In Vitro Cell.Dev.Biol.
—Plant (2012) 48:172–179
DOI 10.1007/s11627-012-9429-0
MICROPROPAGATION
Adventitious shoot regeneration of oriental lily
(Lilium orientalis) and genetic stability evaluation
based on ISSR marker variation
Xiaomei Liu & Guochen Yang
Received: 14 September 2011 / Accepted: 6 February 2012 / Published online: 7 March 2012 / Editor: J. Forster
# The Society for In Vitro Biology 2012
Abstract A regeneration system was developed for oriental
lily (Lilium orientalis) based on both leaf and bulb scale.
Adventitious shoots were regenerated from leaves of in vitro
cultures on Murashige and Skoog medium containing thi-
diazuron (TDZ) or 6-benzylaminopurine (BA) and naphtha-
leneacetic acid (NAA). The highest percent regeneration
from leaf explants was 74.2%, being observed on medium
containing 10.8 μM TDZ and 0.54 μM NAA. The highest
mean number of shoots generated was 4.4 and was obtained
from bulb scale explants on medium containing 0.54 μM
TDZ and 0.54 μM NAA. Adventitious shoots were success-
fully rooted at rates ranging from 79.2% to 100%. The
rooted plantlets survived after acclimatization in the green-
house. The effect of kanamycin concentration on adventi-
tious shoot regeneration was also evaluated, a value of
100 mg l−1 being suggested as a lethal dose for lily trans-
formation. Eighteen ISSR markers were employed to deter-
mine the genetic stability of the regenerated shoots in
comparison to their mother plant. Eleven primers in total
produced 70 clear and reproducible bands. Genetic similar-
ity indicators among the clonal derivatives and the mother
plant ranged from 0.92 to 1.0. All 15 micropropagated
progenies and the mother plant could be grouped together
in one major cluster with a similarity level of 92%. The
somaclonal variation rate across the plantlets was estimat-
ed as 4.2%, indicating that direct shoot formation from
explant regeneration is a safe method for multiplication of
“true-to-type” plants.
X. Liu : G. Yang (*)
Department of Natural Resources and Environmental Design,
North Carolina A&T State University,
Greensboro, NC 27411, USA
e-mail: yangg@ncat.edu
Keywords Acclimatization . Growth regulators .
Kanamycin . Rooting
Introduction
Lilies (Lilium spp.) are monocotyledonous ornamental plants
belonging to the family Liliaceae. They provide one of the
three major types of commercial market flower bulbs—the
others being rose and tulip. The lily is one of the most valuable
floricultural crops in the southern USA, Holland, Japan, and
Mexico (Roh and Lawson 1988), valued for its ornamental
function as a cut flower or a potted plant. Many species are
widely grown in temperate and sub-tropical regions. A large
number of ornamental hybrids have been also developed.
They can be used in herbaceous borders, woodland, and shrub
plantings and as a patio plant. Some lilies, especially Lilium
bulbs, are starchy and edible, although the bulbs of some
species may be very bitter. The non-bitter bulbs such as Lilium
lancifolium, Lilium pumilum, and especially Lilium brownii
are grown on a large scale in China as a luxury or health food
and most often sold in a dry form. They are eaten especially in
the summer to reduce internal body heat.
Despite wide prevalence, many cultivars suffer from viral
and microbial diseases. Conventional breeding methods have
not been successful for control of many of these problems, so
alternative breeding methods may provide an effective solu-
tion. Genetic engineering is a useful tool for introduction into
plants of desirable traits such as modified flower quality,
flower color, flower structure, resistance to diseases and
insects, and stress tolerance. A reliable and efficient in vitro
system to regenerate phenotypically normal plants is a prereq-
uisite for establishment of transformation techniques.
There have been several reports of in vitro regeneration in
lily using various tissues such as leaves, stem nodes,
 ADVENTITIOUS SHOOT REGENERATION OF ORIENTAL LILY 173

