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Plant Cell, tissue and Organ Culture 41: 237-241, 1995. 237 0 1995 KluwerAcademicPublishers. Printedin the Netherlands. Cryopreservation of in vitro-grown apical meristems of lily by vitrification Toshikazu Matsumoto 1, Akira Sakai 2 & Kazuto Yamada 1 IShimane Agricultural Experiment Station, Ashiwata 2440, Izumo, Shimane 693 Japan; 2Asabucho 1-5-23, Kitaku, Sapporo 001 Japan Received25 May 1994;accepted in revisedform 9 February 1995 Key words: Lilium L., cold-hardening Abstract Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Liliumjaponicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25 °C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily. Abbreviations: DMSO - dimethylsulfoxide, EG- ethylene glycol, LN- liquid nitrogen, MS medium - Murashige & Skoog (1962) medium, PVS2 - vitrification solution Introduction Lily is economically very important with tremendous horticultural popularity. As such, various tissue cul- ture methods have been applied to develop new culti- vars and for clonal micropropagation of lily (Stimart & Ascher 1978; Novak & Petru 1981; Fukui etal. 1989). In recent years, cryopreservation of plant cells, meris- tems and organs has become an important tool for the long-term preservation of germplasm or experimental materials without genetic alteration. With the increas- ing interest in genetic engineering of plants, preser- vation of cultured cells and meristems with unique attributes, particularly, genetically modified cells and plants, is assuming greater importance. In our previous report, we succeeded in developing a method for cryopreservation of cultured meristems of wasabi (Wasabia japonica Matsumura) by vitrifi- cation (Matsumoto et al. 1994). Vitrification refers to the physical process by which a concentrated aqueous solution solidifies into metastable glass (glass solid) at sufficiently low temperatures without crystallization. At sufficiently low temperatures, a highly concentrat- ed cryoprotective solution becomes so viscous that it solidifies into a metastable glass at a practical cool- ing rate (Fahy et al. 1984). A vitrification procedure eliminates the need for controlled slow freezing and permits cells and meristems to be cryopreserved by a direct transfer to liquid nitrogen (Sakai et al. 1990, 1991). To our knowledge, there is no report on the cry- opreservation of lily. Here we report the successful cryopreservation of meristems from adventitious buds, which were induced by culturing bulb-scale segments of lily, with subsequent shoot formation and regenera- tion of whole plants.

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

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Page 1: Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Plant Cell, tissue and Organ Culture 41: 237-241, 1995. 237 0 1995 KluwerAcademic Publishers. Printed in the Netherlands.

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

T o s h i k a z u M a t s u m o t o 1, Ak i r a Sakai 2 & K a z u t o Y a m a d a 1 I Shimane Agricultural Experiment Station, Ashiwata 2440, Izumo, Shimane 693 Japan; 2Asabucho 1-5-23, Kitaku, Sapporo 001 Japan

Received 25 May 1994; accepted in revised form 9 February 1995

Key words: Lilium L., cold-hardening

Abstract

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Liliumjaponicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25 °C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.

Abbreviations: DMSO - dimethylsulfoxide, E G - ethylene glycol, L N - liquid nitrogen, MS medium - Murashige & Skoog (1962) medium, PVS2 - vitrification solution

Introduction

Lily is economically very important with tremendous horticultural popularity. As such, various tissue cul- ture methods have been applied to develop new culti- vars and for clonal micropropagation of lily (Stimart & Ascher 1978; Novak & Petru 1981; Fukui etal. 1989). In recent years, cryopreservation of plant cells, meris- tems and organs has become an important tool for the long-term preservation of germplasm or experimental materials without genetic alteration. With the increas- ing interest in genetic engineering of plants, preser- vation of cultured cells and meristems with unique attributes, particularly, genetically modified cells and plants, is assuming greater importance.

In our previous report, we succeeded in developing a method for cryopreservation of cultured meristems of wasabi (Wasabia japonica Matsumura) by vitrifi-

cation (Matsumoto et al. 1994). Vitrification refers to the physical process by which a concentrated aqueous solution solidifies into metastable glass (glass solid) at sufficiently low temperatures without crystallization. At sufficiently low temperatures, a highly concentrat- ed cryoprotective solution becomes so viscous that it solidifies into a metastable glass at a practical cool- ing rate (Fahy et al. 1984). A vitrification procedure eliminates the need for controlled slow freezing and permits cells and meristems to be cryopreserved by a direct transfer to liquid nitrogen (Sakai et al. 1990, 1991).

