ORIGINAL PAPER
An efficient, widely applicable cryopreservation of Lilium shoottips by droplet vitrification
Zhen-Fang Yin • Wen-Lu Bi • Long Chen •
Bing Zhao • Gayle M. Volk • Qiao-Chun Wang
Received: 11 November 2013 / Revised: 13 April 2014 / Accepted: 14 April 2014
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014
Abstract We report a straightforward and widely appli-
cable cryopreservation method for Lilium shoot tips. This
method uses adventitious shoots that were induced from
leaf segments cultured for 4 weeks on a shoot regeneration
medium containing 1 mg/l a-naphthaleneacetic acid and
0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length)
including 2–3 leaf primordia were precultured on Murash-
ige and Skoog (MS; 1962) medium with 0.5 M sucrose for
1 day and then treated with a loading solution containing
0.4 M sucrose and 2 M glycerol for 20 min, followed by a
Plant Vitrification Solution 2 (PVS2) treatment for 4 h at
0 �C. Dehydrated shoot tips were transferred onto 2.5 ll
PVS2 droplets on aluminum foil strips, prior to a direct
immersion into liquid nitrogen for 1 h. Frozen shoot tips
were re-warmed in MS medium containing 1.2 M sucrose
for 20 min at room temperature, followed by post-thaw
culture for shoot regrowth. Shoot regrowth levels ranged
from 42.5 % for L. longiflorum 9 Oriental ‘Triumphator’
to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean
shoot regrowth level of 67.1 % across the six diverse Lilium
genotypes tested. Histological observations found that the
survival patterns were similar in cryopreserved shoot tips of
‘Triumphator’ and ‘Siberia’. Assessments using inter-sim-
ple sequence repeat markers found no differences in re-
generants recovered from the control stock cultures and
from cryopreserved shoot tips in ‘Triumphator’ and ‘Sibe-
ria’. This Lilium droplet-vitrification cryopreservation
method is efficient, simple and widely applicable for the
long-term conservation of lily genetic resources.
Keywords Adventitious shoot � Genetic stability � Lilies �Histological study
Abbreviations
NAA a-Naphthaleneacetic acid
TDZ Thidiazuron
MS Murashige and Skoog (1962)
PVS2 Plant Vitrification Solution 2
ISSR Inter-simple sequence repeat
SMM Shoot maintenance medium
BM Basal medium
BA 6-Benzylaminopurine
LPs Leaf primordia
DMSO Dimethylsulfoxide
LN Liquid nitrogen
RM Rooting medium
AC Activated charcoal
FAA Formaldehyde acetic acid
fixative
TB Toluidine blue
AD Apical dome
Communicated by B. Borkowska.
Z.-F. Yin � W.-L. Bi � L. Chen � Q.-C. Wang (&)
State Key Laboratory of Crop Stress Biology for Arid Areas,
Key Laboratory of Genetic Improvement of Horticultural Crops
of Northwest China, College of Horticulture, Northwest A&F
University, Yangling 712100, Shaanxi,
People’s Republic of China
e-mail: [email protected]
B. Zhao
State Key Laboratory of Crop Stress Biology for Arid Areas,
Key Laboratory of Genetic Improvement of Horticultural Crops
of Northwest China, College of Forestry, Northwest A&F
University, Yangling 712100, Shaanxi,
People’s Republic of China
G. M. Volk
USDA-ARS National Center for Genetic Resources
Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA
123
Acta Physiol Plant
DOI 10.1007/s11738-014-1543-7
Introduction
Lilium is one of the most economically important orna-
mental species worldwide with tremendous horticultural
popularity as a result of its large, attractive flowers and
medicinal functions (Nhut et al. 2001; Bacchetta et al.
2003). The genus Lilium includes many ornamental species
and more than 3,000 cultivars (Godo and Mii 2001). Lilium
longiflorum, L. Oriental hybrids, L. Asiatic hybrids, L.
davidii var. unicolor, L. 9 formolongi and L. longiflo-
rum 9 Oriental are among the most economically impor-
tant groups of lily (Chinestra et al. 2010).