filaments, pistils, pedicels, and bulblet scales (Tribulato et
al. 1997; Irifune et al. 2003; Yang et al. 2007; Mi and Liu
2008). It has been reported that leaves are good for regen-
eration of plantlets by organogenesis and that filament and
bulb scales are effective for direct regeneration (Mi and Liu
2008). Ectopic expression of two MADS-box genes from
orchid and lily alters flower transition and formation in lisian-
thus, revealing the potential for floral modification in orna-
mentals through genetic transformation (Thiruvengadam and
Yang 2009).
Plant tissue culture is recognized as one of the key
components of biotechnology methods because of its poten-
tial to regenerate elite genotypes and to conserve valuable
plant genetic resources. However, regeneration methodolo-
gy also incurs the risk of inducing genetic variability, name-
ly somaclonal variation between sub-clones of a single
parental line (Larkin and Scowcroft 1981). In vitro cultures
pose a problem for recovery of “true-to-type” regenerated
plants due to chromosomal rearrangement, gene amplifica-
tion, gene mutation (Saker et al. 2000), and retrotransposon
activation (Hirochika 1993).
Clonal variation can be detected by DNA profiling using
different types of molecular genetic markers such as random
amplification of polymorphic DNA (RAPD), inter-simple
sequence repeat (ISSR), or restriction fragment length poly-
morphism. ISSR has proven to be effective and reproducible
for detection of genetic uniformity (Martins et al. 2004).
Compared to RAPD amplification primers, ISSR-directed
primers are longer and so offer advantages for the detection
of somaclonal variation. The advantages of using ISSR
primers include a higher degree of sensitivity, increased
stringency, and higher reproducibility of polymorphic ge-
netic alleles. ISSR markers have been successfully applied
to the analysis of genetic fidelity in cauliflower (Leroy et al.
2000), almond (Martins et al. 2004), banana (Lakshman et
al. 2007; Rout et al. 2009; Lu et al. 2011), Cymbopogon
martinii var motia (Bhattacharya et al. 2010), gerbera (Bhatia
et al. 2009), and Swertia chirayita (Joshi and Dhavan 2007).
The objective of the present study was to establish a regen-
eration system based on use of different explants (leaves and/
or bulb scales) for oriental lily (Lilium orientalis) and to also
evaluate genetic stability of plantlets derived from the mother
plant. The results will provide important information for our
long-term objective to genetically transform oriental lily with
an RNAi vector to silence the recently cloned AGAMOUS
homolog gene from lily (gene accession no. is JN314420) in
order to modify flower structure.
Materials and Methods
Plant materials and culture conditions. Stock oriental lily
(L. orientalis) plants were grown in the glasshouse, and bulb
scales were excised. The outer bulb scales were removed,
and the innermost scales were used as explant materials. The
scale bulbs were washed thoroughly under running tap
water, then surface-sterilized with 70% (v/v) ethanol for
30 s, followed by a 20% (v/v) bleach solution for 20 min,
and finally rinsed with sterile water five times over. The
prepared explants of bulb scales were placed on Petri dishes
containing Murashige and Skoog (MS) medium (Murashige
and Skoog 1962) containing 3% (v/v) sucrose, 0.7% (w/v)
Difco–Bacto agar, and different concentrations of thidia-
zuron (TDZ) and naphthaleneacetic acid (NAA; Table 1).
Leaves harvested from in vitro grown lily shoots were
excised to 0.5 cm in length and used as explants. Leaf
explants were then placed on the same culture medium
treatments as for the bulb scale explants. The pH of the
medium was adjusted to 5.7 before addition of agar and
autoclaved at 121°C for 20 min. For each treatment, at least
two Petri dishes (100×15 mm2) containing 20 ml of each
treatment medium were prepared with six leaf explants or

Table 1. Adventitious shoot

regeneration from oriental lily Plant growth regulators Bulb scales Leaf
explant sources leaves and bulb
scales Regeneration Mean shoot no. Regeneration Mean
rate (%) rate (%) shoot no.