To our knowledge, there is no report on the cry- opreservation of lily. Here we report the successful cryopreservation of meristems from adventitious buds, which were induced by culturing bulb-scale segments of lily, with subsequent shoot formation and regenera- tion of whole plants.

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Materials and methods

Plant material

Tissue-cultured lily, Lilium japonicum Thunb. cv. Japanese Pink Lily, was mainly used in the present study. Stock cultures of five other Oriental lily culti- vars; Golden-banded Lily (Lilium platyphyllum Maki- no), 'Star Gazer ' (Lilium x 'Star Gazer'), 'Summer Dress' (Lilium x 'Summer Dress'), Show Lily (Lil- ium specisum Thunb.), 'Kraetzeri' (Lilium specisum f. album novum Mallet), were also used. The medium used for this study was Murashige & Skoog (MS) medi- um containing 3% sucrose and solidified with 0.2% gellan gum (Wako Chemical Co. Japan), and adjusted to pH 5.8 prior to autoclaving at 121°C for 15 min. This medium was used as a basal medium for all the experiments. Bulb-scale segments (about 10 mm in length) of lily were cut to about 5 mm x 5 mm seg- ments. Then, 20 segments of lily were placed on 25 ml basal medium (described above) in a sterilized plastic petri dish (9 cm in diameter) under white fluorescent light (60 #mol m -2 s - l ) , with a 12 h/day photoperiod at 25°C. Adventitious buds with 2 or 3 leaf primor- dia were regenerated from the surface of the explants about 40 days after transfer.

Cold-hardening and preculture

Scale segments with adventitious buds in petri dish- es were cold-hardened at 0°C for 7-30 days under a 12 h/day photoperiod (60 #mol m -2 s- l ) . Meris- tems of about 1.0 mm in length were dissected from the cold-hardened and non-hardened segments. About 100 meristems were placed on the basal medium sup- plemented with 0.3 M sucrose in a petri dish (9 cm in diameter) and precultured at 25°C for 1 day at the same light condition.

Vitrification procedure

Ten precultured meristems were placed in a 1.8 ml cryotube and then loaded with 1.8 ml of various cry- oprotective solutions for 20 min at 25°C. They were sufficiently dehydrated by subsequent exposure to a concentrated vitrification solution (PVS2 Sakai et al. 1990) at 0°C and 25°C for various lengths of time. PVS2 solution contains 30% (w/v) glycerol, 15% (w/v) EG and 15% (w/v) DMSO in 0.4 M sucrose solution (pH 5.8). Ten precultured and loaded meristems were placed in a 1.8 ml cryotube and then 1.8 ml PVS2

Table 1. Effect of loading solutions on the shoot formation of vitrified meristems cooled to -196 ° C following dehydration with PVS2.

Loading solution Shoot formation 1 (% 5: S.E.)

Non-treated 3.3 -t- 1.1 2 M glycerol + 0.4 M sucrose 55.2 5:3.2 0.5 M glycerol + 10% DMSO 60.0 5:3.1 +0.3 M sucrose 10% DMSO + 0.7 M sucrose 0 20% PVS22 23.3 5:2.7 1.5 M glycerol + 5% DMSO 40.0 5:3.1 +0.4 M sucrose

Material: Japanese Pink Lily (L. japonicum Thunb.) l Shoot formation (%): percent of the meristems producing normal shoots 28 days after plating. 220% of the stock PVS2. Approximately 10 meristems were tested for each of four replicates.

was added and mixed. The cryotubes in which meris- tems were finally suspended in 1.0 ml of fresh PVS2 and directly plunged in LN and held there for at least 60 min. The cooling rate was about 200°C/min. Cry- otubes were warmed in a water bath at 40°C (warming rate: about 250°C/min).