Successful plant genetic improvement programs are
dependent upon the availability of novel genetic resources
(Wang et al. 2012). Genebanks can provide researchers
access to diverse materials, but clonally maintained field
collections are both expensive and vulnerable to threats
from diseases and natural disasters. In vitro tissue culture
conservation is also expensive and is susceptible to con-
tamination and somaclonal variation (Scowcroft 1984).
Cryopreservation has become an effective method for the
long-term conservation of plant genetic resources (Benson
2008; Wang et al. 2012).
Bouman and de Klerk (1990) were the first to cryopre-
serve shoot tips of Lilium speciosum using a two-step
freezing method. They obtained survival level of 8 %.
Bouman et al. (2003) reported a vitrification procedure and
obtained survival levels that ranged from 5 to 93 % across
the six lily species tested. Matsumoto et al. (1995) reported
successful cryopreservation of shoot tips of Lilium using
vitrification, which resulted in shoot regrowth levels
between 40 % for L. specisum f. album novum Malle and
85 % for L. platyphyllum. Recently, Chen et al. (2011)
published a method for Lilium cryopreservation that used
droplet vitrification with regrowth levels of 67.6, 62.0 and
35.0 % for L. lancifolium, Lilium 9 Siberia ‘Siberia’ and
Lilium 9 longiflorum ‘Snow Queen’, respectively.
There are still several important issues that need to be
addressed with regard to cryopreservation of lily shoot tips.
First, a widely applicable cryopreservation procedure for
Lilium is still lacking, severely inhibiting the use of cryo-
preservation for long-term preservation of genetic resour-
ces (Matsumoto et al. 1995; Chen et al. 2011) as well as the
use of cryotherapy for production of pathogen-free plants
(Wang et al. 2009; Wang and Valkonen 2009). Second, in
all of the previous studies, in vitro stock shoots were cold-
hardened at 0–5 �C for 5–7 days (Bouman and de Klerk
1990; Matsumoto et al. 1995; Bouman et al. 2003; Chen
et al. 2011) to obtain high level of regrowth from cryo-
preserved shoot tips. Cold hardening requires a tempera-
ture-controlled growth chamber and is also time
consuming. Third, genetic stability of the plants recovered
from cryopreserved shoot tips is an important concern
(Harding 2004). All of the previous studies used shoot tips
harvested from adventitious shoots derived from bulb-scale
segments. Somoclonal variation may occur during the
formation of adventitious shoots (Scowcroft 1984). In
addition, some steps, such as preculture with high sugar
concentrations and plant vitrification solution (PVS) treat-
ment may result in genetic alterations in regenerants fol-
lowing cryopreservation (Harding 2004). Therefore,
assessment of genetic integrity in regenerants following
cryopreservation is desirable. However, information of
genetic stability in regenerants recovered from cryopre-
served shoot tips of lily has been lacking. Fourth, in the
previous studies, shoot tips were excised from bulb-scale
segments that had been cultured for 40–75 days (Mat-
sumoto et al. 1995; Chen et al. 2011). However, efficiency
of adventitious shoot regeneration, which significantly
affects the rate in which materials can be processed for
cryopreservation, has never been evaluated.
The objective of the present study was to develop an
efficient, widely applicable method for cryopreserving
shoot tips of diverse Lilium species and hybrids using a
droplet-vitrification technique. Genetic stability of regen-
erants after cryopreservation was assessed using inter-
simple sequence repeat (ISSR) markers.
Materials and methods
Plant materials
Six lily genotypes belonging to five Lilium species and
hybrids were used in the present study. They are L. Oriental
hybrid ‘Siberia’, L. Asiatic hybrids ‘Elite’ and ‘Pollyanna’,
L. davidii var. unicolor, L. 9 formolongi and L. longiflo-
rum 9 Oriental ‘Triumphator’. L. Oriental hybrid ‘Siberia’
was primarily used for the initial optimization of the key
parameters that affect the droplet-vitrification cryopreser-
vation method. The other five genotypes were subsequently
used to test the established cryopreservation procedures.