0.44 μM BA+0.54 μM NAA 43.2±6.7a 2.9±1.1a 26.2±2.0de 1.2±0.4a

1.1 μM BA+0.54 μM NAA 43.7±6.0 a
3.0±1.1a 61.6±7.0ab 1.6 ±0.6a
2.2 μM BA+0.54 μM NAA 43.3±18.3a 4.2±1.8a 42.0±14.5c 1.5±0.6a
4.4 μM BA+0.54 μM NAA 48.2±18.9a 3.3±1.8a 39.2±9.8cd 1.9±0.8a
8.8 μM BA+0.54 μM NAA 36.4±12.0a 3.3±1.6a 45.4±10.5c 1.2±0.4a
0.54 μM TDZ+0.54 μM NAA 53.4±21.8a 4.4±2.4a 50.0±4.5bc 2.7±1.2a
Values (means±SE) followed by
the same letter in the same col- 1.35 μM TDZ+0.54 μM NAA 33.4±8.3a 3.8±1.8a 24.1±7.0e 2.0±0.8a
umn are not significantly differ- 2.7 μM TDZ+0.54 μM NAA 56.3±6.8a 3.6±1.6a 51.9±1.7bc 1.8±0.7a
ent at α00.05 5.4 μM TDZ+0.54 μM NAA 49.0±8.8a 3.1±1.5a 36.9±10.0cde 1.6±0.7a
BA 6-benzylaminopurine, NAA 10.8 μM TDZ+0.54 μM NAA 51.2±15.0a 3.6±1.9a 74.2±4.3a 2.3±0.9a
naphthaleneacetic acid
174 LIU AND YANG
bulb scale pieces. Cultures were incubated in the dark at 25°C
for 4 wk before exposure to light with a 16-h photoperiod
(100–140 μmol m−2 s−1). After 4 wk on MS, cultures were
transferred to MS medium containing different concentrations
of 6-benzylaminopurine (BA), or TDZ and 0.54 μM NAA for
shoot elongation. This experiment was conducted three times
(total of 72 explants per treatment). Regeneration percentage
and number of shoots per explant were recorded after 8 wk.
Rooting of regenerated shoots. Shoots (2–3 cm in length)
were excised and then placed in Magenta GA-7 vessels
containing MS medium with 2% (w/v) sucrose and 0.7%
(w/v) Difco–Bacto agar with different concentrations of
indole-3-butyric acid (IBA; Table 2). Cultures were placed
in the dark for 4 d prior to transfer to a 16-h photoperiod
(100–140 μmol m−2 s−1). There were 20 shoots for each
replication, conducted three times for a total of 60 shoots.
The number of roots, root length, and percent rooting were
recorded 4 wk after shoots were placed on root induction
medium. Rooted shoots were gradually acclimatized into the
greenhouse.
Sensitivity to kanamycin. Leaf explants were placed on the
regeneration medium with the highest regeneration rate (MS
containing 9.08 μM TDZ and 0.54 μM NAA) based on the
outcomes of our previous experiments and cultured again on
regeneration medium with kanamycin at 0, 25, 50, 75, or
100 mg l−1 for 28 d in the dark. Twenty explants were used
for each treatment and replicated three times. The response
of leaves was determined by counting the number of shoots
initiated.
DNA extraction and PCR amplification conditions. Total
genomic DNA was extracted from 100 mg young leaf tissue
of 15 randomly chosen in vitro-derived clones in addition to
the mother plant, using the DNeasy Plant Mini Kit (Qiagen,
Valencia, CA). Quality and quantity of genomic DNA was
assessed by NanoDrop spectrophotometer ND-2000
(Thermo Scientific, Suwanee, GA). Eighteen ISSR primers
(XXIDT Integrated DNA Technologies Int., Coralville, IA)
were used for standardization of optimum annealing tem-
perature. All the PCR components used in this study were
purchased from Thermo Scientific. PCR amplification was
Table 2. Rooting of adventi-
tious lily shoots Rooting medium
Values (means±SE) followed by ½ MS+0.49 μM IBA
the same letter in the same col- ½ MS+1.23 μM IBA
umn are not significantly differ- ½ MS+2.45 μM IBA
ent at α00.05
½ MS+4.9 μM IBA
IBA indole-3-butyric acid
performed in an Eppendorf thermal cycler (Eppendorf,
Hamburg, Germany). PCR was performed in a 25-μl mix-
ture containing 25–50 ng DNA, 2.5 μl 10× Taq buffer,
2.5 mM MgCl2, 0.2 mM dNTP, 1.6 μM primer, and 1 U
Taq DNA polymerase. PCR reaction conditions consisted of
an initial denaturation step at 94°C for 3 min, 40 cycles of
30 s at 94°C, 50 s at Tm, 1 min at 72°C, and a final extension
step at 72°C for 10 min. Amplified products were resolved
by electrophoresis on 2.0% (w/v) agarose gels in TAE (1×)
buffer, stained with ethidium bromide for 20 min, and
photographs were taken by using a Gel Documentation
system (Bio-Rad, Hercules, CA).
All reactions were repeated at least twice, and only those
bands that were reproducible across all runs were considered
for analysis. Only distinct, reproducible, and well-resolved
fragments ranging from 200 to 2,000 bp were considered for
the analysis. These bands were scored either as present (1)
or absent (0) for each of the ISSR markers across the 15
micropropagated plants. Electrophoretic DNA features of
low visual intensity, which could not be readily distin-
guished as present or absent, were considered to be ambig-
uous markers and were consequently not scored. The size of
the amplification products was estimated using a 1-kb plus
molecular size marker (Thermo Scientific). The similarity
matrix was subjected to the cluster analysis of unweighted
pair group method with arithmetic average (UPGMA) and a
dendrogram was generated by NTSYSpc (Exeter Software,
Setauket, NY) that was used to perform the distance matrix
and clustering analysis of the complete data set (Rohlf 2000).
