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: qiaochunwang@nwsuaf.edu.cn

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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