In vitro stock shoots were maintained on shoot mainte-
nance medium composed of a basal medium (BM) sup-
plemented with 1 mg/l 6-benzylaminopurine (BA) and
0.2 mg/l a-naphthaleneacetic acid (NAA), according to
Yin et al. (2013). BM consisted of half-strength Murashige
and Skoog (MS; 1962) medium containing 30 g/l sucrose
and 7 g/l agar. The pH of the medium was adjusted to 5.8
prior to autoclaving at 121 �C for 20 min. The cultures
were grown in a growth chamber at 23 ± 2 �C under a
16-h photoperiod with a light intensity of 45 lmol m-2 -
s-1 provided by cool-white fluorescent tubes. Subculture
was performed every 4 weeks.
Acta Physiol Plant
123
Adventitious shoot regeneration from leaf segments
Adventitious shoot regeneration was performed as descri-
bed in our previous report (Yin et al. 2013). In brief, leaf
segments, each 0.8–1.0 cm long and 0.4 cm wide, were
excised from the third to sixth nodes from 4-week-old
in vitro stock shoots. Three transverse cuts were made
across the segments in 1 mm intervals. Next, the leaf
segments were cultured on half-strength MS medium
containing 1 mg/l NAA and 0.5 mg/l thidiazuron (TDZ) in
the light conditions as described for in vitro stock shoots.
After 4 weeks of culture, adventitious shoots regenerated
directly, without callus formation.
Cryopreservation using droplet vitrification
Shoot tips (1.5–2 mm in length) containing 2–3 leaf pri-
mordia (LPs) (Fig. 1a) were excised from 4-week-old
adventitious shoots (Fig. 1b) and cultured on BM for 1 day
for stabilization. Shoot tips were precultured onto solid MS
containing different concentrations of sucrose (0.25, 0.5,
0.75 or 1.0 M) for 0, 1, 2, 3 or 4 days in the same growth
conditions as were used for the in vitro stock shoots.
Precultured shoot tips were osmoprotected in a loading
solution comprised MS containing 2 M glycerol in com-
bination with different sucrose concentrations (0, 0.2, 0.4,
0.6, 0.8 or 1.0 M) for 0–100 min at room temperature.
Then, loaded shoot tips were exposed to plant vitrification
solution 2 (PVS2) (Sakai et al. 1990) at 0 �C for varying
lengths of time between 0 and 6 h. PVS2 contains MS
supplemented with 30 % (w/v) glycerol, 15 % (w/v) eth-
ylene glycol, 15 % (w/v) dimethylsulfoxide and 0.4 M
sucrose (Sakai et al. 1990).
Ten droplets, each containing 2.5 ll fresh PVS2, were
placed on a sterile aluminum foil strip (7 9 20 mm) and a
single shoot tip was transferred into each droplet (Fig. 1c).
Each strip was then directly immersed into liquid nitrogen
(LN). After 3 min, each foil strip was transferred into a 1.8-
ml cryovial, which had been previously filled with LN, and
stored in LN for 1 h. Thawing was performed by taking the
Fig. 1 Cryopreservation of shoot tips of Lilium Oriental hybrid
‘Siberia’ by droplet vitrification. a An excised shoot tip (1.5–2 mm)
used for cryopreservation. b Adventitious shoots regenerated from
basal leaf segments of L. Oriental hybrid ‘Siberia’. c Droplets with
shoot tips on an aluminum foil strip (7 9 20 mm). d A surviving
shoot tip 7 days after cryopreservation. e A dead shoot tip 7 days after
cryopreservation. f A regenerated shoot 4 weeks after cryopreserva-
tion. g A rooted plantlet obtained after 4 weeks on RM. Bars = 1 mm
Acta Physiol Plant
123
cryovials out of the LN and then foil strips were removed
from the cryovials and immediately transferred into an
unloading solution composed of MS with 1.2 M sucrose
and incubated at room temperature for 20 min.
Unloaded shoot tips were then placed in a Petri dish
(90 mm in diameter) containing 25 ml BM supplemented
with 1 mg/l NAA and 0.2 mg/l TDZ. The cultures were
placed at 23 ± 2 �C in the dark for 3 days and then trans-
ferred to the light for survival and shoot regrowth. Shoot tips
exhibiting green coloration after 7 days of post-thaw culture
were considered as survival, while shoot regrowth was
defined as surviving shoot tips that regenerated to shoots
(C5 mm in length) after 4 weeks of post-thaw culture.