Data analysis. Shoot numbers are presented as the mean±
standard error (SE) of shoots regenerated from leaf explants
and bulb scales. Regeneration frequency was expressed as
the average percentage of leaves that developed shoots
divided by the total number of leaf explants. To understand
the relationship between factors, regeneration percentage,
and mean shoot number, data representing dependent vari-
ables were regressed upon the TDZ or BA concentration as
the independent variable. The same method was applied for
analysis of rooting data. Linear and quadratic models were
tested to fit the data at α00.05 level of significance. When
the quadratic model was not significant, the linear model
was used. If regression did not fit, the data were analyzed
Days to root Mean root Mean root Rooting rate
initiation number length (cm) (%)
14 3.47±1.9b 2.36±0.53b 81.1±2.5c
13 4.55±2.6b 2.06±0.72c 86.9±1.8b
13 5.16±2.8b 2.67±0.63a 79.2±2.0c
12 7.82±4.1a 2.80±0.70a 100a
 ADVENTITIOUS SHOOT REGENERATION OF ORIENTAL LILY
with an analysis of variance using the GLM procedure of SAS
(software version 9.2). The Duncan’s test was used to distin-
guish differences between treatment means at α00.05 level.
Results and Discussion
Shoot regeneration. After 2 wk of incubation, most leaf
explants showed elongation and enlargement. Formation of
adventitious shoots was observed after 3 to 4 wk, around the
wounded area (Fig. 1A). Shoots developed through both
indirect (involved in callus formation) and direct organo-
genesis. Most shoots developed directly from explants via
direct organogenesis, while only a few shoots developed via
indirect organogenesis. Shoots turned green when trans-
ferred to the shoot elongation medium after 2 wk (Fig. 1B)
and elongated 4 wk after culture on this medium (Fig. 1C).
A significant difference in the shoot regeneration rate was
observed among ten treatments. The regeneration rate
ranged from 24.1% to 74.2%. The most superior shoot
regeneration rate (74.2%) was achieved from leaf explants
cultured on medium containing 10.08 μM TDZ and
0.54 μM NAA (Table 1). However, there was no significant
difference between treatments for mean number of shoots.
Most of the leaf explants only developed one to two shoots,
the mean number of shoots ranging from 1.2 to 2.7 per
explant. Also, shoot organogenesis occurred from explants
cultured on control medium in the absence of plant growth
regulators, but the regeneration rate was very low (15%).
When bulb scales were used as explants, new bulblets
appeared 4 wk after culture on the treatment media. Several
bulblets developed directly from single explants. The color
Figure 1. Adventitious shoot regeneration of oriental lily from leaf and
bulb scale explants. (A) New bulblets induced from leaf; (B) adventitious
shoots arising from leaf explants; (C) elongated adventitious shoots; (D)
175
of the new bulblets was white (Fig. 1D). The regeneration
rate across treatment conditions ranged from 43.2% to 56%.
There were no significant differences for shoot regeneration
between treatments. In addition, the medium that produced
the highest mean number of shoots (4.4) did not produce the
highest regeneration rate (74.2%). Additionally, shoot elon-
gation and new shoot development were observed when
bulblets were transferred to the shoot elongation medium
(Fig. 1E). Adventitious shoot regeneration of oriental lily
has been previously reported, with similar results. Bacchetta
et al. (2003) investigated adventitious shoot regeneration
from leaf explants derived from two cultivars of Lilium
species with very different regeneration rates. The highest
regeneration rates were, respectively, 83.3% for the first
cultivar when using half-strength MS medium supple-
mented with BA and IAA and 33.3% for the second cultivar,
with a very large standard deviation of 19.1%. Kumar et al.
(2005) achieved a regeneration rate of 67% when using bulb
scale as explants for oriental lily hybrid cultivar (Lilium
auratum×Lilium speciosum×Lilium rubellum) ‘Star Gazer.’
In the present study, variations in regeneration rates were
observed from different replications for the same culture
medium. This effect was probably due to different cell
competency. When using bulb scales, cell competency
may differ across several layers. An alternative explanation
for the experimental variation was contamination when us-
ing bulb scales. Contamination rates varied from one repli-
cation to another. Since fresh bulb scales from the soil were
used for this experiment, a sterilization procedure had to be
involved. When leaf explants were used, the results from
different replications were consistent with much smaller
variation because actively growing young leaves were
new bulblets regenerated from bulb scale explants; (E) adventitious
shoots elongating from bulb scale; (F) elongated shoots rooted; (G)
acclimatized oriental lily seedlings in the green house.
176 LIU AND YANG