Viability and plant regrowth

After rapid warming in a water bath at 40 ° C, PVS2 was drained from the cryotubes and replaced with 1.8 ml of 1.2 M sucrose solution and held for 20 min. Approxi- mately 60 meristems were transferred onto two sheets of sterilized filter paper discs (5 cm in diameter) over the basal medium in a petri dish, then cultured under the standard conditions described previously. After one day, the meristems were transferred onto fresh filter paper discs in a petri dish containing the same medi- um.

Recovering meristems were observed at weekly intervals. Shoot formation was recorded as percent of total number of meristems forming normal shoots 28 days after plating. Ten meristems were tested in each of four replicates for each experiment.

Results

Cold-hardened meristems at 0°C for 28 days were precultured on the basal medium containing 0.3 M

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Table 2. Effect of the period of cold-hardening on the shoot forma- tion of vitrified meristems cooled to -196 ° C following dehydration with PVS2.

Period of cold-hardening Shoot formation t

(days) (% 4- S.E.)

0 40.0 4-3.1

7 72.5 4- 2.8

14 74.0 4- 3.1

21 77.3 4- 3.0

30 76.7 4- 2.3

Material: Japanese Pink Lily (L. japonicum Thunb.) 1Shoot formation(%): percent of the meristems producing normal shoots 28 days after plating. Approximately 10 meristems were tested for each of four replicates.

sucrose at 25°C for 1 day, and were then loaded with four different cryoprotective solutions for 20 min at 25°C before being dehydrated with PVS2 at 0°C for 60 min. As shown in Table 1, most of the loading solutions tested were very effective at improving the survival of vitrified meristems cooled to -196°C. A higher rate of shoot formation was observed in the meristems loaded with a mixture of 2 M glycerol plus 0.4 M sucrose or of 0.5 M glycerol, 10% DMSO and 0.3 M sucrose. These two loading solutions provided similar levels of shoot formation. However, in the pre- vious report (Matsumoto etal. 1994), a mixture of 2 M glycerol plus 0.4 M sucrose provided the highest rate of shoot formation in wasabi meristems. Thus, a mix- ture of 2 M glycerol plus 0.4 M sucrose was adopted as the loading solution for lily meristems in subse- quent experiments. Cold-hardened meristems at 0°C for 7 days significantly increased the rate of shoot for- mation from 40% (non-hardened) to 72%. However, a longer hardening treatment resulted in only a slight increase (Table 2).

The effects of preculturing and loading on the shoot formation of vitrified meristems are summarized in Table 3. Cold-hardened meristems which were loaded with a mixture of 2 M glycerol plus 0.4 M sucrose produced the highest shoot formation (82.5%) after cooling to - 196 ° C.

To determine the optimal time of exposure to PVS2 at 25 or 0°C, precultured, loaded meristems were dehy- drated with PVS2 for various lengths of time prior to a plunge into LN. Exposure to PVS2 produced treatment time-dependent shoot formation (Fig. 1). The highest rate of shoot formation was obtained with the meris- terns treated with PVS2 for up to 100 to 110 min at 0°C

Table 3. Effects of precultufing and loading on the shoot formation of cold-hardened lily meristems cooled to - 196 ° C by vitrification.

Preeulture Loading Shoot formation 1

(% 4- S.E.)

- - 5 . 0 4 - 1 . 4

+ - 28.2 4- 2.8

- + 50.0 4- 3.2

+ + 82.5 q- 2.4

Material: Japanese Pink Lily (L. japonicum Thunb.) 1Shoot formation(%): percent of the meristems producing normal shoots 28 days after plating. Approximately 10 meristems were tested for each of four replicates.

I00 100

v g

m ~(

o

' . . ~ . . o . . a . . . ' ' o

25'(3

. o . . ~ . ' o . . o .cx. T r e a t e d c o n t r o l "~z-. ,o.-o, .o '"

~ L N

10 20 30 50 100 T i m e ( s i n ) T i m e ( t h i n )

Fig. 1. Effect of exposure lime to PVS2 at 0 or 25°C on the shoot formation of meristems cooled to -196°C by vitrification. Materi- al: Japanese Pink Lily (L. japonicum Thunh.) Meristems following preculture and loading were treated with PVS2 at 25 or 0°C for various lengths of lime before being immersed into LN(o). Approx- imately 10 meristems were tested for each of four replicates. The bar represents the standard error. Treated control(o): same as treated with PVS2 without cooling to -196°C.

or 20 min at 25 ° C, respectively. Meristems treated with PVS2 for up to 130 min at 0°C or up to 30 min at 25°C without-cooling in LN (treated control) retained high levels of shoot formation (about 90%).