Application of the droplet-vitrification procedure
to other Lilium species and hybrids
The droplet-vitrification cryopreservation procedure
developed for L. Oriental hybrid ‘Siberia’ was applied to
five additional lily genotypes including L. Asiatic hybrids
‘Elite’ and ‘Pollyanna’, L. davidii var. unicolor, L. 9 for-
molongi and L. longiflorum 9 Oriental ‘Triumphator’.
Histological studies of cryopreserved shoot tips
Histological studies were conducted to reveal pattern of
cell survival in cryopreserved shoot tips (?LN) of two
lilies, hoping to understand why they produced different
shoot regrowth rates following cryopreservation. Two lilies
were selected: ‘Siberia’ and ‘Triumphator’ that produced
the highest (87.5 %) and lowest (42.5 %) shoot regrowth
rates, respectively. After 24 h of post-thaw culture, shoot
tips were fixed in formaldehyde: acetic acid fixative (FAA,
50 % ethanol: formalin: acetic acid = 18:1:1) for 24 h,
stepwise dehydrated through an incremental ethanol series
(50, 70, 85, 90 and 95 %, each for 2 h), and stored in
100 % ethanol overnight. After embedding in paraffin,
5-lm-thick section was cut with a microtome (Leica
RM2235, Germany) and stained with 0.1 % toluidine blue
(TB; Sakai 1973). The sections were observed with a light
microscope (Leica DM2000, Germany). Shoot tips freshly
excised from 4-week-old adventitious shoots were served
as a positive control, while those that were freshly excised
and directly immersed in LN, re-warmed and post-thaw
cultured for 1 day were served as a negative control. Both
the positive control and the negative control underwent the
same histological processes as described above for the
cryopreserved shoot tips.
Assessment of genetic stability
Two lilies: ‘Siberia’ and ‘Triumphator’ that produced the
highest (87.5 %) and lowest (42.5 %) shoot regrowth rates,
respectively, were selected and used to determine if levels
of shoot regrowth rate affect genetic stability in the re-
generants. After 12 months of shoot regeneration, 30 re-
generants and 30 in vitro stock shoots (controls) were
randomly selected from a population of 150 regenerated
plants and 200 in vitro stock shoots and subjected to an
assessment of genetic stability using ISSR. DNA extraction
and ISSR analysis were performed as described in our
previous report (Yin et al. 2013).
Experimental design and data statistical analyses
For cryopreservation experiments, at least ten shoot tips
were used in each of three replicates. All experiments were
repeated twice. Data expressed as percentage with their
standard error were transformed using arc sine prior to
analysis of variance (Compton 1994) and Student’s t test.
Least significant differences were calculated at P \ 0.05.
For ISSR analyses, ISSR fingerprint was manually scored
for the presence (1) and absence (0) of each band. Bands of
equal molecular weight and mobility generated by the same
primer were considered to represent the same locus. Both
distinct monomorphic bands and polymorphic bands were
scored. The experiment was repeated twice to confirm their
repeatability, and only reliable and repeatable bands were
included in the data analysis.
Results
Adventitious shoot regeneration
All of the leaf segments from the six lily genotypes
belonging to five lily species and hybrids produced
adventitious shoots after 4 weeks of culture, without any
callus formation. The average number of shoots per explant
was 7.0, 5.6, 5.0, 5.5, 4.7 and 4.8 for L. Oriental hybrid
‘Siberia’, L. Asiatic hybrids ‘Elite’, L. Asiatic hybrids
‘‘Pollyanna’, L. davidii var. unicolor, L. 9 formolongi and
L. longiflorum 9 Oriental ‘Triumphator’.
Overall process of shoot regeneration
from cryopreserved shoot tips
After 7 days of post-thaw culture, surviving cryopreserved
shoot tips showed green color (Fig. 1d), while dead ones
became white (Fig. 1e). Surviving shoot tips started to
elongate and produce new leaves after 2 weeks of post-
thaw culture. Shoots formed directly without callus for-
mation (Fig. 1f) after 4 weeks of post-thaw culture. When
transferred onto MS containing 0.5 mg/l NAA and 4 g/l
activated charcoal (AC) for rooting, almost all shoots
developed roots after 4 weeks of culture (Fig. 1g). Rooted
Acta Physiol Plant
123
shoots (more than 95 %) were successfully established in
soil under the greenhouse conditions within 4 weeks (data
not shown).