Table 3. Kanamycin sensitivity of oriental lily for leaf explants transformed plants. It is important to evaluate and minimize
−1
Kan (mg l ) Regeneration rate Mean no. of shoots the effect that this antibiotic could have on adventitious
shoot regeneration. After obtaining regeneration results
0 86.9±13.5a 2.4±0.8a from both leaf and bulb scales, the leaf explants were chosen
25 83.3±2.4ab 2.0±0.8a for kanamycin sensitivity experiments. Two reasons dictated
50 58.4±19.9b 1.9±0.9a this choice: Firstly, the leaf explants, as stated earlier, are
75 9.1±3.6c 1.5±0.7a from in vitro cultures and therefore sterile and abundant, and
100 0 0 secondly, these explants exhibited a higher regeneration rate
than the bulb scale explants.
Values (means±SE) followed by the same letter in the same column are Adventitious shoots were still able to regenerate from leaf
not significantly different at α00.05
explants cultured on media containing kanamycin at 25, 50,
Kan kanamycin
or 75 mg l−1. However, no adventitious shoot regeneration
was observed when leaf explants were cultured on media
selected as explants from very similar positions on the mother containing kanamycin at or greater than 100 mg l−1. Both
plants. Furthermore, these leaf explants were obtained from in the regeneration rate and the mean number of shoots de-
vitro growing plantlets that were 100% sterile. creased when the concentration of kanamycin in the culture
medium increased. This regression showed that the quadrat-
Rooting. Lily shoots developed roots as early as 2 wk after ic model fitted well. Duncan’s tests showed that there was a
culture on rooting media containing IBA (0.49, 1.23, 2.45, significant difference among different kanamycin treatments
or 4.8 μM; Fig. 1F). The rooting rate among treatments (Table 3). Only a few shoots (1.5± 0.7) regenerated on
ranged from 79.2% to 100% (Table 2). The mean number medium containing kanamycin at 75 mg l−1, and these
of roots increased as IBA concentration increased. There shoots became chlorotic, did not elongate, and failed to root.
was a significant difference for all three rooting parameters Shoot regeneration efficiency from the control of kanamycin
(rooting rate, mean root number, and mean root length) sensitivity experiments was higher than that obtained from
between the four treatments. experiments involving control by plant growth regulators.
The explanation was probably due to the season, since
Effects of kanamycin on shoot regeneration. The antibiotic the alternative experiments were conducted at different
kanamycin is commonly used for selection of adventitious times of the yr. Plant cell competency varies depending

