Plant Cell Reports (1994) 13:442-446 Plant Cell Reports �9 Springer-Verlag 1994

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabiajaponica) by vitrification and subsequent high plant regeneration

Fir. M a t s u m o t o 1, A. S a k a i 2, and K. Y a m a d a 1

Shimane Agricultural Experiment Station, izumo, 693 Japan 2 Asabucho 1-5-23, Kitaku, Sapporo, 001 Japan

Received 7 October 1993/Revised version received 11 February 1994 - Communicated by E Constabel

S u m m a r y . In v i tro-grown apical meristems of wasabi ( W a s a b i a j a p o n i c a Matsumura) were successfully cryopreserved by vitrification. Excised

apical meristems precultured on solidified M S

medium containing 0.3M sucrose at 20~ for 1 day were

loaded with a mixture of 2M glycerol and 0.4M sucrose

for 20 min at 25~ Cryoprotected meristems were

then sufficiently dehydrated with a highly concentrated

vitrification solution (designated PVS2) for 10 rain at

25~ prior to a plunge into liquid nitrogen. After rapid

warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 rain and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation

a m o u n t e d to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for

cryopreserving meristems of wasabi.

K e y words: cryopreservation - apical meristems -

vitrification - wasabi (Wasabiajaponica)

A b b r e v i a t i o n s : BA: 6-benzylaminopurine; DMSO: dimethylsulfoxide; EG: ethylene glycol; LN: liquid nitrogen; MS medium: Murashige and Skoog medium

(1962); PVS2: vitrification solution

Correspondence to." T. Matsumoto

In troduct ion

Wasabi, Japanese horseradish (faro. Cruciferae), is an important crop in Japan. The roots contain a pungent ingredient (sinigrin). Myrosinase is responsible for the development of the flavor and pungency of wasabi roots (Ohtsuru and Kawatani 1979). Wasabi also contains other useful compounds such as peroxidasc which is widely used as a label enzyme in clinical diagnosis and immunoassay analysis (Taniguchi et al. 1988). The plant occurs in remote and inaccessible cool mountain springs in Japan. The optimal temperature range is 14

to 15~ and the growth is inhibited at temperatures

over 18~

Since seeds are sensitive to desiccation, the enltivars and strains of wasabi are maintained as living material in the field of some repositories in Japan. Recently, a meristem culture of wasabi has been developed for clonal propagation Oramada and Haruki 1992) and in

vitro-cultured plantlets are maintained by subculturing. However, cryopreservation may be an alternative approach for long-term storage of germplasm and valuable cell lines using a minimum of space and maintenance.

The vitrification (glass formation) procedure for cryopreservation eliminates the need for controlled slow freezing and permits cells and meristems to be cryopreserved by direct transfer into LN (Fahy et al. 1984; Chen et al. 1984; Sakai et al. 1991; Sakai 1993). It does not require controlled freezing equipment or sophisticated, expensive apparatus. Thus, vitrification is a potent ia l ly valuable cryogenic protocol for cryopreserving meristems. The vitrification procedure has been applied to a wide range of meristems (Dereuddre et al. 1988; Langis et al. 1990; Yamada et al. 1991;

Ni ino et al. 1992 a,b; Towill and Jarret 1992; Schnabel-Preikstas et al. a,b,c 1992). In these reports, a high level of sugar or sorbitol during preculture was reported to be essential in achieving high survival rates of cryopreserved meristems. However, such preculture did not lead to substantial increase in the survival of

wasabi meristems cooled to -196~ The reduction in

survival rate following dehydration suggests that dehydration stress is a major limitation (Langis et al.

1990, Nishizawa et al. 1993). We observed that the limitation could be overcome by treatment of vitrified wasabi meristems with a mixture of 2M glycerol plus

0.4M sucrose for 10 to 20 min at 25~ We report here

a simple and effective method for cryopreservation of meristems of in vitro-grown plants of wasabi.