Effects of preculture
The sucrose concentration in the preculture medium and
the preculture duration exerted strong effects on shoot
regrowth. In both treated (-LN) and cryopreserved shoot
tips (?LN), 0.5 M sucrose resulted in the highest level of
shoot regrowth (Fig. 2). Lower and higher sucrose con-
centrations dramatically decreased the level of shoot
regrowth. A 1 day preculture resulted in the highest level of
shoot regrowth for both treated (-LN) and cryopreserved
shoot tips (?LN) (Fig. 3).
Effects of loading
The sucrose concentration in the loading solution did not
affect the level of shoot regrowth in the treated control
(-LN), but did significantly influence the level of shoot
regrowth in cryopreserved shoot tips (?LN). After LN
exposure, the highest level of shoot regrowth was achieved
with 0.4 M sucrose in the loading solution (Fig. 4). Higher
and lower sucrose concentrations resulted in markedly
reduced levels of shoot regrowth for the cryopreserved
shoot tips (?LN). Treated control (-LN) showed a
reduced level of shoot regrowth as the loading time interval
increased from 0 to 40 min. The level of shoot regrowth for
cryopreserved shoot tips (?LN) was significantly higher
when the loading duration was 20 min (Fig. 5).
Effects of dehydration with PVS2
The level of shoot regrowth was strongly affected by the
PVS2 exposure time in the treated (-LN) and
Fig. 2 Effect of sucrose concentration in preculture medium on shoot
regrowth of cryopreserved shoot tips of Lilium Oriental hybrid
‘Siberia’ by droplet vitrification. Shoot tips were precultured on BM
containing different sucrose concentrations (0.25, 0.5, 0.75 and
1.0 M) for 1 day. Precultured shoot tips were loaded and dehydrated
by PVS2 on ice for 4 h, followed by a direct immersion into LN for
1 h. Vertical bars represent SE. Different letters indicate significant
differences at P \ 0.05 by the Student’s t test
Fig. 3 Effect of duration of preculture on shoot regrowth of
cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’ by
droplet vitrification. Shoot tips were precultured on BM containing
0.5 M sucrose for 0–4 days. Precultured shoot tips were loaded and
dehydrated by PVS2 on ice for 4 h, followed by a direct immersion
into LN for 1 h. Vertical bars represent SE. Different letters indicate
significant differences at P \ 0.05 by the Student’s t test
Fig. 4 Effect of sucrose concentration in loading solution on shoot
regrowth of cryopreserved shoot tips of Lilium Oriental hybrid
‘Siberia’ by droplet vitrification. Shoot tips were precultured on BM
containing 0.5 M sucrose for 1 day. Precultured shoot tips were
loaded with loading solutions containing different concentrations of
sucrose (0, 0.2, 0.4, 0.6 and 0.8 M) for 20 min and dehydrated by
PVS2 on ice for 4 h, followed by a direct immersion into LN for 1 h.
Vertical bars represent SE. Different letters indicate significant
differences at P \ 0.05 by the Student’s t test
Acta Physiol Plant
123
cryopreserved (?LN) shoot tips. In the treated control
(-LN), shoot regrowth levels were high with 0–5 h of
PVS2 treatment, and then declined significantly (Fig. 6). In
contrast cryopreserved shoot tips (?LN) had the highest
levels of regrowth after 3–4 h (Fig. 6).
Testing of the established protocol for other four Lilium
species and hybrids
The droplet-vitrification procedure developed above was
tested for other five lily genotypes belonging to four Lilium
species and hybrids. Results showed that all lilies tested
produced high survival rates (60–95 %) and high level of
shoot regrowth (42.5–87.5 %), with mean survival and
shoot regrowth level of 75.4 % and 67.1 % obtained for
total six lily genotypes belonging to five Lilium species and
hybrids (Table 1).