Table 4. List of primers, their sequence, numbers, and size of the amplified fragments generated by ISSR primers in oriental lily

Primer name Sequence Tm (°C) Reaction to template DNA No. of monomorphic No. of polymorphic Size range
bands bands (bp)

1 (AG)8YA 48.9 Positive, reproducible 5 0 200–1,500

2 (AG)8RA 48.7 Negative
3 (AG)8YT 49.2 Positive, reproducible 4 0 200–600
4 (AG)8RT 48.8 Positive, not reproducible 3 0 300–500
5 (GA)8RT 47.4 Positive, reproducible 3 0 200–600
6 (GA)8RG 48.4 Positive, reproducible 2 0 200–700
7 (GA)8RC 48.9 Positive, not reproducible 4 0 200–1,000
8 (GA)8YG 48.8 Negative
9 (GT)8RA 51.4 Positive, reproducible 5 1 200–1,000
10 (AC)8RG 54.3 Positive, reproducible 4 0 200–1,000
11 (CT)8YA 47.1 Negative
12 (CT)8RT 47.1 Positive, reproducible 4 0 300–800
13 (CT)8RG 48.1 Positive, reproducible 9 1 200–1,000
14 (CT)8RC 48.6 Positive, reproducible and monomorphic 10 0 250–1,300
15 (CA)8RC 53.1 Positive, reproducible 5 0 200–1,000
16 (CA)8RA 51.7 Positive, reproducible 7 2 300–1,200
17 (GT)8RA 51.4 Negative
18 (GT)8YA 51.4 Positive, reproducible 5 0 200–700
 ADVENTITIOUS SHOOT REGENERATION OF ORIENTAL LILY 177

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1500 bp Genetic stability assessment by ISSR marker. The regenera-
1000 bp tion protocol used in this study involved formation of ad-
700 bp ventitious shoots from in vitro-generated explants (leaves)
500 bp and bulb scales. To validate the efficacy of this approach, it
was essential to ascertain the genetic status of resultant
progeny. ISSR markers were employed to characterize the
100 bp
genetic similarity of the mother plant and its derivatives. Of 18
primers used, 12 generated clear, reproducible, and distin-
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1500 bp guishable pattern of bands in the size range of 0.2 to 1.3 kb
1000 bp for the mother plant and its clonal derivatives (Table 4). Ten
700 bp primers produced monomorphic bands. Three primers were
500 bp polymorphic showing dissimilarity between the control moth-
er plants and their clonal derivatives. Primer numbers 13, 14,
and 16 proved to be highly reproducible. Overall, the number
100 bp
of bands per primer ranged from 3 to 9. A total of 70 scorable
bands were produced; the average number of bands per primer
Figure 2. PCR amplification products obtained by ISSR primer 13 was 5. However, four primers produced no amplification
(above) and 14 (bottom). Primer 13 produced a pattern with poly-
morphic bands, primer 14 produced monomorphic bands. Lane M
products at all. Modification of PCR amplification and stain-
samples from the mother plant; lanes 1–15 in vitro raised clones; lane ing conditions did not substantially improve the patterns, and
16 1bk plus DNA ladder. so the sub-optimal results were probably due to characteristics
of the primers, or the relative abundance of priming sites in
lily genomic DNA. Primers based on (CT)n arrays (numbers
on the season, and so timing of the in vitro experiments 13 and 14) produced highly distinguishable and reproducible
could have affected the regeneration efficiency, even profile patterns, suggesting that this repeat type is highly
though the medium components were identical (Amin and abundant in oriental lily. Profile quality depends on the motif
Jaiswal 1987). abundance, as has been confirmed by several other reports.

