium
bulb scales grown in-vitro. Effects of various
cultural conditions. Physiol Plant .1979; 46:184.
[14] Takayama S, Misawa M. Differentiation in Lilium
bulb scales grown in-vitro. Effects of activated
charcoal, physiological age of bulbs and sucrose
concentration on differentiation and scale leaf
formation in-vitro. Physiol Plant. 1980; 48:
121-125.
[15] Takayama S, Misawa M. The mass propagation of
Lilium in-vitro by stimulation of multiple
adventitious bulb -scale formation and by shake
culture. Can J Bot .1983; 61:224~228.
[16] UIrich MR, Davies FT, Koh YC, Duray SA, Egilla
JN. Micropropagation of Crinum Ellen Bosanquet
by triscales.Sci Hortic .1999; 82:95-102.
[17] Van Tuyl, J.M, Van De Sande, K , Van Dien, R, Van
Holsteijn, H.M.C. Overcoming interspecific
crossing arriers in Lilium by ovary and embryo.
Acta Hort. 1990; 266, 317322.
[18] Wozniewski
T,Blaschek
W,
Franz
G.In-vitropropagation of Lilium testaceum and
structural
investigation of the storage
-1.4-glucomannan. Plant Cell Rep. 1991;10:
457-460.
[19] Woodcock HBD, Stearn WT.Lilies of the world.
Scribners,New York .1950.
[20] Yun SY, Lee MS, Lim SC, Shin JD.
Micropropagation of Heloniopsis orientalis
(Thunb.).C. Tanaka in-vitro. Kor J Plant Tiss
Cult.2000; 27:197-202.

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