Histological studies on the shoot tips
In the positive control shoot tips (living shoot tips), dense
TB-stained and well-preserved cytoplasm was observed in
the cells of both the leaf primordia (LPs) and the apical
dome (AD), with the nucleolus enclosed in the nucleus
(Fig. 7a). These were characteristics of living cells (Helliot
et al. 2002, 2003). In contrast, in the negative control shoot
tips (dead shoot tips), reduced levels of TB-stained cyto-
plasm were observed in the cells in both the LPs and the
AD (Fig. 7b), which is a typical sign of dead cells. In
cryopreserved shoot tips (?LN) of the two lilies tested,
TB-stained and well-preserved cytoplasm was observed
mainly in the upper part of AD (Fig. 7c, d) and in the
youngest LPs (Fig. 7e, f). Cells in the lower part of AD and
in the older LPs were mostly damaged (data not shown).
Both lilies showed a similar pattern of cell survival fol-
lowing cryopreservation.
Assessment of genetic stability by ISSR
In both ‘Siberia’ and ‘Triumphator’, out of 40 primers
tested, 8 primers produced strong, clear and reproducible
Fig. 5 Effect of duration of loading solution on shoot regrowth of
cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’ by droplet
vitrification. Shoot tips were precultured on BM containing 0.5 M
sucrose for 1 day. Precultured shoot tips were loaded with loading
solutions containing 0.4 M sucrose and 2 M glycerol for 0–50 min
and dehydrated by PVS2 on ice for 4 h, followed by a direct
immersion into LN for 1 h. Vertical bars represent SE. Different
letters indicate significant differences at P \ 0.05 by the Student’s
t test
Fig. 6 Effect of exposure time to PVS2 on shoot regrowth of
cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’ by droplet
vitrification. Shoot tips were precultured on BM containing 0.5 M
sucrose for 1 day. Precultured shoot tips were loaded and dehydrated
by PVS2 on ice for 0–6 h, followed by a direct immersion into LN for
1 h. Vertical bars represent SE. Different letters indicate significant
differences at P \ 0.05 by the Student’s t test
Table 1 Survival and shoot regrowth of cryopreserved shoot tips of
six Lilium genotypes by droplet vitrification
Species and hybrids Survival
(% ± SE)*
Shoot regrowth
(% ± SE)*
L. Oriental hybrid ‘Siberia’ 95.0 ± 2.5a 87.5 ± 4.2a
L. davidii var. unicolor 82.5 ± 4.2ab 80.0 ± 6.2ab
L. 9 formolongi 62.5 ± 4.2cd 52.5 ± 4.2c
L. Asiatic hybrids
‘Elite’ 72.5 ± 4.2ab 72.5 ± 7.4bc
‘Pollyanna’ 80.0 ± 3.6bc 67.5 ± 4.2b
L. longiflorum 9 Oriental
‘Triumphator’
60.0 ± 6.2d 42.5 ± 7.4c
Average 75.4 67.1
* Data represent mean ± SE. Values followed by the different letters
within the same column are significantly different at P \ 0.05 by the
Student’s t test
Acta Physiol Plant
123
bands, which differed in the two lilies (Table 2; Fig. 8). In
‘Siberia’, a total of 40 bands were scorable and the number
of bands for each primer varied between 3 and 7, with an
average of 5 bands per primer obtained (Table 2; Fig. 8). A
total of 1,200 bands were scored in 30 plantlets analyzed
(data not shown). In ‘Triumphator’, a total of 44 scorable
bands were obtained (Table 2). The number of bands for
each primer varied between 4 and 8, with an average of 5.5
bands per primer (Table 2; Fig. 8). There were no poly-
morphic bands when 30 representative original source
plants were compared with 30 regenerants after cryopres-
ervation for both ‘Siberia’ and ‘Triumphator’ (Table 2;
Fig. 8). Thus, we were unable to detect any signs of genetic
variation resulting from the cryopreservation process.
Discussion
In this study, a straightforward, widely applicable cryo-
preservation using droplet vitrification was described for
shoot tips of Lilium. With our optimized parameters, shoot
regrowth levels ranged from 42.5 % for L. longiflo-
rum 9 Oriental ‘Triumphator’ to 87.5 % for L. Oriental
hybrid ‘Siberia’, with an average shoot regrowth level of
67.1 % obtained across the six lily genotypes belonging to
five Lilium species and hybrids tested, which are among the
commercially important groups of lily. To the best of our
knowledge, this publication represents the widest applica-
tion of a cryopreservation method for Lilium.