14

15

10

11

12

13

0.90 0.93 0.95 0.97 1.00

Coefficient

Figure 3. UPGMA-based dendrogram showing the similarity among in vitro-generated plants of lily and the mother plant. M mother plant, 1 to 15
the derived plants.
178 LIU AND YANG
Blair et al. (1999) reported that (GA)n motifs were more
common than (GT)n in rice. Primers based on the (GA)n
motif produced significantly more bands on average (57.3±
4.9) than primers based on the (GT)n motif (12.6±7.1).
Lakshman et al. (2007) reported that in banana, both
(GA)n and (GT)n were abundant, as primers based on them
produced high quality DNA profiles. Similarly, Joshi and
Dhavan (2007) reported that in S. chirayita, (AG)n, (GA)n,
and (GT)n repeat array types were most abundant.
Cluster analysis was performed to assess the coefficients
of similarity derived from the ISSR data of the 70 scored
bands. Comparison of the bands exhibited a similarity range
from 0.92 to 1 among the 15 clonal derivatives and the
mother plant (Fig. 2). The analysis demonstrated that based
on the ISSR generated coefficients, all 15 clonal derivatives
and the mother plant could be grouped together in a single
cluster with a 92% level of similarity (Fig. 3). This value
indicated a very low level of genetic divergence for clonal
shoot regeneration.
Extensive research has been conducted into the mecha-
nism of somaclonal variation. The low level of genetic
instability that was detected may be attributed to the accu-
mulation of somatic mutations during the in vitro culture
period. In most cases, such variation is considered to be an
undesirable side effect of in vitro propagation (Larkin and
Scowcroft 1981). It was recognized that the presence or
absence of variations during in vitro propagation depends
upon the source of explants and the mode of regeneration
(Goto et al. 1998), including levels of growth substances
that are used (Martin et al. 2006). Axillary branch applica-
tion and somatic embryogenesis are better methods for
generation of genetically stable clones. However, organo-
genic differentiation is very prone to somatic variation
(Pontaroli and Camadro 2005).
Considering the importance of ensuring the genetic sta-
bility of in vitro plants in any conservation program, it is
important to choose a procedure that does not induce vari-
ation. The experiments presented in this study have shown
that the direct shoot formation (direct organogenesis) can be
induced in oriental lily from both leaf and bulb scale
explants. As there is no callus formation, this regeneration
process is genetically stable. This finding supports the ob-
servation that a meristem-based micropropagation system is
genetically stable. The present data are similar to reports of
others. Plants regenerated from adventitious buds derived
from well-developed meristematic tissues exhibited the lowest
tendency for genetic variation (Goto et al. 1998; Joshi and
Dhavan 2007). Greater variation was found among plants
derived from callus, as compared to those raised from em-
bryogenic tissue (Al-Zahim et al. 1999; Yang et al. 1999).
Our results revealed that direct regeneration is effective at
maintaining regeneration frequency with morphogenetic
competence. Both leaf and bulb scales can be used as
explants for lily regeneration via direct organogenesis. The
highest regeneration rate for leaf explants was higher than that
for bulb scale explants. However, the highest number of
regenerated shoots was lower. Use of in vitro regenerated
leaves as explants for oriental lily regeneration and future
transformation is recommended because of their ready avail-
ability. Leaf explants produced the most superior regeneration
rate (74.2%), with much smaller experimental variations com-
pared to bulb scale explants. The best regeneration medium
was MS supplemented with 10.8 μM TDZ+0.54 μM NAA.
For oriental lily, direct shoot formation from explant regener-
ation is a safe mode for multiplication of true-to-type plants.
The regeneration system described in this study will be
used for transformation of oriental lily and provide an alter-
native approach to achieve desirable breeding objectives.
The long-term goal of this research is to develop a reliable
system for genetic modification of the flower morphology of
the oriental lily by modification of the Lily AGAMOUS gene
by RNAi silencing. It may be hypothesized that this ap-
proach will result in deletion of the stamen from the lily
flower and will improve its commercial value, since pollen
spillage may be damaging to clothing fabrics and may also
produce undesirable allergenic effects in people. The pro-
posed technique could be also be used to target other aller-
genic pollen-producing plants.
Acknowledgments The authors wish to thank Dr. Ken Gruber and
Laurie Gengenbach for their review and editing of the manuscript. This
research was jointly supported by the Center of Excellence for Post-
Harvest Technology and the Agricultural Research Program, North
Carolina A&T State University, USA.
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