Material and Methods

Plant material In vitro-grown plantlets of wasabi (Wasabiajaponica Matsumura)

cv. SHIMANE No.3 were used in the present study. Stock cultures of wasabi plants were maintained on Murashige & Skoog (1962) basal medium (half-strength of ammonium nitrate and potassium nitrate, termed 1/2 MS medium) containing 0.ling 1 ~ BA, 3% sucrose and 0.2% Gellan gum at pH 5.8 (Yamada and Haruki 1992). Stock cultures were subcultured every 35 to 40 days. These tissue-cultured plantlets were grown on 5 ml medium in test tubes (11 mm

diameter) under white fluorescent light (52 /zmol s "~ m-2), 16 hr

photoperiod at 20~ Apical meristems of about 1.0 mm in length were dissected from

about 30 mm long 30 to 40-day-old plantlets. Shoot tips were

precultured on a solidified 1/2 MS medium containing various

concentrations of sucrose in Petri dishes (9 cm diameter) at 20~C under continuous light for 1 to 3 days. Precnltured meristems were

loaded with various cryoprotectants for 5 to 20 min at 25"~C before dehydration with a vitrification solution.

Vitrg~eation procedure Briefly, the vitrification procedure involves the following steps : (a)

loading (cryoprotect ion)of the meristems with a mixture of eryoproteetive solutions; (b) dehydration of the meristems by exposing them to a concentrated vitrification solution (PVS2, Sakai et al. 1990); (c) plunging meristems into LN; (d) rapid warming; (e) unloading PVS2 by transferring the meristems to 1.2M sucrose solution; and (f) plating.

Ten precultured meristems were placed in a 1.8 ml eryotube and

then loaded with eryoprotective solutions for 20 min at 25"C. After removing the cryoprotective solution using a Pasteur pipette, 2.0 ml of PVS2 were added and gently mixed. After removing the PVS2 using a Pasteur pipette, this step was repeated once and held at 25 or

0*C for various lengths of time. The cryotubes in which meristems were finally suspended in 1.0 ml of PVS2 were plunged into LN and held there for at least 60 rain. PVS2 contains 30%(w/v) glycerol, 15%(w/v) EG and 15%(w/v) DMSO in 0.4M sucrose solution (pH 5.8)

(Sakai et al. 1990). The cooling rate was about 200*C/rain.

Cryotubes were warmed in a water bath at 40"C (warming rate : about

250*C/rain).

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Viability and plant growth After rapid warming, PVS2 was drained from the cryotubes and

replaced with 1.2M sucrose solution and held for 20 min. Meristems were transferred onto sterilized filter paper discs over solidified 1/2 MS basal medium containing 0.1 rag t 1BA, 3% sucrose, 0.2% Gellan gum and cultured under standard conditions described above. After one day, the meristems were transferred onto a fresh filter paper disc in a Peltri dish containing the same medium. Recovering meristems were observed at weekly intervals. Shoot formation was recorded as percent of total number of meristems forming normal shoots 21 days after plating. Ten apical meristems were tested for each o f f our replicates for each experiment.

Results

In preliminary experiments, apical meristems were precultured on solidified 1/2 MS medium supplemented with different concentrations (0.3 to 0.TM) of sucrose at

20~ for 1 day and then dehydrated with PVS2 for 30 to

40 min at 0~ before being immersed into LN. Little or

no survival was observed in the vitrified and warmed

apical meristems cooled to -196~ To enhance the

survival, precultured meristems with 0.TM sucrose for 1 day were loaded with various cryoprotective solutions for

20 rain at 25~ before dehydration with PVS2. As

shown in Table 1, the loading treatment (Steponkus et

al. 1992; Nishizawa et al. 1992,1993) was very effective in improving the survival of vitrified meristems cooled

to -196~ The highest rate of shoot formation was

observed in the meristems loaded with a mixture of 2M glycerol and 0.4M sucrose. Based on this result, this mixture was adopted as the loading solution for wasabi apical meristems. The optimum sucrose concentration for preculture was determined. The shoot formation was highest in the vitrified apical meristems precultured with 0.3M sucrose (data not shown).

Table 1. Effect of loading solutions on the shoot formation of

vitrified apical meristems cooled to -196~ following dehydration with PVS2.

Loading solution Shoot formation

(% + S.E.)

None-treated 1 2.5 5= 2.1

2.0M glyceroi+0.4M sucrose 87.1 4" 2.1

1.5M glycerol+0.4M sucrose 54.8 5= 3.2 +5% DMSO

20% PVS2 a 65 .0 5= 3 .0

Precuitured apical meristems were loaded with various loading

solutions for 20 rain at 2 5 ~ before being dehydrated with PVS2 for 50

rain at 0*C. Preculture : on solidified medium supplemented with

0.7M sucrose.at 20'~ for 1 day. a: 20% of the stock PVS2; Shoot formation(%): percent of the apical rneristems producing normal shoot 21 days after plating. Approximately 10 meristems were tested for each of four duplicates. Material: cv. SHIMANE No.3.