The present study reports several improvements over
previously published protocols for cryopreservation of Li-
lium shoot tips. Our method of adventitious shoot regen-
eration from leaf segments produced a high number of
shoot tips for all the tested Lilium species and hybrids
within a relatively short period of time (4 weeks). This
improves the efficiency of producing shoot tips for the
cryopreservation procedures. This study eliminated the use
of cold hardening of in vitro stock shoots prior to excise
shoot tips, thus shortening the processing time for cryo-
preservation. In addition, an assessment was performed of
Fig. 7 Histological observation in longitudinal sections of shoot tips
of Lilium Oriental hybrid ‘Siberia’ and Lilium longiflorum 9 Oriental
‘Triumphator’. a A freshly excised shoot tip of Lilium Oriental hybrid
‘Siberia’. Surviving cells (as indicated by black arrow) in the apical
dome showed dense, TB-stained and well-preserved cytoplasm, with
nucleolus enclosed in nucleus. b A shoot tip of Lilium Oriental hybrid
‘Siberia’ directly immersed in LN shortly after excision (dead
control). Dead cells (as indicated by white arrow) in the apical dome
showed much weaker TB-stained cytoplasm. Nuclei were heavily
condensed. c A cryopreserved shoot tip of Lilium Oriental hybrid
‘Siberia’ after precultured on 0.5 M sucrose-enriched BM for 1 day.
d A cryopreserved shoot tip of Lilium longiflorum 9 Oriental
‘Triumphator’ after precultured on 0.5 M sucrose-enriched BM for
1 day. e A leaf primordium of cryopreserved shoot tip of Lilium
Oriental hybrid ‘Siberia’ after precultured on 0.5 M sucrose-enriched
BM for 1 day. f A leaf primordium of cryopreserved shoot tip of
Lilium longiflorum 9 Oriental ‘Triumphator’ after precultured on
0.5 M sucrose-enriched BM for 1 day. Bars = 10 lm
Acta Physiol Plant
123
the genetic integrity in the regenerated lily plants after
cryopreservation using ISSR molecular markers. The
results demonstrated that regenerants from cryopreserved
shoot tips were genetically stable. Finally, this droplet-
vitrification procedure was applied to six genotypes
belonging to five Lilium species and hybrids, thus leading
to establishment of a widest cryopreservation protocol so
far reported for diverse lily genetic resources.
For successful cryopreservation, shoot tips are often
treated with sucrose preculture to improve desiccation and
cryotolerance. Preculture with 0.3 M sucrose-enriched
medium for 1 day (Matsumoto et al. 1995) or 2 days (Chen
et al. 2011) was found optimal for producing the highest
shoot regrowth level of cryopreserved shoot tips of Lilium.
In this study, we found that a preculture treatment with
0.5 M sucrose for 1 day resulted in the highest shoot
regrowth level of cryopreserved shoot tips. In both studies
of Matsumoto et al. (1995) and Chen et al. (2011), cold-
hardened stock cultures were used, while non-cold-hard-
ened ones were applied in our study. This may result in
different requirements for optimal sucrose concentration in
the preculture medium.
Table 2 Name of primers,
primer sequence and number of
amplified bands generated in the
plantlets regenerated from leaf
segments of Lilium Oriental
hybrid ‘Siberia’ and Lilium
longiflorum 9 Oriental
‘Triumphator’ by ISSR marker
Species or hybrids Name of
Primers
Primer sequence
(50–30)Number of
amplified bands
Number of
polymorphic bands
L. Oriental hybrid ‘Siberia’ 3A30 (CT)7GAA 5 0
3A37 (CA)7TGA 7 0
3A35 (CT)7TGA 3 0
3A59 (CT)7GTG 4 0
UBC 843 (CT)8 RA 5 0
UBC 844 (CT)8RC 6 0
UBC 815 (CT)8G 4 0
UBC 857 (AC)8YG 6 0
Total 40 0
L. longiflorum 9 Oriental
‘Triumphator’
3A30 (CT)7GAA 5 0
3A37 (CA)7TGA 6 0
3A35 (CT)7TGA 4 0
3A59 (CT)7GTG 4 0
UBC 840 (GA)8 YT 5 0
UBC 844 (CT)8RC 6 0
UBC 845 (CT)8RG 6 0
UBC 857 (AC)8YG 8 0
Total 44 0
Fig. 8 ISSR banding pattern in the mother plants and plantlets
regenerated after cryopreservation of Lilium. a ISSR banding pattern
of Lilium Oriental hybrid ‘Siberia’ by the primer 3A37. b ISSR
banding pattern of Lilium Oriental hybrid ‘Siberia’ by the primer
UBC857. c ISSR banding pattern of Lilium longiflorum 9 Oriental
‘Triumphator’ by the primer 3A37. d ISSR banding pattern of Lilium
longiflorum 9 Oriental ‘Triumphator’ by the primer UBC857.