Shoot formation using the same vitrification pro- cedure with or without cold-hardening was compared in five other Oriental cultivars of lily. Two cultivars considerably improved the rate of shoot formation by hardening (Table 4). However, in three other Oriental cultivars, little or no increase was observed.

Figure 2 shows shoot formation from vitrified and warmed meristems after 28 days of reculture. A rooted bulblet developed from a cryopreserved meristem by vitrification is shown in Fig. 3. Nearly all the shoots were grown to bulblets and formed roots on plant- growth-regulator-free MS medium and were success- fully transferred to soil in pots. Variations were some- times observed on regenerated leaves. However, this

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Table 4. Shoot formation of cold-hardened or non-hardened meris- terns from five Oriental eultivars of lily cooled to -196°C by vit- rification.

Cultivar Shoot formation (% 4- S.E.) 1

Hardening Non-hardening

Golden-banded Lily 85.0 q- 2.3 85.0 4- 2.3

(L. platyphyllum Makino)

'Star Gazer' 52.5 4- 3.2 59.0 4- 3.1

(L. x 'Star Gazer')

'Summer Dress' 73.3 4- 2.8 55.0 4- 3.1

(L. x ' Summer Dress') Show Lily 45.0 4- 3.1 20.0 4- 2.5

(L. specisum Thtmb.) 'Kraetzeri' (L. specisum f. 40.0 4- 3.1 40.0 -4- 3.1

album novum Mallet)

1 Shoot formation(%): percent of the meristems producing normal shoot 28 days after plating. Approximately 10 meristems were tested for each of four repli- cates.

Fig. 2. Shoots formed from culturing cryopreserved meristems by vitrification, 14 days after reculture. Material: Japanese Pink Lily (L. japonicum Thunb.) Bar - 5 ram.

phenomenon disappeared on the 4th or 5th leaves and the bulblets grew normally. Except for this observation, no morphological abnormalities were observed in the plants developed from cryopreserved meristems.

Discussion

For successful cryopreservation, it is necessary to avoid lethal intracellular freezing, which occurs dur- ing rapid cooling in liquid nitrogen (Sakai & Yoshida 1967). Thus, cells and meristems have to be suffi-

Fig. 3. Plantlet developed from a meristem cooled to -196°C by vitrification, 60 days after reculture. Material: Japanese Pink Lily (L. japonicum Thunb.) Bar - 20ram.

ciently dehydrated to be capable of vitrifying before being immersed into liquid nitrogen (Sakai 1993). In the vitrification method, cell and meristems are dehy- drated by a highly concentrated vitrification solution. However, the direct exposure of less tolerant cells and meristems to a vitrification solution results in harm- ful effects due to osmotic stress and chemical toxicity. The harmful effects due to dehydration can be alleviat- ed or eliminated by adequate preconditionings, such as cold-hardening (Reed 1990), preculture with high con- centration of sucrose or sorbitol (Yamada et al. 1991; Niino et al. 1992 a,b) and cryoprotective treatment (loading) (Langis & Steponkus 1990, Nishizawa et al.

1992, 1993; Matsumoto et al. 1994). In our previous study using in v i tro-grown apical

meristems of wasabi (Matsumoto e ta l . 1994), this lim- itation was completely overcome by the cryoprotective treatment (loading) with a mixture of 2 M glycerol plus 0.4 M sucrose following preculture with 0.3 M sucrose for one day. This loading solution was reported to be very effective in enhancing freeze-dehydration toler- ance or dehydration tolerance in many cells and meris- tems (Sakai et al. 1991; Nishizawa et al. 1992, 1993). In the present study, the same protocol -preculture and loading- gave high levels of shoot formation (85 to 55%) for 3 species or cultivars of the 5 lilies test- ed, but did not for 2 cultivars (20 or 40%). However, cold-hardening improved the rate of shoot formation from 20 to 40% in one cultivar, but did not in other cultivars. The method for preconditioning for enhanc- ing dehydration or freezing tolerance appears species- specific.