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The effects of preculturing and loading on the survival

of vitrified apical meristems were summarized in Table 2.

The apical meristems loaded with a mixture of 2M

glycerol and 0.4M sucrose fol lowing preculture with

0.3M sucrose for 1 day produced the highest shoot

formation (100%) after cooling t o - 1 9 6 ~ .

Table 2. Effects of preeulturing and loading on the shoot formation of wasabi apical meristems cooled to -196"C by vitrification.

, i i i

Period of preculture Loading Shoot formation (day) ( 96 4- S.E. )

- - 1 0 . 0 4- 1.4

- + 73.3 4- 2.4

1 - 6 1 . 2 4- 2.7

I + 100 4 -0

3 - 25.0 4- 2.7

3 + 40.0 4- 2.7

Preeulturing: at 250C for 1 or 3 days on solidified 1/2 MS medium supplemented with 0.3M sucrose ; Loading: treated with a mixture of

2M glycerol and 0.4M sucrose for 20 rain at 250C; Shoot formation (%): percent of apical meristems producing normal shoots 21 days after plating. Approximately 10 meristems were tested for each of four replicates. Material: cv. SHIMANE No.3

100

# - , ,

50 o

g

0 l0 20 30 40 50 60 0 10 20 30 E x p o s u r e t o PVS2(min)

Fig.1. Effect of exposure to PV $2 at 25 or 0*C on the shoot formation o f apical meristems cooled to - 196 *C by vitrification. Material : wasabi ev. SHIMANE No.3. Excised apical meristems were preeultured with 0.3M sucrose for 1 day and then loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25"C. Apical meristems following preculture and loading were dehydrated with PVS2 at 25 or 0*C for various lengths of time before being immersed into LN(o). Approximately 10 meristems were tested for each of four replicates. Bar represents standard error. Control(o): same as treated with PVS2 without cooling to -196"(;.

formation (Fig.l). The highest rate of shoot formation

was obtained with meristems treated with PVS2 for 10

min at 25"C or for 30 - 50 min at 0*C, respectively.

Apical meristems treated with PVS2 for up to 20 min at

25"C or for up to about 60 min at 0~ without cooling

in L N (control) retained high levels of shoot formation

(about 90%).

Successfully vitrified and warmed meristems remained

green cont inuously after plating, resumed growth in

about 3 days and developed shoots within two weeks

wi thout intermediary callus formation. Fluorescence

microscopic examination of longitudinal sections through

the meristematic dome of vitrified meristems after 3 days

o f re, culture revealed that in most of the meristems, the

domes appeared to be viable based on FDA staining

(Widholm 1972) (data not shown).

Fig. 2 shows shoots formed from the vitrified and wanned apical meristems after 21 days of reeulture. A

rooted plantlet developed from cryopreserved meristems

by vitrification is shown in Fig.3. Almost all of the

shoots formed roots on hormone free solidified 1/2 MS

medium and were successfully transferred to soil in pots.

No morphological abnormalities were observed in the

plants developed from cryopreserved apical meristems.

Fig.2. Shoots formed from eryopreserved meristems by vitrification, 20 days after reeulture. Material: wasabi cv. SHIMANE No.3. Bar = 5mm

To determine the optimum time of exposure to PVS2

at 25 or 0*G, precultured, loaded meristems were

dehydrated with PVS2 for various lengths of time prior

to a plunge into LN. Exposure to PVS2 for various length o f time resulted in a variable rate of shoot

Fig.3. Plantlet developed from an apical meristem cooled to -196"C by vitrification 60 days after reculture. Material: cv. SHIMANE No.3. Bar = 20mm

Shoot formation using the same vitrification procedure was compared for three other cultivars of wasabi. High levels of shoot formation were obtained for each cultivar tested (Fable 3).

Shoot formation of wasabi meristems cooled to

-196~ was compared with different cryogenic protocols.

In the conventional slow freezing method, apical

meristems were frozen at rate of 0.5~ to -40'C in

the presence of a mixture of 2M glycerol plus 0.4M sucrose or 10% DMSO plus 0.7M sucrose. The levels of shoot formation obtained in these protocols were nearly the same, but the time used for these procedures was greatly different (Table 4).

Table 3. Shoot formation of vitrified apical mefistems from four

eultivars of wasabi cooled to -196"C.