M marker; lane 1–2 the mother plants; lanes 3–10 the plantlets
regenerated after cryopreservation
Acta Physiol Plant
123
In many cases, preculture alone is not sufficient to
induce LN tolerance in shoot tips. Therefore, an osmo-
protection step using a loading solution was deemed
essential (Matsumoto et al. 1995). In the present study, we
found that a loading treatment for 20 min with a solution
containing 2 M glycerol and 0.4 M sucrose produced the
highest level of shoot regrowth in cryopreserved shoot tips.
This was consistent with the results obtained in Lilium
(Matsumoto et al. 1995; Chen et al. 2011) and other bul-
bous monocots such as Allium sativum (Kim et al. 2009)
and Galanthus elwesii (Maslanka et al. 2013).
Time duration and temperature of exposure of the
samples to PVS2 are the two important factors that affect
shoot regrowth of cryopreserved shoot tips. Over-exposure
of the samples to PVS2 can cause chemical toxicity and
excessive osmotic stress (Matsumoto et al. 1995). Com-
pared with room temperature (22 �C), low temperature
(0 �C) reduces toxicity of the vitrification solution and
often enhances the regrowth of cryopreserved shoot tips of
Lilium (Matsumoto et al. 1995; Chen et al. 2011). These
data were in line with the results reported here.
Histological studies indicated that the cells in the apical
dome and the youngest LPs could survive the cryo-treat-
ment more readily than cells located below the apical dome
and in the older LPs. These results on Lilium were con-
sistent with those on other monocots such as Musa (Helliot
et al. 2002, 2003). In our present study, both ‘Siberia’ and
‘Triumphator’ showed a similar pattern of cell survival
following cryopreservation. However, their shoot regrowth
rates significantly differed from each other. This may be
attributed to genotype-specific response to the recovery
medium, as is a common phenomenon in shoot tip culture
(Bhojwani and Razdan 1996).
ISSR markers have been employed to assess genetic
integrity in the plants that regenerated after cryopreserva-
tion (Harding 2004). To date, there have not been any
assessments of the genetic stability in Lilium plants that
regenerate after cryopreservation. We previously reported
assessment of genetic integrity in the adventitious shoots
from leaf segments of L. Oriental hybrid ‘Siberia’ by ISSR
and AFLP (Yin et al. 2013), and found that the regenerants
were genetically stable. In this study, we further tested
genetic stability in the shoots regenerated from cryopre-
served shoot tips of two lilies: ‘Siberia’ that produced the
highest shoot regrowth rate and ‘Triumphator’ that pro-
duced the lowest shoot regrowth. The results showed that
both lilies were genetically stable for the primers that we
employed, indicating that the level of shoot regrowth did
not have detectable influence on genetic stability in this
experimental system.
In conclusion, a droplet-vitrification cryopreservation
was successfully described for Lilium shoot tips. The pro-
tocol showed to be efficient and widely applicable to the
six Lilium tested in the present study. This protocol is being
tested for its potential applications to long-term preserva-
tion of more lily genetic resources and to lily virus
eradication.
Author contribution The first author Zhen-Fang Yin
implemented the main experiments involved in the present
study, data collection and analysis, and prepared the
manuscript. Wen-Lu Bi was responsible for tissue culture
work, histological studies, and data collection and analysis.
Long Chen and Bing Zhao carried out the assessments of
genetic stability in the regenerants by ISSR analysis. Dr.
Gayle M. Volk revised the manuscript critically. The cor-
responding author Dr. Qiao-Chun Wang worked as a chief
scientist for the present study, including making research
plan, experimental design, data analysis and revision of the
manuscript.
Acknowledgments The present study is financially supported by
the President Foundation of Northwest A&F University, China.
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