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The vitrification method is a simple and preferable method for cryopreservation of meristems (Matsumoto et al. 1994; Niino et al. 1992a,b; Reed 1990; Yamada et al. 1991). The keys of success to cryopreservation by vitrification are to carefully control the procedures for dehydration and cryoprotectant permeation and to prevent injury by excess osmotic stresses or chemical toxicity during dehydration (Rail 1987). To expand the applicability of cryogenic protocols to a wide range of plant cultured cells and meristems, it is essential to car- ry out basic studies on preconditioning for enhancing the freezing or dehydration tolerance.

It is particularly important that cryopreserved meristems be capable of producing true-to-type plants identical to the non-treated phenotype. Lily meristems cryopreserved by vitrification lost green color within one day after plating, but put forth green leaves within 5 days and developed shoots directly within 3 weeks without intermediary callus formation. These shoots grew to normal bulblets with roots and acclimatizated to transfer to soil in pots. Some variation was observed in the 1st to 3rd leaves of regenerated plantlets from cryopreserved meristems. However, the variation was not observed in the subsequent leaves. Thus, this abnormality seems to be temporary and not genetic based. No other morphological changes were observed the plants developed from cryopreserved meristems. However, further study is necessary to confirm their phenotype by cytological, biochemical and morpho- logical analyses.

Acknowledgements

The authors wish to express their cordial thanks to Mr. K Haruki, Shimane Agricultural Experiment Station for supplying lily stock culture. This study was sup- ported by a special research grant 'Applied Biotech- nology Program for Prefectural Agriculture' from the Ministry of Agriculture, Forestry and Fisheries, Japan.

Langis R & Steponkus PL (1990) Cryopreservation of rye protoplasts by vitrification. Plant Physiol. 92:666-671

Matsumoto T, Sakai A & Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep. 13:442-446

Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497

Niino T, Sakai A, Yakuwa H & Nojiri K (1992 a) Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell Tiss Org Cult. 28:261-266

Niino T, Sakai A, Magoshi J & Kato S (1992 b) Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. Cryo- Letters 13:303-312

Nishizawa S, Sakai A, Amano Y & Matsuzawa T (1992) Cryo- preservation of asparagus (Asparagus officinalis L.) embryogenic cells and subsequent plant regeneration by a simple freezing method. Cryo-Letters 13:379-388

Nishizawa S, Sakai A, Amano Y & Matsuzawa T (1993) Cryo- preservation of asparagus (Asparagtts officinalis L.) embryogenic cells and subsequent plant regeneration by vitrification. Plant Sci. 91:67-73

Novak FJ & Petru E (1981) Tissue culture propagation of Lilium hybrids. Sci. Hort. 14:191-199

Rail WF (1987) Factors affecting the survival of mouse embryo cryopreserved by vitrification. Cryobiology. 24:367-402

Reed BM (1990) Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25:15-113

Sakai A (1993) Cryogenic strategies for survival of plant cultured cells and meristems cooled to -196°C. JICA GRP, REF, No. 6: 5-26

Sakai A, Kobayashi S & Oiyama I (1990) Cryopreservation of nucel- lar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9:30-33

Sakai A, Kobayashi S & Oiyama I (1991) Survival by vitrification of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) cooled to -196°C. J. Plant Physiol. 137:465-470

Sakai A & Yoshida S (1967) Survival of plant tissue at super-low temperatures. I. Effects of cooling and rewarming rates on sur- vival. Plant Physiol. 42:1695-1701

Stimart DP & Ascher D (1978) Tissue culture of bulb scale sections for asexual propagation of Lilium longiflorum Thunb. J. Amer. Hort. Sci. 103(2): 182-184

Yamada T, Sakai A, Matsumura T & Higuchi S (1991) Cryopreser- vation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Sci. 78:81-87

References

Fahy GM, MacFarlene DR, Angell CA & Meryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21: 407--426

Fukui H, Adachi N, Hara T & Nakamura M (1989) hz vitro growth and rapid multiplication of Lilium japoniclnn Thunb. Plant Tiss. Cult. Lett. 6(3): 119-124