I iiiiiiiiiii IIIIIIIII

Cultivar Shoot formation

(% 4- S.E,)

SrlIMANE No3 92.2 + 1.7 IWAMI 78.5 4- 7.9 SANBE 81.8 + 2.7 RAKAN No.2 84.9 4- 2.6

Apical meristems following preculturing and loading were

dehydrated with PVS2 for 50 min at 0*C and then immersed in LN.

Preeulturing: 0.3M sucrose at 20'C for 1day; loading: a mixture of

2M glycerol plus 0AM sucrose for 20 min at 25*(;. Approximately 10 meristems were tested for each of four duplicates.

Table 4. Shoot formation of apical meristems cooled to -196~ by two different cryogenic protocols.

IIIIIIII III I I iiiiiiiiiiiiiii I I I IIIIII I H

Cryogenie protocol shoot formation Time used

(% + S~E.) (rain)

Slow freezing method a 97.5 + 1.0 130 Slow freezing method b 95.0 + 1.4 130 Vitrification e 97.5 + 0.6 50 Vitrificaaon d 97.5 + 1.0 90

a,b:Meristems were cryoprotected with 2M glycerol plus 0.4M

suerose(a) or 10%DMSO plus 0.7M sucrose(b) at 250C for 20 rain,

then frozen to -40"C at 0.5*C/rain prior to a plunge into LN.

e,d : Preeultnred meristems were loaded with a mixture of 2M glycerol

plus 0.4M sucrose for 20rain at 25'E: and then dehydrated with PVS2

at 25"C( c ) for 10min or 0*C( d ) for 50 rain, respectively before

cooling in LN; Time used: total time used for eryoproteetion, freeze-

dehydration or dehydration with PVS2 before cooling in LN.

Material: cv. SHIMANE No.3.

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Discussion

For successful cryopreservation, it is necessary to avoid lethal intracellular freezing, which occurs during rapid cooling in LN. Thus, cells and meristems have to be sufficiently dehydrated to be capable of vitrifying before being immersed into LN (Sakai 1993). In the vitrification method, cells and meristems are dehydrated by a highly concentrated vitrification solution. However, direct exposure of meristems to a vitrification solution may lead to harmful effects due to osmotic stress or chemical toxicity. Thus, decrease in survival following dehydration in a concentrated vitrification solution and unloading in 1.2M sucrose can be a major limitation in cryopreservation by vitrification. In the present study, this limitation was almost completely overcome by the cryoprotective treatment (loading) with a mixture of 2M

glycerol plus 0.4M sucrose for 10 to 20 min at 25~

A mixture of 1.6 - 2.0M glycerol and 0.4M sucrose was reported to be very effective in inducing freeze- dehydration or dehydration tolerance for asparagus embryogenic cells (Nishizawa et al. 1992, 1993). More recently, in vitro-cultured multiple bud dusters of melon (Ogawa, unpublished), chicory (Nishizawa, unpublished) and chrysanthemum (Kohmura, unpublished) were also successfully cryopreserved by vitrification. In these materials, a loading treatment following precnlturing seems to be essential to produce a high rate of shoot formation.

Rates of shoot formation of wasabi apical meristems

treated with PVS2 at 25~ or 0~ without cooling in

LN (control) was similar to the vitrified shoot tips. Nearly the same results were reported in the vitrified apical meristems of white clover (Yamada et al. 1991), apple, pear and mulberry (Niino et al. 1992 a,b). Thus, vitrification under adequately controlled conditions does not cause additional loss beyond that produced during dehydrat ion by the vitr if ication solution. Cryopreservatiou by the complete vitrification of cultured cells and meristems eliminates concern for potentially damaging effects of intra- or extracellular crystallization (]Rail 1987). Thus, vitrification is a simple and effect method for cryopreserving meristems.

It is particularly important that cryopreserved meristems be capable of producing true-to-type plants identical to the non-treated phenotype. Cryopreserved meristems of wasabi by vitrification remained green after plating and developed shoots directly within 2 weeks without intermediary callus formation. Almost all of the shoots forming ix)ors were successfully transferred to pots. No morphological abnormalities were observed in

446

the plants developed from cryopreserved meristems. However, further study is necessary to confirm their phenotype by cytological, biochemical and morphological analysis.

Acknowledgments, The authors wish to express their cordial thanks to Mr. H.Kohmura, Hiroshima Prefectural Agricultural Research Center for technical cooperation. This study was supported by a special research grant 'Applied Bio-technology Program for Prefectural Agriculture' from Ministry of Agriculture, Forestry and Fisheries, Japan.

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